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Sample records for splicing regulatory protein

  1. Overexpression of KH-type splicing regulatory protein regulates proliferation, migration, and implantation ability of osteosarcoma.

    Science.gov (United States)

    Pruksakorn, Dumnoensun; Teeyakasem, Pimpisa; Klangjorhor, Jeerawan; Chaiyawat, Parunya; Settakorn, Jongkolnee; Diskul-Na-Ayudthaya, Penchatr; Chokchaichamnankit, Daranee; Pothacharoen, Peraphan; Srisomsap, Chantragan

    2016-09-01

    Osteosarcoma is a common malignant bone tumor in children and adolescents. The current 5-year survival rate is ~60% and that seems to be reaching a plateau. In order to improve treatment outcomes of osteosarcoma, a better understanding of tumorigenesis and underlying molecular mechanisms is required for searching out possible new treatment targets. This study aimed to identify the potential proteins involving the pathogenesis of osteosarcoma using a proteomics approach. Proteins extracted from primary cell culture of osteosarcoma (n=7) and osteoblasts of cancellous bone (n=7) were studied. Using 2-DE based proteomics and LC-MS/MS analysis, we successfully determined seven differentially expressed protein spots. Four upregulated proteins and three downregulated proteins were observed in this study in which KH-type splicing regulatory protein (KSRP) was selected for further exploration. KSRP was significantly upregulated in osteosarcoma cells compared to osteoblasts using western blot assay. In addition, immunohistochemistry demonstrated that KSRP was also highly expressed in osteosarcoma tissue of independent cases from the experimental group. More importantly, KSRP silencing of osteosarcoma cell lines significantly decreased cell proliferation, migration ability, as well as implantation and growth ability in chick chorioallantoic membrane assay. Taken together, these findings demonstrate, that KSRP plays important roles in regulatory controls of osteosarcoma pathogenesis and serves as a potentially therapeutic target of osteosarcoma.

  2. KH-type splicing regulatory protein is involved in esophageal squamous cell carcinoma progression.

    Science.gov (United States)

    Fujita, Yuji; Masuda, Kiyoshi; Hamada, Junichi; Shoda, Katsutoshi; Naruto, Takuya; Hamada, Satoshi; Miyakami, Yuko; Kohmoto, Tomohiro; Watanabe, Miki; Takahashi, Rizu; Tange, Shoichiro; Saito, Masako; Kudo, Yasusei; Fujiwara, Hitoshi; Ichikawa, Daisuke; Tangoku, Akira; Otsuji, Eigo; Imoto, Issei

    2017-11-24

    KH-type splicing regulatory protein (KHSRP) is a multifunctional RNA-binding protein, which is involved in several post-transcriptional aspects of RNA metabolism, including microRNA (miRNA) biogenesis. It affects distinct cell functions in different tissues and can have an impact on various pathological conditions. In the present study, we investigated the oncogenic functions of KHSRP and their underlying mechanisms in the pathogenesis of esophageal squamous cell carcinoma (ESCC). KHSRP expression levels were elevated in ESCC tumors when compared with those in non-tumorous tissues by immunohistochemistry, and cytoplasmic KHSRP overexpression was found to be an independent prognosticator for worse overall survival in a cohort of 104 patients with ESCC. KHSRP knockdown inhibited growth, migration, and invasion of ESCC cells. KHSRP knockdown also inhibited the maturation of cancer-associated miRNAs, such as miR-21, miR-130b, and miR-301, and induced the expression of their target mRNAs, such as BMP6, PDCD4, and TIMP3, resulting in the inhibition of epithelial-to-mesenchymal transition. Our findings uncover a novel oncogenic function of KHSRP in esophageal tumorigenesis and implicate its use as a marker for prognostic evaluation and as a putative therapeutic target in ESCC.

  3. HIV-1 splicing is controlled by local RNA structure and binding of splicing regulatory proteins at the major 5' splice site

    NARCIS (Netherlands)

    Mueller, Nancy; Berkhout, Ben; Das, Atze T.

    2015-01-01

    The 5' leader region of the human immunodeficiency virus 1 (HIV-1) RNA genome contains the major 5' splice site (ss) that is used in the production of the many spliced viral RNAs. This splice-donor (SD) region can fold into a stable stem-loop structure and the thermodynamic stability of this RNA

  4. Regulatory mechanisms for 3'-end alternative splicing and polyadenylation of the Glial Fibrillary Acidic Protein, GFAP, transcript

    DEFF Research Database (Denmark)

    Blechingberg, Jenny; Lykke-Andersen, Søren; Jensen, Torben Heick

    2007-01-01

    (PTB) protein enhanced both exon 7a polyadenylation and exon 7a splicing. Finally, increasing transcription by the VP16 trans-activator did not affect the frequency of use of the exon 7a polyadenylation signal whereas the exon 7a splicing frequency was decreased. Our data suggest a model...

  5. Overexpression of KH-type splicing regulatory protein regulates proliferation, migration, and implantation ability of osteosarcoma

    OpenAIRE

    Pruksakorn, Dumnoensun; Teeyakasem, Pimpisa; Klangjorhor, Jeerawan; Chaiyawat, Parunya; Settakorn, Jongkolnee; Diskul-Na-Ayudthaya, Penchatr; Chokchaichamnankit, Daranee; Pothacharoen, Peraphan; Srisomsap, Chantragan

    2016-01-01

    Osteosarcoma is a common malignant bone tumor in children and adolescents. The current 5-year survival rate is ~60% and that seems to be reaching a plateau. In order to improve treatment outcomes of osteosarcoma, a better understanding of tumorigenesis and underlying molecular mechanisms is required for searching out possible new treatment targets. This study aimed to identify the potential proteins involving the pathogenesis of osteosarcoma using a proteomics approach. Proteins extracted fro...

  6. Splicing regulatory factors, ageing and age-related disease.

    Science.gov (United States)

    Latorre, Eva; Harries, Lorna W

    2017-07-01

    Alternative splicing is a co-transcriptional process, which allows for the production of multiple transcripts from a single gene and is emerging as an important control point for gene expression. Alternatively expressed isoforms often have antagonistic function and differential temporal or spatial expression patterns, yielding enormous plasticity and adaptability to cells and increasing their ability to respond to environmental challenge. The regulation of alternative splicing is critical for numerous cellular functions in both pathological and physiological conditions, and deregulated alternative splicing is a key feature of common chronic diseases. Isoform choice is controlled by a battery of splicing regulatory proteins, which include the serine arginine rich (SRSF) proteins and the heterogeneous ribonucleoprotein (hnRNP) classes of genes. These important splicing regulators have been implicated in age-related disease, and in the ageing process itself. This review will outline the important contribution of splicing regulator proteins to ageing and age-related disease. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Protein splicing and its evolution in eukaryotes

    Directory of Open Access Journals (Sweden)

    Starokadomskyy P. L.

    2010-02-01

    Full Text Available Inteins, or protein introns, are parts of protein sequences that are post-translationally excised, their flanking regions (exteins being spliced together. This process was called protein splicing. Originally inteins were found in prokaryotic or unicellular eukaryotic organisms. But the general principles of post-translation protein rearrangement are evolving yielding different post-translation modification of proteins in multicellular organisms. For clarity, these non-intein mediated events call either protein rearrangements or protein editing. The most intriguing example of protein editing is proteasome-mediated splicing of antigens in vertebrates that may play important role in antigen presentation. Other examples of protein rearrangements are maturation of Hg-proteins (critical receptors in embryogenesis as well as maturation of several metabolic enzymes. Despite a lack of experimental data we try to analyze some intriguing examples of protein splicing evolution.

  8. The role of polypyrimidine tract-binding proteins and other hnRNP proteins in plant splicing regulation

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    Andreas eWachter

    2012-05-01

    Full Text Available Alternative precursor mRNA splicing is a widespread phenomenon in multicellular eukaryotes and represents a major means for functional expansion of the transcriptome. While several recent studies have revealed an important link between splicing regulation and fundamental biological processes in plants, many important aspects, such as the underlying splicing regulatory mechanisms, are so far not well understood. Splicing decisions are in general based on a splicing code that is determined by the dynamic interplay of splicing-controlling factors and cis-regulatory elements. Several members of the group of heterogeneous nuclear ribonucleoprotein (hnRNP proteins are well-known regulators of splicing in animals and the comparatively few reports on some of their plant homologues revealed similar functions. This also applies to polypyrimidine tract-binding proteins (PTBs, a thoroughly investigated class of hnRNP proteins with splicing regulatory functions in both animals and plants. Further examples from plants are auto- and cross-regulatory splicing circuits of glycine-rich RNA-binding proteins (GRPs and splicing enhancement by oligouridylatebinding proteins. Besides their role in defining splice site choice, hnRNP proteins are also involved in multiple other steps of nucleic acid metabolism, highlighting the functional versatility of this group of proteins in higher eukaryotes.

  9. Histone and RNA-binding protein interaction creates crosstalk network for regulation of alternative splicing.

    Science.gov (United States)

    Kim, Yong-Eun; Park, Chungoo; Kim, Kyoon Eon; Kim, Kee K

    2018-04-30

    Alternative splicing is an essential process in eukaryotes, as it increases the complexity of gene expression by generating multiple proteins from a single pre-mRNA. However, information on the regulatory mechanisms for alternative splicing is lacking, because splicing occurs over a short period via the transient interactions of proteins within functional complexes of the spliceosome. Here, we investigated in detail the molecular mechanisms connecting alternative splicing with epigenetic mechanisms. We identified interactions between histone proteins and splicing factors such as Rbfox2, Rbfox3, and splicing factor proline and glutamine rich protein (SFPQ) by in vivo crosslinking and immunoprecipitation. Furthermore, we confirmed that splicing factors were bound to specific modified residues of histone proteins. Additionally, changes in histone methylation due to histone methyltransferase inhibitor treatment notably affected alternative splicing in selected genes. Therefore, we suggested that there may be crosstalk mechanisms connecting histone modifications and RNA-binding proteins that increase the local concentration of RNA-binding proteins in alternative exon loci of nucleosomes by binding specific modified histone proteins, leading to alternative splicing. This crosstalk mechanism may play a major role in epigenetic processes such as histone modification and the regulation of alternative splicing. Copyright © 2018 Elsevier Inc. All rights reserved.

  10. Alanine repeats influence protein localization in splicing speckles and paraspeckles.

    Science.gov (United States)

    Chang, Shuo-Hsiu; Chang, Wei-Lun; Lu, Chia-Chen; Tarn, Woan-Yuh

    2014-12-16

    Mammalian splicing regulatory protein RNA-binding motif protein 4 (RBM4) has an alanine repeat-containing C-terminal domain (CAD) that confers both nuclear- and splicing speckle-targeting activities. Alanine-repeat expansion has pathological potential. Here we show that the alanine-repeat tracts influence the subnuclear targeting properties of the RBM4 CAD in cultured human cells. Notably, truncation of the alanine tracts redistributed a portion of RBM4 to paraspeckles. The alanine-deficient CAD was sufficient for paraspeckle targeting. On the other hand, alanine-repeat expansion reduced the mobility of RBM4 and impaired its splicing activity. We further took advantage of the putative coactivator activator (CoAA)-RBM4 conjoined splicing factor, CoAZ, to investigate the function of the CAD in subnuclear targeting. Transiently expressed CoAZ formed discrete nuclear foci that emerged and subsequently separated-fully or partially-from paraspeckles. Alanine-repeat expansion appeared to prevent CoAZ separation from paraspeckles, resulting in their complete colocalization. CoAZ foci were dynamic but, unlike paraspeckles, were resistant to RNase treatment. Our results indicate that the alanine-rich CAD, in conjunction with its conjoined RNA-binding domain(s), differentially influences the subnuclear localization and biogenesis of RBM4 and CoAZ. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  11. Reflections on protein splicing: structures, functions and mechanisms

    Science.gov (United States)

    Anraku, Yasuhiro; Satow, Yoshinori

    2009-01-01

    Twenty years ago, evidence that one gene produces two enzymes via protein splicing emerged from structural and expression studies of the VMA1 gene in Saccharomyces cerevisiae. VMA1 consists of a single open reading frame and contains two independent genetic information for Vma1p (a catalytic 70-kDa subunit of the vacuolar H+-ATPase) and VDE (a 50-kDa DNA endonuclease) as an in-frame spliced insert in the gene. Protein splicing is a posttranslational cellular process, in which an intervening polypeptide termed as the VMA1 intein is self-catalytically excised out from a nascent 120-kDa VMA1 precursor and two flanking polypeptides of the N- and C-exteins are ligated to produce the mature Vma1p. Subsequent studies have demonstrated that protein splicing is not unique to the VMA1 precursor and there are many operons in nature, which implement genetic information editing at protein level. To elucidate its structure-directed chemical mechanisms, a series of biochemical and crystal structural studies has been carried out with the use of various VMA1 recombinants. This article summarizes a VDE-mediated self-catalytic mechanism for protein splicing that is triggered and terminated solely via thiazolidine intermediates with tetrahedral configurations formed within the splicing sites where proton ingress and egress are driven by balanced protonation and deprotonation. PMID:19907126

  12. Involvement of the catalytic subunit of protein kinase A and of HA95 in pre-mRNA splicing

    International Nuclear Information System (INIS)

    Kvissel, Anne-Katrine; Orstavik, Sigurd; Eikvar, Sissel; Brede, Gaute; Jahnsen, Tore; Collas, Philippe; Akusjaervi, Goeran; Skalhegg, Bjorn Steen

    2007-01-01

    Protein kinase A (PKA) is a holoenzyme consisting of two catalytic (C) subunits bound to a regulatory (R) subunit dimer. Stimulation by cAMP dissociates the holoenzyme and causes translocation to the nucleus of a fraction of the C subunit. Apart from transcription regulation, little is known about the function of the C subunit in the nucleus. In the present report, we show that both Cα and Cβ are localized to spots in the mammalian nucleus. Double immunofluorescence analysis of splicing factor SC35 with the C subunit indicated that these spots are splicing factor compartments (SFCs). Using the E1A in vivo splicing assay, we found that catalytically active C subunits regulate alternative splicing and phosphorylate several members of the SR-protein family of splicing factors in vitro. Furthermore, nuclear C subunits co-localize with the C subunit-binding protein homologous to AKAP95, HA95. HA95 also regulates E1A alternative splicing in vivo, apparently through its N-terminal domain. Localization of the C subunit to SFCs and the E1A splicing pattern were unaffected by cAMP stimulation. Our findings demonstrate that the nuclear PKA C subunit co-locates with HA95 in SFCs and regulates pre-mRNA splicing, possibly through a cAMP-independent mechanism

  13. Pre-mRNA Splicing in Plants: In Vivo Functions of RNA-Binding Proteins Implicated in the Splicing Process

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    Katja Meyer

    2015-07-01

    Full Text Available Alternative pre-messenger RNA splicing in higher plants emerges as an important layer of regulation upon exposure to exogenous and endogenous cues. Accordingly, mutants defective in RNA-binding proteins predicted to function in the splicing process show severe phenotypic alterations. Among those are developmental defects, impaired responses to pathogen threat or abiotic stress factors, and misregulation of the circadian timing system. A suite of splicing factors has been identified in the model plant Arabidopsis thaliana. Here we summarize recent insights on how defects in these splicing factors impair plant performance.

  14. An Engineered Split Intein for Photoactivated Protein Trans-Splicing.

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    Stanley Wong

    Full Text Available Protein splicing is mediated by inteins that auto-catalytically join two separated protein fragments with a peptide bond. Here we engineered a genetically encoded synthetic photoactivatable intein (named LOVInC, by using the light-sensitive LOV2 domain from Avena sativa as a switch to modulate the splicing activity of the split DnaE intein from Nostoc punctiforme. Periodic blue light illumination of LOVInC induced protein splicing activity in mammalian cells. To demonstrate the broad applicability of LOVInC, synthetic protein systems were engineered for the light-induced reassembly of several target proteins such as fluorescent protein markers, a dominant positive mutant of RhoA, caspase-7, and the genetically encoded Ca2+ indicator GCaMP2. Spatial precision of LOVInC was demonstrated by targeting activity to specific mammalian cells. Thus, LOVInC can serve as a general platform for engineering light-based control for modulating the activity of many different proteins.

  15. Human papillomavirus type 16 E2 and E6 are RNA-binding proteins and inhibit in vitro splicing of pre-mRNAs with suboptimal splice sites

    International Nuclear Information System (INIS)

    Bodaghi, Sohrab; Jia Rong; Zheng Zhiming

    2009-01-01

    Human papillomavirus type 16 (HPV16) genome expresses six regulatory proteins (E1, E2, E4, E5, E6, and E7) which regulate viral DNA replication, gene expression, and cell function. We expressed HPV16 E2, E4, E6, and E7 from bacteria as GST fusion proteins and examined their possible functions in RNA splicing. Both HPV16 E2, a viral transactivator protein, and E6, a viral oncoprotein, inhibited splicing of pre-mRNAs containing an intron with suboptimal splice sites, whereas HPV5 E2 did not. The N-terminal half and the hinge region of HPV16 E2 as well as the N-terminal and central portions of HPV16 E6 are responsible for the suppression. HPV16 E2 interacts with pre-mRNAs through its C-terminal DNA-binding domain. HPV16 E6 binds pre-mRNAs via nuclear localization signal (NLS3) in its C-terminal half. Low-risk HPV6 E6, a cytoplasmic protein, does not bind RNA. Notably, both HPV16 E2 and E6 selectively bind to the intron region of pre-mRNAs and interact with a subset of cellular SR proteins. Together, these findings suggest that HPV16 E2 and E6 are RNA binding proteins and might play roles in posttranscriptional regulation during virus infection

  16. Sexy splicing: regulatory interplays governing sex determination from Drosophila to mammals.

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    Lalli, Enzo; Ohe, Kenji; Latorre, Elisa; Bianchi, Marco E; Sassone-Corsi, Paolo

    2003-02-01

    A remarkable array of strategies is used to produce sexual differentiation in different species. Complex gene hierarchies govern sex determination pathways, as exemplified by the classic D. melanogaster paradigm, where an interplay of transcriptional, splicing and translational mechanisms operate. Molecular studies support the hypothesis that genetic sex determination pathways evolved in reverse order, from downstream to upstream genes, in the cascade. The recent identification of a role for the key regulatory factors SRY and WT1(+KTS) in pre-mRNA splicing indicates that important steps in the mammalian sex determination process are likely to operate at the post-transcriptional level.

  17. Splicing Regulatory Elements and mRNA-abundance of dlg1 and capt, Genetically Interacting with dFMRP in Drosophila Brain

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    Maria Petrova

    2014-09-01

    Full Text Available To further understand the molecular and cellular mechanisms underlying the disease, we used the Drososphila FraX model and investigated a not well studied role of Drosophila Fragile X Mental Retardation Protein (dFMRP in alternative splicing of neuronal mRNAs to which it binds via a G-quartet sequence. By means of qRT-PCR we established the relative abundance of some isoforms of the gene dlg1, resulting from alternative exon skipping nearby a G-quartet and an exonic ESE-sequence, both acting as exonic splicing enhancers. We also investigated the relative mRNA-abundance of all capt-isoforms and the pre-mRNAs of both genes. We proposed a possible involvement of dFMRP in alternative splicing of genes, interacting with dfmr1. In the absence of dFMRP in larval and pupal brains, we found a change in the mRNA-level of one of the studied isoforms of dlg1 and of its pre-mRNA.We also established previously reported splicing regulatory elements and predicted computationally novel hexamere sequences in the exonic/intronic ends of both genes with p upative regulatory roles in alternative splicing.

  18. The connection between splicing and cancer

    OpenAIRE

    Srebrow, Anabella; Kornblihtt, Alberto Rodolfo

    2017-01-01

    Alternative splicing is a crucial mechanism for generating protein diversity. Different splice variants of a given protein can display different and even antagonistic biological functions. Therefore, appropriate control of their synthesis is required to assure the complex orchestration of cellular processes within multicellular organisms. Mutations in cisacting splicing elements or changes in the activity of regulatory proteins that compromise the accuracy of either constitutive or alternativ...

  19. Mechanism of protein splicing of the Pyrococcus abyssi lon protease intein

    International Nuclear Information System (INIS)

    O'Brien, Kevin M.; Schufreider, Ann K.; McGill, Melissa A.; O'Brien, Kathryn M.; Reitter, Julie N.; Mills, Kenneth V.

    2010-01-01

    Research highlights: → The Pyrococcus abyssi lon protease intein promotes efficient protein splicing. → Inteins with mutations that interfere with individual steps of splicing do not promote unproductive side reactions. → The intein splices with Lys in place of the highly conserved penultimate His. → The intein is flanked by a Gly-rich region at its C terminus that may increase the efficiency of the third step of splicing, Asn cyclization coupled to peptide bond cleavage. -- Abstract: Protein splicing is a post-translational process by which an intervening polypeptide, the intein, excises itself from the flanking polypeptides, the exteins, coupled to ligation of the exteins. The lon protease of Pyrococcus abyssi (Pab) is interrupted by an intein. When over-expressed as a fusion protein in Escherichia coli, the Pab lon protease intein can promote efficient protein splicing. Mutations that block individual steps of splicing generally do not lead to unproductive side reactions, suggesting that the intein tightly coordinates the splicing process. The intein can splice, although it has Lys in place of the highly conserved penultimate His, and mutants of the intein in the C-terminal region lead to the accumulation of stable branched-ester intermediate.

  20. SON connects the splicing-regulatory network with pluripotency in human embryonic stem cells.

    Science.gov (United States)

    Lu, Xinyi; Göke, Jonathan; Sachs, Friedrich; Jacques, Pierre-Étienne; Liang, Hongqing; Feng, Bo; Bourque, Guillaume; Bubulya, Paula A; Ng, Huck-Hui

    2013-10-01

    Human embryonic stem cells (hESCs) harbour the ability to undergo lineage-specific differentiation into clinically relevant cell types. Transcription factors and epigenetic modifiers are known to play important roles in the maintenance of pluripotency of hESCs. However, little is known about regulation of pluripotency through splicing. In this study, we identify the spliceosome-associated factor SON as a factor essential for the maintenance of hESCs. Depletion of SON in hESCs results in the loss of pluripotency and cell death. Using genome-wide RNA profiling, we identified transcripts that are regulated by SON. Importantly, we confirmed that SON regulates the proper splicing of transcripts encoding for pluripotency regulators such as OCT4, PRDM14, E4F1 and MED24. Furthermore, we show that SON is bound to these transcripts in vivo. In summary, we connect a splicing-regulatory network for accurate transcript production to the maintenance of pluripotency and self-renewal of hESCs.

  1. The implications of alternative splicing in the ENCODE protein complement

    DEFF Research Database (Denmark)

    Tress, Michael L.; Martelli, Pier Luigi; Frankish, Adam

    2007-01-01

    suggested as one explanation for the discrepancy between the number of human genes and functional complexity. Here, we carry out a detailed study of the alternatively spliced gene products annotated in the ENCODE pilot project. We find that alternative splicing in human genes is more frequent than has...

  2. Benzo[a]pyrene treatment leads to changes in nuclear protein expression and alternative splicing

    Energy Technology Data Exchange (ETDEWEB)

    Yan Chunlan; Wu Wei [Department of Toxicology, Zhejiang University School of Public Health, 388 Yu-Hang-Tang Road, Hangzhou, Zhejiang 310058 (China); Li Haiyan [Department of Toxicology, Zhejiang University School of Public Health, 388 Yu-Hang-Tang Road, Hangzhou, Zhejiang 310058 (China); Huzhou Maternity and Child Care Hospital, Huzhou, Zhejiang 313000 (China); Zhang Guanglin [Department of Toxicology, Zhejiang University School of Public Health, 388 Yu-Hang-Tang Road, Hangzhou, Zhejiang 310058 (China); Duerksen-Hughes, Penelope J. [Department of Basic Sciences, Loma Linda University School of Medicine, Loma Linda, CA 92354 (United States); Zhu Xinqiang, E-mail: zhuxq@zju.edu.cn [Department of Toxicology, Zhejiang University School of Public Health, 388 Yu-Hang-Tang Road, Hangzhou, Zhejiang 310058 (China); Yang Jun, E-mail: gastate@zju.edu.cn [Department of Toxicology, Zhejiang University School of Public Health, 388 Yu-Hang-Tang Road, Hangzhou, Zhejiang 310058 (China); Zhejiang-California International Nanosystems Institute, Hangzhou, Zhejiang 310029 (China)

    2010-04-01

    Benzo[a]pyrene (BaP) is a potent pro-carcinogen generated from the combustion of fossil fuel and cigarette smoke. Previously, using a proteomic approach, we have shown that BaP can induce changes in the expression of many cellular proteins, including transcription regulators. In the present study, using a similar approach, we examined the nuclear protein response to BaP in HeLa cells and found that BaP treatment caused expression changes in many nuclear proteins. Twenty-four of these proteins were successfully identified, several of which are involved in the alternative splicing of mRNA, DNA replication, recombination, and repair. The changed expression levels were further confirmed by immunoblot analysis using specific antibodies for two proteins, Lamin A and mitotic checkpoint protein Bub3. The nuclear localization of these two proteins was also confirmed by confocal microscopy. To determine whether alternative splicing was activated following BaP treatment, we examined Fas and CD44, two genes previously shown to be targets of alternative splicing in respond to DNA damage. While no significant activation of alternative splicing was observed for Fas, CD44 splicing variants were found after BaP treatment. Together, these data show that DNA damage induces dramatic changes in nuclear protein expression, and that alternative splicing might be involved in the cellular response to DNA damage.

  3. Intrasplicing coordinates alternative first exons with alternative splicing in the protein 4.1R gene

    Energy Technology Data Exchange (ETDEWEB)

    Conboy, John G.; Parra, Marilyn K.; Tan, Jeff S.; Mohandas, Narla; Conboy, John G.

    2008-11-07

    In the protein 4.1R gene, alternative first exons splice differentially to alternative 3' splice sites far downstream in exon 2'/2 (E2'/2). We describe a novel intrasplicing mechanism by which exon 1A (E1A) splices exclusively to the distal E2'/2 acceptor via two nested splicing reactions regulated by novel properties of exon 1B (E1B). E1B behaves as an exon in the first step, using its consensus 5' donor to splice to the proximal E2'/2 acceptor. A long region of downstream intron is excised, juxtaposing E1B with E2'/2 to generate a new composite acceptor containing the E1B branchpoint/pyrimidine tract and E2 distal 3' AG-dinucleotide. Next, the upstream E1A splices over E1B to this distal acceptor, excising the remaining intron plus E1B and E2' to form mature E1A/E2 product. We mapped branch points for both intrasplicing reactions and demonstrated that mutation of the E1B 5' splice site or branchpoint abrogates intrasplicing. In the 4.1R gene, intrasplicing ultimately determines N-terminal protein structure and function. More generally, intrasplicing represents a new mechanism whereby alternative promoters can be coordinated with downstream alternative splicing.

  4. HOLLYWOOD: a comparative relational database of alternative splicing.

    Science.gov (United States)

    Holste, Dirk; Huo, George; Tung, Vivian; Burge, Christopher B

    2006-01-01

    RNA splicing is an essential step in gene expression, and is often variable, giving rise to multiple alternatively spliced mRNA and protein isoforms from a single gene locus. The design of effective databases to support experimental and computational investigations of alternative splicing (AS) is a significant challenge. In an effort to integrate accurate exon and splice site annotation with current knowledge about splicing regulatory elements and predicted AS events, and to link information about the splicing of orthologous genes in different species, we have developed the Hollywood system. This database was built upon genomic annotation of splicing patterns of known genes derived from spliced alignment of complementary DNAs (cDNAs) and expressed sequence tags, and links features such as splice site sequence and strength, exonic splicing enhancers and silencers, conserved and non-conserved patterns of splicing, and cDNA library information for inferred alternative exons. Hollywood was implemented as a relational database and currently contains comprehensive information for human and mouse. It is accompanied by a web query tool that allows searches for sets of exons with specific splicing characteristics or splicing regulatory element composition, or gives a graphical or sequence-level summary of splicing patterns for a specific gene. A streamlined graphical representation of gene splicing patterns is provided, and these patterns can alternatively be layered onto existing information in the UCSC Genome Browser. The database is accessible at http://hollywood.mit.edu.

  5. Semi-supervised Learning Predicts Approximately One Third of the Alternative Splicing Isoforms as Functional Proteins

    Directory of Open Access Journals (Sweden)

    Yanqi Hao

    2015-07-01

    Full Text Available Alternative splicing acts on transcripts from almost all human multi-exon genes. Notwithstanding its ubiquity, fundamental ramifications of splicing on protein expression remain unresolved. The number and identity of spliced transcripts that form stably folded proteins remain the sources of considerable debate, due largely to low coverage of experimental methods and the resulting absence of negative data. We circumvent this issue by developing a semi-supervised learning algorithm, positive unlabeled learning for splicing elucidation (PULSE; http://www.kimlab.org/software/pulse, which uses 48 features spanning various categories. We validated its accuracy on sets of bona fide protein isoforms and directly on mass spectrometry (MS spectra for an overall AU-ROC of 0.85. We predict that around 32% of “exon skipping” alternative splicing events produce stable proteins, suggesting that the process engenders a significant number of previously uncharacterized proteins. We also provide insights into the distribution of positive isoforms in various functional classes and into the structural effects of alternative splicing.

  6. Impaired RNA splicing of 5'-regulatory sequences of the astroglial glutamate transporter EAAT2 in human astrocytoma

    NARCIS (Netherlands)

    Münch, C.; Penndorf, A.; Schwalenstöcker, B.; Troost, D.; Ludolph, A. C.; Ince, P.; Meyer, T.

    2001-01-01

    A loss of the glutamate transporter EAAT2 has been reported in the neoplastic transformation of astrocytic cells and astrocytoma. The RNA expression of EAAT2 and five 5'-regulatory splice variants was investigated to identify alterations of the post-transcriptional EAAT2 gene regulation in human

  7. Genetics of alternative splicing evolution during sunflower domestication.

    Science.gov (United States)

    Smith, Chris C R; Tittes, Silas; Mendieta, J Paul; Collier-Zans, Erin; Rowe, Heather C; Rieseberg, Loren H; Kane, Nolan C

    2018-06-11

    Alternative splicing enables organisms to produce the diversity of proteins necessary for multicellular life by using relatively few protein-coding genes. Although differences in splicing have been identified among divergent taxa, the shorter-term evolution of splicing is understudied. The origins of novel splice forms, and the contributions of alternative splicing to major evolutionary transitions, are largely unknown. This study used transcriptomes of wild and domesticated sunflowers to examine splice differentiation and regulation during domestication. We identified substantial splicing divergence between wild and domesticated sunflowers, mainly in the form of intron retention. Transcripts with divergent splicing were enriched for seed-development functions, suggesting that artificial selection impacted splicing patterns. Mapping of quantitative trait loci (QTLs) associated with 144 differential splicing cases revealed primarily trans -acting variation affecting splicing patterns. A large proportion of identified QTLs contain known spliceosome proteins and are associated with splicing variation in multiple genes. Examining a broader set of wild and domesticated sunflower genotypes revealed that most differential splicing patterns in domesticated sunflowers likely arose from standing variation in wild Helianthus annuus and gained frequency during the domestication process. However, several domesticate-associated splicing patterns appear to be introgressed from other Helianthus species. These results suggest that sunflower domestication involved selection on pleiotropic regulatory alleles. More generally, our findings indicate that substantial differences in isoform abundances arose rapidly during a recent evolutionary transition and appear to contribute to adaptation and population divergence.

  8. Protein trans-splicing of multiple atypical split inteins engineered from natural inteins.

    Directory of Open Access Journals (Sweden)

    Ying Lin

    Full Text Available Protein trans-splicing by split inteins has many uses in protein production and research. Splicing proteins with synthetic peptides, which employs atypical split inteins, is particularly useful for site-specific protein modifications and labeling, because the synthetic peptide can be made to contain a variety of unnatural amino acids and chemical modifications. For this purpose, atypical split inteins need to be engineered to have a small N-intein or C-intein fragment that can be more easily included in a synthetic peptide that also contains a small extein to be trans-spliced onto target proteins. Here we have successfully engineered multiple atypical split inteins capable of protein trans-splicing, by modifying and testing more than a dozen natural inteins. These included both S1 split inteins having a very small (11-12 aa N-intein fragment and S11 split inteins having a very small (6 aa C-intein fragment. Four of the new S1 and S11 split inteins showed high efficiencies (85-100% of protein trans-splicing both in E. coli cells and in vitro. Under in vitro conditions, they exhibited reaction rate constants ranging from ~1.7 × 10(-4 s(-1 to ~3.8 × 10(-4 s(-1, which are comparable to or higher than those of previously reported atypical split inteins. These findings should facilitate a more general use of trans-splicing between proteins and synthetic peptides, by expanding the availability of different atypical split inteins. They also have implications on understanding the structure-function relationship of atypical split inteins, particularly in terms of intein fragment complementation.

  9. Co-evolution of SNF spliceosomal proteins with their RNA targets in trans-splicing nematodes.

    Science.gov (United States)

    Strange, Rex Meade; Russelburg, L Peyton; Delaney, Kimberly J

    2016-08-01

    Although the mechanism of pre-mRNA splicing has been well characterized, the evolution of spliceosomal proteins is poorly understood. The U1A/U2B″/SNF family (hereafter referred to as the SNF family) of RNA binding spliceosomal proteins participates in both the U1 and U2 small interacting nuclear ribonucleoproteins (snRNPs). The highly constrained nature of this system has inhibited an analysis of co-evolutionary trends between the proteins and their RNA binding targets. Here we report accelerated sequence evolution in the SNF protein family in Phylum Nematoda, which has allowed an analysis of protein:RNA co-evolution. In a comparison of SNF genes from ecdysozoan species, we found a correlation between trans-splicing species (nematodes) and increased phylogenetic branch lengths of the SNF protein family, with respect to their sister clade Arthropoda. In particular, we found that nematodes (~70-80 % of pre-mRNAs are trans-spliced) have experienced higher rates of SNF sequence evolution than arthropods (predominantly cis-spliced) at both the nucleotide and amino acid levels. Interestingly, this increased evolutionary rate correlates with the reliance on trans-splicing by nematodes, which would alter the role of the SNF family of spliceosomal proteins. We mapped amino acid substitutions to functionally important regions of the SNF protein, specifically to sites that are predicted to disrupt protein:RNA and protein:protein interactions. Finally, we investigated SNF's RNA targets: the U1 and U2 snRNAs. Both are more divergent in nematodes than arthropods, suggesting the RNAs have co-evolved with SNF in order to maintain the necessarily high affinity interaction that has been characterized in other species.

  10. Primate-specific spliced PMCHL RNAs are non-protein coding in human and macaque tissues

    Directory of Open Access Journals (Sweden)

    Delerue-Audegond Audrey

    2008-12-01

    Full Text Available Abstract Background Brain-expressed genes that were created in primate lineage represent obvious candidates to investigate molecular mechanisms that contributed to neural reorganization and emergence of new behavioural functions in Homo sapiens. PMCHL1 arose from retroposition of a pro-melanin-concentrating hormone (PMCH antisense mRNA on the ancestral human chromosome 5p14 when platyrrhines and catarrhines diverged. Mutations before divergence of hylobatidae led to creation of new exons and finally PMCHL1 duplicated in an ancestor of hominids to generate PMCHL2 at the human chromosome 5q13. A complex pattern of spliced and unspliced PMCHL RNAs were found in human brain and testis. Results Several novel spliced PMCHL transcripts have been characterized in human testis and fetal brain, identifying an additional exon and novel splice sites. Sequencing of PMCHL genes in several non-human primates allowed to carry out phylogenetic analyses revealing that the initial retroposition event took place within an intron of the brain cadherin (CDH12 gene, soon after platyrrhine/catarrhine divergence, i.e. 30–35 Mya, and was concomitant with the insertion of an AluSg element. Sequence analysis of the spliced PMCHL transcripts identified only short ORFs of less than 300 bp, with low (VMCH-p8 and protein variants or no evolutionary conservation. Western blot analyses of human and macaque tissues expressing PMCHL RNA failed to reveal any protein corresponding to VMCH-p8 and protein variants encoded by spliced transcripts. Conclusion Our present results improve our knowledge of the gene structure and the evolutionary history of the primate-specific chimeric PMCHL genes. These genes produce multiple spliced transcripts, bearing short, non-conserved and apparently non-translated ORFs that may function as mRNA-like non-coding RNAs.

  11. Conditional protein splicing: a new tool to control protein structure and function in vitro and in vivo.

    Science.gov (United States)

    Mootz, Henning D; Blum, Elyse S; Tyszkiewicz, Amy B; Muir, Tom W

    2003-09-03

    Protein splicing is a naturally occurring process in which an intervening intein domain excises itself out of a precursor polypeptide in an autocatalytic fashion with concomitant linkage of the two flanking extein sequences by a native peptide bond. We have recently reported an engineered split VMA intein whose splicing activity in trans between two polypeptides can be triggered by the small molecule rapamycin. In this report, we show that this conditional protein splicing (CPS) system can be used in mammalian cells. Two model constructs harboring maltose-binding protein (MBP) and a His-tag as exteins were expressed from a constitutive promoter after transient transfection. The splicing product MBP-His was detected by Western blotting and immunoprecipitation in cells treated with rapamycin or a nontoxic analogue thereof. No background splicing in the absence of the small-molecule inducer was observed over a 24-h time course. Product formation could be detected within 10 min of addition of rapamycin, indicating the advantage of the posttranslational nature of CPS for quick responses. The level of protein splicing was dose dependent and could be competitively attenuated with the small molecule ascomycin. In related studies, the geometric flexibility of the CPS components was investigated with a series of purified proteins. The FKBP and FRB domains, which are dimerized by rapamycin and thereby induce the reconstitution of the split intein, were fused to the extein sequences of the split intein halves. CPS was still triggered by rapamycin when FKBP and FRB occupied one or both of the extein positions. This finding suggests yet further applications of CPS in the area of proteomics. In summary, CPS holds great promise to become a powerful new tool to control protein structure and function in vitro and in living cells.

  12. Two CRM protein subfamilies cooperate in the splicing of group IIB introns in chloroplasts.

    Science.gov (United States)

    Asakura, Yukari; Bayraktar, Omer Ali; Barkan, Alice

    2008-11-01

    Chloroplast genomes in angiosperms encode approximately 20 group II introns, approximately half of which are classified as subgroup IIB. The splicing of all but one of the subgroup IIB introns requires a heterodimer containing the peptidyl-tRNA hydrolase homolog CRS2 and one of two closely related proteins, CAF1 or CAF2, that harbor a recently recognized RNA binding domain called the CRM domain. Two CRS2/CAF-dependent introns require, in addition, a CRM domain protein called CFM2 that is only distantly related to CAF1 and CAF2. Here, we show that CFM3, a close relative of CFM2, associates in vivo with those CRS2/CAF-dependent introns that are not CFM2 ligands. Mutant phenotypes in rice and Arabidopsis support a role for CFM3 in the splicing of most of the introns with which it associates. These results show that either CAF1 or CAF2 and either CFM2 or CFM3 simultaneously bind most chloroplast subgroup IIB introns in vivo, and that the CAF and CFM subunits play nonredundant roles in splicing. These results suggest that the expansion of the CRM protein family in plants resulted in two subfamilies that play different roles in group II intron splicing, with further diversification within a subfamily to accommodate multiple intron ligands.

  13. Polypyrimidine Tract Binding Protein Homologs from Arabidopsis Are Key Regulators of Alternative Splicing with Implications in Fundamental Developmental Processes[W

    Science.gov (United States)

    Rühl, Christina; Stauffer, Eva; Kahles, André; Wagner, Gabriele; Drechsel, Gabriele; Rätsch, Gunnar; Wachter, Andreas

    2012-01-01

    Alternative splicing (AS) generates transcript variants by variable exon/intron definition and massively expands transcriptome diversity. Changes in AS patterns have been found to be linked to manifold biological processes, yet fundamental aspects, such as the regulation of AS and its functional implications, largely remain to be addressed. In this work, widespread AS regulation by Arabidopsis thaliana Polypyrimidine tract binding protein homologs (PTBs) was revealed. In total, 452 AS events derived from 307 distinct genes were found to be responsive to the levels of the splicing factors PTB1 and PTB2, which predominantly triggered splicing of regulated introns, inclusion of cassette exons, and usage of upstream 5′ splice sites. By contrast, no major AS regulatory function of the distantly related PTB3 was found. Dependent on their position within the mRNA, PTB-regulated events can both modify the untranslated regions and give rise to alternative protein products. We find that PTB-mediated AS events are connected to diverse biological processes, and the functional implications of selected instances were further elucidated. Specifically, PTB misexpression changes AS of PHYTOCHROME INTERACTING FACTOR6, coinciding with altered rates of abscisic acid–dependent seed germination. Furthermore, AS patterns as well as the expression of key flowering regulators were massively changed in a PTB1/2 level-dependent manner. PMID:23192226

  14. A novel protein-protein interaction in the RES (REtention and Splicing) complex.

    Science.gov (United States)

    Tripsianes, Konstantinos; Friberg, Anders; Barrandon, Charlotte; Brooks, Mark; van Tilbeurgh, Herman; Seraphin, Bertrand; Sattler, Michael

    2014-10-10

    The retention and splicing (RES) complex is a conserved spliceosome-associated module that was shown to enhance splicing of a subset of transcripts and promote the nuclear retention of unspliced pre-mRNAs in yeast. The heterotrimeric RES complex is organized around the Snu17p protein that binds to both the Bud13p and Pml1p subunits. Snu17p exhibits an RRM domain that resembles a U2AF homology motif (UHM) and Bud13p harbors a Trp residue reminiscent of an UHM-ligand motif (ULM). It has therefore been proposed that the interaction between Snu17p and Bud13p resembles canonical UHM-ULM complexes. Here, we have used biochemical and NMR structural analysis to characterize the structure of the yeast Snu17p-Bud13p complex. Unlike known UHMs that sequester the Trp residue of the ULM ligand in a hydrophobic pocket, Snu17p and Bud13p utilize a large interaction surface formed around the two helices of the Snu17p domain. In total 18 residues of the Bud13p ligand wrap around the Snu17p helical surface in an U-turn-like arrangement. The invariant Trp(232) in Bud13p is located in the center of the turn, and contacts surface residues of Snu17p. The structural data are supported by mutational analysis and indicate that Snu17p provides an extended binding surface with Bud13p that is notably distinct from canonical UHM-ULM interactions. Our data highlight structural diversity in RRM-protein interactions, analogous to the one seen for nucleic acid interactions. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. TAPAS: tools to assist the targeted protein quantification of human alternative splice variants.

    Science.gov (United States)

    Yang, Jae-Seong; Sabidó, Eduard; Serrano, Luis; Kiel, Christina

    2014-10-15

    In proteomes of higher eukaryotes, many alternative splice variants can only be detected by their shared peptides. This makes it highly challenging to use peptide-centric mass spectrometry to distinguish and to quantify protein isoforms resulting from alternative splicing events. We have developed two complementary algorithms based on linear mathematical models to efficiently compute a minimal set of shared and unique peptides needed to quantify a set of isoforms and splice variants. Further, we developed a statistical method to estimate the splice variant abundances based on stable isotope labeled peptide quantities. The algorithms and databases are integrated in a web-based tool, and we have experimentally tested the limits of our quantification method using spiked proteins and cell extracts. The TAPAS server is available at URL http://davinci.crg.es/tapas/. luis.serrano@crg.eu or christina.kiel@crg.eu Supplementary data are available at Bioinformatics online. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  16. Spliced X-box binding protein 1 couples the unfolded protein response to hexosamine biosynthetic pathway.

    Science.gov (United States)

    Wang, Zhao V; Deng, Yingfeng; Gao, Ningguo; Pedrozo, Zully; Li, Dan L; Morales, Cyndi R; Criollo, Alfredo; Luo, Xiang; Tan, Wei; Jiang, Nan; Lehrman, Mark A; Rothermel, Beverly A; Lee, Ann-Hwee; Lavandero, Sergio; Mammen, Pradeep P A; Ferdous, Anwarul; Gillette, Thomas G; Scherer, Philipp E; Hill, Joseph A

    2014-03-13

    The hexosamine biosynthetic pathway (HBP) generates uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) for glycan synthesis and O-linked GlcNAc (O-GlcNAc) protein modifications. Despite the established role of the HBP in metabolism and multiple diseases, regulation of the HBP remains largely undefined. Here, we show that spliced X-box binding protein 1 (Xbp1s), the most conserved signal transducer of the unfolded protein response (UPR), is a direct transcriptional activator of the HBP. We demonstrate that the UPR triggers HBP activation via Xbp1s-dependent transcription of genes coding for key, rate-limiting enzymes. We further establish that this previously unrecognized UPR-HBP axis is triggered in a variety of stress conditions. Finally, we demonstrate a physiologic role for the UPR-HBP axis by showing that acute stimulation of Xbp1s in heart by ischemia/reperfusion confers robust cardioprotection in part through induction of the HBP. Collectively, these studies reveal that Xbp1s couples the UPR to the HBP to protect cells under stress. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. Pre-mRNA mis-splicing of sarcomeric genes in heart failure.

    Science.gov (United States)

    Zhu, Chaoqun; Chen, Zhilong; Guo, Wei

    2017-08-01

    Pre-mRNA splicing is an important biological process that allows production of multiple proteins from a single gene in the genome, and mainly contributes to protein diversity in eukaryotic organisms. Alternative splicing is commonly governed by RNA binding proteins to meet the ever-changing demands of the cell. However, the mis-splicing may lead to human diseases. In the heart of human, mis-regulation of alternative splicing has been associated with heart failure. In this short review, we focus on alternative splicing of sarcomeric genes and review mis-splicing related heart failure with relatively well studied Sarcomeric genes and splicing mechanisms with identified regulatory factors. The perspective of alternative splicing based therapeutic strategies in heart failure has also been discussed. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. A dual-specificity isoform of the protein kinase inhibitor PKI produced by alternate gene splicing.

    Science.gov (United States)

    Kumar, Priyadarsini; Walsh, Donal A

    2002-03-15

    We have previously shown that the protein kinase inhibitor beta (PKIbeta) form of the cAMP-dependent protein kinase inhibitor exists in multiple isoforms, some of which are specific inhibitors of the cAMP-dependent protein kinase, whereas others also inhibit the cGMP-dependent enzyme [Kumar, Van Patten and Walsh (1997), J. Biol. Chem. 272, 20011-20020]. We have now demonstrated that the switch from a cAMP-dependent protein kinase (PKA)-specific inhibitor to one with dual specificity arises as a consequence of alternate gene splicing. We have confirmed using bacterially produced pure protein that a single inhibitor species has dual specificity for both PKA and cGMP-dependent protein kinase (PKG), inhibiting each with very high and closely similar inhibitory potencies. The gene splicing converted a protein with 70 amino acids into one of 109 amino acids, and did not change the inhibitory potency to PKA, but changed it from a protein that had no detectable PKG inhibitory activity to one that now inhibited PKG in the nanomolar range.

  19. Meta-Analysis of Multiple Sclerosis Microarray Data Reveals Dysregulation in RNA Splicing Regulatory Genes

    Directory of Open Access Journals (Sweden)

    Elvezia Maria Paraboschi

    2015-09-01

    Full Text Available Abnormalities in RNA metabolism and alternative splicing (AS are emerging as important players in complex disease phenotypes. In particular, accumulating evidence suggests the existence of pathogenic links between multiple sclerosis (MS and altered AS, including functional studies showing that an imbalance in alternatively-spliced isoforms may contribute to disease etiology. Here, we tested whether the altered expression of AS-related genes represents a MS-specific signature. A comprehensive comparative analysis of gene expression profiles of publicly-available microarray datasets (190 MS cases, 182 controls, followed by gene-ontology enrichment analysis, highlighted a significant enrichment for differentially-expressed genes involved in RNA metabolism/AS. In detail, a total of 17 genes were found to be differentially expressed in MS in multiple datasets, with CELF1 being dysregulated in five out of seven studies. We confirmed CELF1 downregulation in MS (p = 0.0015 by real-time RT-PCRs on RNA extracted from blood cells of 30 cases and 30 controls. As a proof of concept, we experimentally verified the unbalance in alternatively-spliced isoforms in MS of the NFAT5 gene, a putative CELF1 target. In conclusion, for the first time we provide evidence of a consistent dysregulation of splicing-related genes in MS and we discuss its possible implications in modulating specific AS events in MS susceptibility genes.

  20. Human-specific protein isoforms produced by novel splice sites in the human genome after the human-chimpanzee divergence

    Directory of Open Access Journals (Sweden)

    Kim Dong Seon

    2012-11-01

    Full Text Available Abstract Background Evolution of splice sites is a well-known phenomenon that results in transcript diversity during human evolution. Many novel splice sites are derived from repetitive elements and may not contribute to protein products. Here, we analyzed annotated human protein-coding exons and identified human-specific splice sites that arose after the human-chimpanzee divergence. Results We analyzed multiple alignments of the annotated human protein-coding exons and their respective orthologous mammalian genome sequences to identify 85 novel splice sites (50 splice acceptors and 35 donors in the human genome. The novel protein-coding exons, which are expressed either constitutively or alternatively, produce novel protein isoforms by insertion, deletion, or frameshift. We found three cases in which the human-specific isoform conferred novel molecular function in the human cells: the human-specific IMUP protein isoform induces apoptosis of the trophoblast and is implicated in pre-eclampsia; the intronization of a part of SMOX gene exon produces inactive spermine oxidase; the human-specific NUB1 isoform shows reduced interaction with ubiquitin-like proteins, possibly affecting ubiquitin pathways. Conclusions Although the generation of novel protein isoforms does not equate to adaptive evolution, we propose that these cases are useful candidates for a molecular functional study to identify proteomic changes that might bring about novel phenotypes during human evolution.

  1. Roles of viral and cellular proteins in the expression of alternatively spliced HTLV-1 pX mRNAs

    International Nuclear Information System (INIS)

    Princler, Gerald L.; Julias, John G.; Hughes, Stephen H.; Derse, David

    2003-01-01

    The human T cell leukemia virus type 1 (HTLV-1) genome contains a cluster of at least five open reading frames (ORFs) near the 3' terminus within the pX region. The pX ORFs are encoded by mono- or bicistronic mRNAs that are generated by alternative splicing. The various pX mRNAs result from skipping of the internal exon (2-exon versus 3-exon isofoms) or from the utilization of alternative splice acceptor sites in the terminal exon. The Rex and Tax proteins, encoded by ORFs X-III and X-IV, have been studied intensively and are encoded by the most abundant of the alternative 3-exon mRNAs. The protein products of the other pX ORFs have not been detected in HTLV-1-infected cell lines and the levels of the corresponding mRNAs have not been accurately established. We have used real-time RT-PCR with splice-site specific primers to accurately measure the levels of individual pX mRNA species in chronically infected T cell lines. We have asked whether virus regulatory proteins or ectopic expression of cellular factors influence pX mRNA splicing in cells that were transfected with HTLV-1 provirus clones. In chronically infected cell lines, the pX-tax/rex mRNA was present at 500- to 2500-fold higher levels than the pX-tax-orfII mRNA and at approximately 1000-fold higher levels than pX-rex-orfI mRNA. Chronically infected cell lines that contain numerous defective proviruses expressed 2-exon forms of pX mRNAs at significantly higher levels compared to cell lines that contain a single full-length provirus. Cells transfected with provirus expression plasmids expressed similar relative amounts of 3-exon pX mRNAs but lower levels of 2-exon mRNA forms compared to cells containing a single, full-length provirus. The pX mRNA expression patterns were nearly identical in cells transfected with wild-type, Tax-minus, or Rex-minus proviruses. Cotransfection of cells with HTLV-1 provirus in combination with SF2/ASF expression plasmid resulted in a relative increase in pX-tax/rex m

  2. Alternative splicing affects the targeting sequence of peroxisome proteins in Arabidopsis.

    Science.gov (United States)

    An, Chuanjing; Gao, Yuefang; Li, Jinyu; Liu, Xiaomin; Gao, Fuli; Gao, Hongbo

    2017-07-01

    A systematic analysis of the Arabidopsis genome in combination with localization experiments indicates that alternative splicing affects the peroxisomal targeting sequence of at least 71 genes in Arabidopsis. Peroxisomes are ubiquitous eukaryotic cellular organelles that play a key role in diverse metabolic functions. All peroxisome proteins are encoded by nuclear genes and target to peroxisomes mainly through two types of targeting signals: peroxisomal targeting signal type 1 (PTS1) and PTS2. Alternative splicing (AS) is a process occurring in all eukaryotes by which a single pre-mRNA can generate multiple mRNA variants, often encoding proteins with functional differences. However, the effects of AS on the PTS1 or PTS2 and the targeting of the protein were rarely studied, especially in plants. Here, we systematically analyzed the genome of Arabidopsis, and found that the C-terminal targeting sequence PTS1 of 66 genes and the N-terminal targeting sequence PTS2 of 5 genes are affected by AS. Experimental determination of the targeting of selected protein isoforms further demonstrated that AS at both the 5' and 3' region of a gene can affect the inclusion of PTS2 and PTS1, respectively. This work underscores the importance of AS on the global regulation of peroxisome protein targeting.

  3. Cytoplasmic tethering of a RING protein RBCK1 by its splice variant lacking the RING domain

    International Nuclear Information System (INIS)

    Yoshimoto, Nobuo; Tatematsu, Kenji; Koyanagi, Tomoyoshi; Okajima, Toshihide; Tanizawa, Katsuyuki; Kuroda, Shun'ichi

    2005-01-01

    RBCC protein interacting with PKC 1 (RBCK1) is a transcription factor belonging to the RING-IBR protein family and has been shown to shuttle between the nucleus and cytoplasm, possessing both the nuclear export and localization signals within its amino acid sequence. RBCK2, lacking the C-terminal half of RBCK1 including the RING-IBR domain, has also been identified as an alternative splice variant of RBCK1. RBCK2 shows no transcriptional activity and instead it represses the transcriptional activity of RBCK1. Here, we show that RBCK2 is present usually in the cytoplasm containing two Leu-rich regions that presumably serve as a nuclear export signal (NES). Moreover, an NES-disrupted RBCK1 that is mostly localized within the nucleus is translocated to the cytoplasm when coexpressed with RBCK2, suggesting that RBCK2 serves as a cytoplasmic tethering protein for RBCK1. We propose a novel and general function of RING-lacking splice variants of RING proteins to control the intracellular localization and functions of the parental RING proteins by forming a hetero-oligomeric complex

  4. Protein tyrosine phosphatases: regulatory mechanisms.

    NARCIS (Netherlands)

    den Hertog, J.; Ostman, A.; Bohmer, F.D.

    2008-01-01

    Protein-tyrosine phosphatases are tightly controlled by various mechanisms, ranging from differential expression in specific cell types to restricted subcellular localization, limited proteolysis, post-translational modifications affecting intrinsic catalytic activity, ligand binding and

  5. Fanconi anemia FANCD2 and FANCI proteins regulate the nuclear dynamics of splicing factors.

    Science.gov (United States)

    Moriel-Carretero, María; Ovejero, Sara; Gérus-Durand, Marie; Vryzas, Dimos; Constantinou, Angelos

    2017-12-04

    Proteins disabled in the cancer-prone disorder Fanconi anemia (FA) ensure the maintenance of chromosomal stability during DNA replication. FA proteins regulate replication dynamics, coordinate replication-coupled repair of interstrand DNA cross-links, and mitigate conflicts between replication and transcription. Here we show that FANCI and FANCD2 associate with splicing factor 3B1 (SF3B1), a key spliceosomal protein of the U2 small nuclear ribonucleoprotein (U2 snRNP). FANCI is in close proximity to SF3B1 in the nucleoplasm of interphase and mitotic cells. Furthermore, we find that DNA replication stress induces the release of SF3B1 from nuclear speckles in a manner that depends on FANCI and on the activity of the checkpoint kinase ATR. In chromatin, both FANCD2 and FANCI associate with SF3B1, prevent accumulation of postcatalytic intron lariats, and contribute to the timely eviction of splicing factors. We propose that FANCD2 and FANCI contribute to the organization of functional domains in chromatin, ensuring the coordination of DNA replication and cotranscriptional processes. © 2017 Moriel-Carretero et al.

  6. Fox-2 protein regulates the alternative splicing of scleroderma-associated lysyl hydroxylase 2 messenger RNA.

    Science.gov (United States)

    Seth, Puneet; Yeowell, Heather N

    2010-04-01

    Scleroderma (systemic sclerosis [SSc]) is a complex connective tissue disorder characterized by hardening and thickening of the skin. One hallmark of scleroderma is excessive accumulation of collagen accompanied by increased levels of pyridinoline collagen crosslinks derived from hydroxylysine residues in the collagen telopeptide domains. Lysyl hydroxylase 2 (LH2), an important alternatively spliced enzyme in collagen biosynthesis, acts as a collagen telopeptide hydroxylase. Changes in the pattern of LH2 alternative splicing, favoring increased inclusion of the alternatively spliced LH2 exon 13A, thereby increasing the levels of the long transcript of LH2 (LH2[long]), are linked to scleroderma disease. This study was undertaken to examine the role played by RNA binding protein Fox-2 in regulating exon 13A inclusion, which leads to the generation of scleroderma-associated LH2(long) messenger RNA (mRNA). Phylogenetic sequence analysis of introns flanking exon 13A was performed. A tetracycline-inducible system in T-Rex 293 cells was used to induce Fox-2 protein, and endogenous LH2(long) mRNA was determined by reverse transcriptase-polymerase chain reaction. An LH2 minigene was designed, validated, and used in Fox-2 overexpression and mutagenesis experiments. Knockdown of Fox-2 was performed in mouse embryonic fibroblasts and in fibroblasts from SSc patients. Overexpression of Fox-2 enhanced the inclusion of exon 13A and increased the generation of LH2(long) mRNA, whereas knockdown of Fox-2 decreased LH2(long) transcripts. Mutational analysis of an LH2 minigene demonstrated that 2 of the 4 Fox binding motifs flanking LH2 exon 13A are required for inclusion of exon 13A. In early passage fibroblasts derived from patients with scleroderma, the knockdown of Fox-2 protein significantly decreased the endogenous levels of LH2(long) mRNA. Our findings indicate that Fox-2 plays an integral role in the regulation of LH2 splicing. Knockdown of Fox-2 and other methods to decrease

  7. Prolyl hydroxylation regulates protein degradation, synthesis, and splicing in human induced pluripotent stem cell-derived cardiomyocytes.

    Science.gov (United States)

    Stoehr, Andrea; Yang, Yanqin; Patel, Sajni; Evangelista, Alicia M; Aponte, Angel; Wang, Guanghui; Liu, Poching; Boylston, Jennifer; Kloner, Philip H; Lin, Yongshun; Gucek, Marjan; Zhu, Jun; Murphy, Elizabeth

    2016-06-01

    Protein hydroxylases are oxygen- and α-ketoglutarate-dependent enzymes that catalyse hydroxylation of amino acids such as proline, thus linking oxygen and metabolism to enzymatic activity. Prolyl hydroxylation is a dynamic post-translational modification that regulates protein stability and protein-protein interactions; however, the extent of this modification is largely uncharacterized. The goals of this study are to investigate the biological consequences of prolyl hydroxylation and to identify new targets that undergo prolyl hydroxylation in human cardiomyocytes. We used human induced pluripotent stem cell-derived cardiomyocytes in combination with pulse-chase amino acid labelling and proteomics to analyse the effects of prolyl hydroxylation on protein degradation and synthesis. We identified 167 proteins that exhibit differences in degradation with inhibition of prolyl hydroxylation by dimethyloxalylglycine (DMOG); 164 were stabilized. Proteins involved in RNA splicing such as serine/arginine-rich splicing factor 2 (SRSF2) and splicing factor and proline- and glutamine-rich (SFPQ) were stabilized with DMOG. DMOG also decreased protein translation of cytoskeletal and sarcomeric proteins such as α-cardiac actin. We searched the mass spectrometry data for proline hydroxylation and identified 134 high confidence peptides mapping to 78 unique proteins. We identified SRSF2, SFPQ, α-cardiac actin, and cardiac titin as prolyl hydroxylated. We identified 29 prolyl hydroxylated proteins that showed a significant difference in either protein degradation or synthesis. Additionally, we performed next-generation RNA sequencing and showed that the observed decrease in protein synthesis was not due to changes in mRNA levels. Because RNA splicing factors were prolyl hydroxylated, we investigated splicing ± inhibition of prolyl hydroxylation and detected 369 alternative splicing events, with a preponderance of exon skipping. This study provides the first extensive

  8. [Genetic diagnostics of pathogenic splicing abnormalities in the clinical laboratory--pitfalls and screening approaches].

    Science.gov (United States)

    Niimi, Hideki; Ogawa, Tomomi; Note, Rhougou; Hayashi, Shirou; Ueno, Tomohiro; Harada, Kenu; Uji, Yoshinori; Kitajima, Isao

    2010-12-01

    In recent years, genetic diagnostics of pathogenic splicing abnormalities are increasingly recognized as critically important in the clinical genetic diagnostics. It is reported that approximately 10% of pathogenic mutations causing human inherited diseases are splicing mutations. Nonetheless, it is still difficult to identify splicing abnormalities in routine genetic diagnostic settings. Here, we studied two different kinds of cases with splicing abnormalities. The first case is a protein S deficiency. Nucleotide analyses revealed that the proband had a previously reported G to C substitution in the invariant AG dinucleotide at the splicing acceptor site of intronl/exon2, which produces multiple splicing abnormalities resulting in protein S deficiency. The second case is an antithrombin (AT) deficiency. This proband had a previously reported G to A substitution, at nucleotide position 9788 in intron 4, 14 bp in front of exon 5, which created a de novo exon 5 splice site and resulted in AT deficiency. From a practical standpoint, we discussed the pitfalls, attentions, and screening approaches in genetic diagnostics of pathogenic splicing abnormalities. Due to the difficulty with full-length sequence analysis of introns, and the lack of RNA samples, splicing mutations may escape identification. Although current genetic testing remains to be improved, to screen for splicing abnormalities more efficiently, it is significant to use an appropriate combination of various approaches such as DNA and/or RNA samples, splicing mutation databases, bioinformatic tools to detect splice sites and cis-regulatory elements, and in vitro and/or in vivo experimentally methods as needed.

  9. Dynamic regulation of genome-wide pre-mRNA splicing and stress tolerance by the Sm-like protein LSm5 in Arabidopsis

    KAUST Repository

    Cui, Peng

    2014-01-07

    Background: Sm-like proteins are highly conserved proteins that form the core of the U6 ribonucleoprotein and function in several mRNA metabolism processes, including pre-mRNA splicing. Despite their wide occurrence in all eukaryotes, little is known about the roles of Sm-like proteins in the regulation of splicing.Results: Here, through comprehensive transcriptome analyses, we demonstrate that depletion of the Arabidopsis supersensitive to abscisic acid and drought 1 gene (SAD1), which encodes Sm-like protein 5 (LSm5), promotes an inaccurate selection of splice sites that leads to a genome-wide increase in alternative splicing. In contrast, overexpression of SAD1 strengthens the precision of splice-site recognition and globally inhibits alternative splicing. Further, SAD1 modulates the splicing of stress-responsive genes, particularly under salt-stress conditions. Finally, we find that overexpression of SAD1 in Arabidopsis improves salt tolerance in transgenic plants, which correlates with an increase in splicing accuracy and efficiency for stress-responsive genes.Conclusions: We conclude that SAD1 dynamically controls splicing efficiency and splice-site recognition in Arabidopsis, and propose that this may contribute to SAD1-mediated stress tolerance through the metabolism of transcripts expressed from stress-responsive genes. Our study not only provides novel insights into the function of Sm-like proteins in splicing, but also uncovers new means to improve splicing efficiency and to enhance stress tolerance in a higher eukaryote. 2014 Cui et al.; licensee BioMed Central Ltd.

  10. LSM Proteins Provide Accurate Splicing and Decay of Selected Transcripts to Ensure Normal Arabidopsis Development[W

    Science.gov (United States)

    Perea-Resa, Carlos; Hernández-Verdeja, Tamara; López-Cobollo, Rosa; Castellano, María del Mar; Salinas, Julio

    2012-01-01

    In yeast and animals, SM-like (LSM) proteins typically exist as heptameric complexes and are involved in different aspects of RNA metabolism. Eight LSM proteins, LSM1 to 8, are highly conserved and form two distinct heteroheptameric complexes, LSM1-7 and LSM2-8,that function in mRNA decay and splicing, respectively. A search of the Arabidopsis thaliana genome identifies 11 genes encoding proteins related to the eight conserved LSMs, the genes encoding the putative LSM1, LSM3, and LSM6 proteins being duplicated. Here, we report the molecular and functional characterization of the Arabidopsis LSM gene family. Our results show that the 11 LSM genes are active and encode proteins that are also organized in two different heptameric complexes. The LSM1-7 complex is cytoplasmic and is involved in P-body formation and mRNA decay by promoting decapping. The LSM2-8 complex is nuclear and is required for precursor mRNA splicing through U6 small nuclear RNA stabilization. More importantly, our results also reveal that these complexes are essential for the correct turnover and splicing of selected development-related mRNAs and for the normal development of Arabidopsis. We propose that LSMs play a critical role in Arabidopsis development by ensuring the appropriate development-related gene expression through the regulation of mRNA splicing and decay. PMID:23221597

  11. SRSF3 represses the expression of PDCD4 protein by coordinated regulation of alternative splicing, export and translation

    Energy Technology Data Exchange (ETDEWEB)

    Park, Seung Kuk; Jeong, Sunjoo, E-mail: sjsj@dankook.ac.kr

    2016-02-05

    Gene expression is regulated at multiple steps, such as transcription, splicing, export, degradation and translation. Considering diverse roles of SR proteins, we determined whether the tumor-related splicing factor SRSF3 regulates the expression of the tumor-suppressor protein, PDCD4, at multiple steps. As we have reported previously, knockdown of SRSF3 increased the PDCD4 protein level in SW480 colon cancer cells. More interestingly, here we showed that the alternative splicing and the nuclear export of minor isoforms of pdcd4 mRNA were repressed by SRSF3, but the translation step was unaffected. In contrast, only the translation step of the major isoform of pdcd4 mRNA was repressed by SRSF3. Therefore, overexpression of SRSF3 might be relevant to the repression of all isoforms of PDCD4 protein levels in most types of cancer cell. We propose that SRSF3 could act as a coordinator of the expression of PDCD4 protein via two mechanisms on two alternatively spliced mRNA isoforms.

  12. PDZ-containing proteins: alternative splicing as a source of functional diversity.

    Science.gov (United States)

    Sierralta, Jimena; Mendoza, Carolina

    2004-12-01

    Scaffold proteins allow specific protein complexes to be assembled in particular regions of the cell at which they organize subcellular structures and signal transduction complexes. This characteristic is especially important for neurons, which are highly polarized cells. Among the domains contained by scaffold proteins, the PSD-95, Discs-large, ZO-1 (PDZ) domains are of particular relevance in signal transduction processes and maintenance of neuronal and epithelial polarity. These domains are specialized in the binding of the carboxyl termini of proteins allowing membrane proteins to be localized by the anchoring to the cytoskeleton mediated by PDZ-containing scaffold proteins. In vivo studies carried out in Drosophila have taught that the role of many scaffold proteins is not limited to a single process; thus, in many cases the same genes are expressed in different tissues and participate in apparently very diverse processes. In addition to the differential expression of interactors of scaffold proteins, the expression of variants of these molecular scaffolds as the result of the alternative processing of the genes that encode them is proving to be a very important source of variability and complexity on a main theme. Alternative splicing in the nervous system is well documented, where specific isoforms play roles in neurotransmission, ion channel function, neuronal cell recognition, and are developmentally regulated making it a major mechanism of functional diversity. Here we review the current state of knowledge about the diversity and the known function of PDZ-containing proteins in Drosophila with emphasis in the role played by alternatively processed forms in the diversity of functions attributed to this family of proteins.

  13. Adipocyte spliced form of X-box-binding protein 1 promotes adiponectin multimerization and systemic glucose homeostasis

    NARCIS (Netherlands)

    Sha, H.; Yang, L.; Liu, M.; Xia, S.; Liu, Y.; Liu, F.; Kersten, A.H.; Qi, L.

    2014-01-01

    The physiological role of the spliced form of X-box–binding protein 1 (XBP1s), a key transcription factor of the endoplasmic reticulum (ER) stress response, in adipose tissue remains largely unknown. In this study, we show that overexpression of XBP1s promotes adiponectin multimerization in

  14. Functional analysis of U1-70K interacting SR proteins in pre-mRNA splicing in Arabidopsis

    International Nuclear Information System (INIS)

    Reddy, A.S.N.

    2008-01-01

    Proteins of a serine/arginine-rich (SR) family are part of the spliceosome and are implicated in both constitutive and alternative splicing of pre-mRNAs. With the funding from DOE we have been studying alternative of splicing of genes encoding serine/arginine-rich (SR) proteins and the roles of SR proteins that interact with U1-70K in regulating basic and alternative splicing. Alternative splicing of pre-mRNAs of Arabidopsis serine/arginine-rich proteins and its regulation by hormones and stresses: We analyzed the splicing of all 19 Arabidopsis genes in different tissues, during different seedling stages and in response to various hormonal and stress treatments. Remarkably, about 90 different transcripts are produced from 15 SR genes, thereby increasing the transcriptome complexity of SR genes by about five fold. Using the RNA isolated from polysomes we have shown that most of the splice variants are recruited for translation. Alternative splicing of some SR genes is controlled in a developmental and tissue-specific manner (Palusa et al., 2007). Interestingly, among the various hormones and abiotic stresses tested, temperature stress (cold and heat) and ultraviolet light dramatically altered alternative splicing of pre-mRNAs of several SR genes whereas hormones altered the splicing of only two SR genes (Palusa et al., 2007). Localization and dynamics of a novel serine/arginine-rich protein that interacts with U1-70K: We analyzed the intranuclear movement of SR45 fused to GFP by fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP). We demonstrate that the movement of GFP-SR45 is ATP-dependent. Interestingly, inhibition of transcription or phosphorylation slowed the mobility of GFP-SR45 (Ali et al., 2006). Our studies have revealed that the nuclear localization signals are located in arg/ser-rich domains (RS) 1 and 2, whereas the speckle targeting signals are exclusively present in RS2 (Ali et al., 2006). The regulation of

  15. Identification of a functionally distinct truncated BDNF mRNA splice variant and protein in Trachemys scripta elegans.

    Directory of Open Access Journals (Sweden)

    Ganesh Ambigapathy

    Full Text Available Brain-derived neurotrophic factor (BDNF has a diverse functional role and complex pattern of gene expression. Alternative splicing of mRNA transcripts leads to further diversity of mRNAs and protein isoforms. Here, we describe the regulation of BDNF mRNA transcripts in an in vitro model of eyeblink classical conditioning and a unique transcript that forms a functionally distinct truncated BDNF protein isoform. Nine different mRNA transcripts from the BDNF gene of the pond turtle Trachemys scripta elegans (tBDNF are selectively regulated during classical conditioning: exon I mRNA transcripts show no change, exon II transcripts are downregulated, while exon III transcripts are upregulated. One unique transcript that codes from exon II, tBDNF2a, contains a 40 base pair deletion in the protein coding exon that generates a truncated tBDNF protein. The truncated transcript and protein are expressed in the naïve untrained state and are fully repressed during conditioning when full-length mature tBDNF is expressed, thereby having an alternate pattern of expression in conditioning. Truncated BDNF is not restricted to turtles as a truncated mRNA splice variant has been described for the human BDNF gene. Further studies are required to determine the ubiquity of truncated BDNF alternative splice variants across species and the mechanisms of regulation and function of this newly recognized BDNF protein.

  16. Identification of a functionally distinct truncated BDNF mRNA splice variant and protein in Trachemys scripta elegans.

    Science.gov (United States)

    Ambigapathy, Ganesh; Zheng, Zhaoqing; Li, Wei; Keifer, Joyce

    2013-01-01

    Brain-derived neurotrophic factor (BDNF) has a diverse functional role and complex pattern of gene expression. Alternative splicing of mRNA transcripts leads to further diversity of mRNAs and protein isoforms. Here, we describe the regulation of BDNF mRNA transcripts in an in vitro model of eyeblink classical conditioning and a unique transcript that forms a functionally distinct truncated BDNF protein isoform. Nine different mRNA transcripts from the BDNF gene of the pond turtle Trachemys scripta elegans (tBDNF) are selectively regulated during classical conditioning: exon I mRNA transcripts show no change, exon II transcripts are downregulated, while exon III transcripts are upregulated. One unique transcript that codes from exon II, tBDNF2a, contains a 40 base pair deletion in the protein coding exon that generates a truncated tBDNF protein. The truncated transcript and protein are expressed in the naïve untrained state and are fully repressed during conditioning when full-length mature tBDNF is expressed, thereby having an alternate pattern of expression in conditioning. Truncated BDNF is not restricted to turtles as a truncated mRNA splice variant has been described for the human BDNF gene. Further studies are required to determine the ubiquity of truncated BDNF alternative splice variants across species and the mechanisms of regulation and function of this newly recognized BDNF protein.

  17. The neurogenetics of alternative splicing.

    Science.gov (United States)

    Vuong, Celine K; Black, Douglas L; Zheng, Sika

    2016-05-01

    Alternative precursor-mRNA splicing is a key mechanism for regulating gene expression in mammals and is controlled by specialized RNA-binding proteins. The misregulation of splicing is implicated in multiple neurological disorders. We describe recent mouse genetic studies of alternative splicing that reveal its critical role in both neuronal development and the function of mature neurons. We discuss the challenges in understanding the extensive genetic programmes controlled by proteins that regulate splicing, both during development and in the adult brain.

  18. A CRM domain protein functions dually in group I and group II intron splicing in land plant chloroplasts.

    Science.gov (United States)

    Asakura, Yukari; Barkan, Alice

    2007-12-01

    The CRM domain is a recently recognized RNA binding domain found in three group II intron splicing factors in chloroplasts, in a bacterial protein that associates with ribosome precursors, and in a family of uncharacterized proteins in plants. To elucidate the functional repertoire of proteins with CRM domains, we studied CFM2 (for CRM Family Member 2), which harbors four CRM domains. RNA coimmunoprecipitation assays showed that CFM2 in maize (Zea mays) chloroplasts is associated with the group I intron in pre-trnL-UAA and group II introns in the ndhA and ycf3 pre-mRNAs. T-DNA insertions in the Arabidopsis thaliana ortholog condition a defective-seed phenotype (strong allele) or chlorophyll-deficient seedlings with impaired splicing of the trnL group I intron and the ndhA, ycf3-int1, and clpP-int2 group II introns (weak alleles). CFM2 and two previously described CRM proteins are bound simultaneously to the ndhA and ycf3-int1 introns and act in a nonredundant fashion to promote their splicing. With these findings, CRM domain proteins are implicated in the activities of three classes of catalytic RNA: group I introns, group II introns, and 23S rRNA.

  19. Structural Insights into RNA Recognition by the Alternate-Splicing Regulator CUG-Binding Protein 1

    Energy Technology Data Exchange (ETDEWEB)

    M Teplova; J Song; H Gaw; A Teplov; D Patel

    2011-12-31

    CUG-binding protein 1 (CUGBP1) regulates multiple aspects of nuclear and cytoplasmic mRNA processing, with implications for onset of myotonic dystrophy. CUGBP1 harbors three RRM domains and preferentially targets UGU-rich mRNA elements. We describe crystal structures of CUGBP1 RRM1 and tandem RRM1/2 domains bound to RNAs containing tandem UGU(U/G) elements. Both RRM1 in RRM1-RNA and RRM2 in RRM1/2-RNA complexes use similar principles to target UGU(U/G) elements, with recognition mediated by face-to-edge stacking and water-mediated hydrogen-bonding networks. The UG step adopts a left-handed Z-RNA conformation, with the syn guanine recognized through Hoogsteen edge-protein backbone hydrogen-bonding interactions. NMR studies on the RRM1/2-RNA complex establish that both RRM domains target tandem UGUU motifs in solution, whereas filter-binding assays identify a preference for recognition of GU over AU or GC steps. We discuss the implications of CUGBP1-mediated targeting and sequestration of UGU(U/G) elements on pre-mRNA alternative-splicing regulation, translational regulation, and mRNA decay.

  20. An unusual spliced variant of DELLA protein, a negative regulator of gibberellin signaling, in lettuce.

    Science.gov (United States)

    Sawada, Yoshiaki; Umetsu, Asami; Komatsu, Yuki; Kitamura, Jun; Suzuki, Hiroyuki; Asami, Tadao; Fukuda, Machiko; Honda, Ichiro; Mitsuhashi, Wataru; Nakajima, Masatoshi; Toyomasu, Tomonobu

    2012-01-01

    DELLA proteins are negative regulators of the signaling of gibberellin (GA), a phytohormone regulating plant growth. DELLA degradation is triggered by its interaction with GID1, a soluble GA receptor, in the presence of bioactive GA. We isolated cDNA from a spliced variant of LsDELLA1 mRNA in lettuce, and named it LsDELLA1sv. It was deduced that LsDELLA1sv encodes truncated LsDELLA1, which has DELLA and VHYNP motifs at the N terminus but lacks part of the C-terminal GRAS domain. The recombinant LsDELLA1sv protein interacted with both Arabidopsis GID1 and lettuce GID1s in the presence of GA. A yeast two-hybrid assay suggested that LsDELLA1sv interacted with LsDELLA1. The ratio of LsDELLA1sv to LsDELLA1 transcripts was higher in flower samples at the late reproductive stage and seed samples (dry seeds and imbibed seeds) than in the other organ samples examined. This study suggests that LsDELLA1sv is a possible modulator of GA signaling in lettuce.

  1. Molecular characterization of a CpTRIM35-like protein and its splice variants from whitespotted bamboo shark (Chiloscyllium plagiosum)

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Xinshang, E-mail: sanmaosound@163.com; Zhao, Heng, E-mail: hengzhao2000@gmail.com; Chen, Yeyu, E-mail: cyyleaf@126.com; Luo, Huiying, E-mail: luohuiying@caas.cn; Yao, Bin, E-mail: binyao@caas.cn

    2014-10-24

    Highlights: • A TRIM gene and three splice variants were firstly cloned from elasmobranch fish. • The genes were constitutively expressed with high levels in spleen and kidney. • The gene products were distributed in cytoplasm alone or cytoplasm and nucleus. • As E3 ubiquitin ligases, the proteins differed in immune responses to challenges. - Abstract: The tripartite motif (TRIM) proteins play important roles in a broad range of biological processes, including apoptosis, cell proliferation and innate immunity response. In this study, a TRIM gene and its three splice variants were cloned from an elasmobranch fish—whitespotted bamboo shark (Chiloscyllium plagiosum Bennett). Phylogenetic analysis indicated that the gene was closely related to TRIM35 homologs, thus termed CpTRIM35-like. Deduced CpTRIM35 has a RBCC-PRY/SPRY structure typical of TRIM proteins, and its splice variants (CpTRIM35-1–3) have different truncations at the C-terminus. The gene products were constitutively expressed in adult sharks with the highest levels in spleen and kidney. The different subcellular locations, upregulation upon LPS and poly I:C stimulation, and significant E3 ubiquitin ligase activities suggested their different roles in immune responses as an E3 ubiquitin ligase. This is the first TRIM protein ever characterized in elasmobranch fish.

  2. Molecular characterization of a CpTRIM35-like protein and its splice variants from whitespotted bamboo shark (Chiloscyllium plagiosum)

    International Nuclear Information System (INIS)

    Zhang, Xinshang; Zhao, Heng; Chen, Yeyu; Luo, Huiying; Yao, Bin

    2014-01-01

    Highlights: • A TRIM gene and three splice variants were firstly cloned from elasmobranch fish. • The genes were constitutively expressed with high levels in spleen and kidney. • The gene products were distributed in cytoplasm alone or cytoplasm and nucleus. • As E3 ubiquitin ligases, the proteins differed in immune responses to challenges. - Abstract: The tripartite motif (TRIM) proteins play important roles in a broad range of biological processes, including apoptosis, cell proliferation and innate immunity response. In this study, a TRIM gene and its three splice variants were cloned from an elasmobranch fish—whitespotted bamboo shark (Chiloscyllium plagiosum Bennett). Phylogenetic analysis indicated that the gene was closely related to TRIM35 homologs, thus termed CpTRIM35-like. Deduced CpTRIM35 has a RBCC-PRY/SPRY structure typical of TRIM proteins, and its splice variants (CpTRIM35-1–3) have different truncations at the C-terminus. The gene products were constitutively expressed in adult sharks with the highest levels in spleen and kidney. The different subcellular locations, upregulation upon LPS and poly I:C stimulation, and significant E3 ubiquitin ligase activities suggested their different roles in immune responses as an E3 ubiquitin ligase. This is the first TRIM protein ever characterized in elasmobranch fish

  3. Regulatory Guide 1.131: Qualification tests of electric cables, field splices, and connections for light-water-cooled nuclear power plants

    International Nuclear Information System (INIS)

    Anon.

    1992-01-01

    Criterion III, ''Design Control,'' of Appendix B, ''Quality Assurance Criteria for Nuclear Power Plants and Fuel Reprocessing Plant,'' to 10 CFR Part 50, ''Licensing of Production and Utilization Facilities,'' requires that, where a test program is used to verify the adequacy of a specific design feature, it include suitable qualification testing of a prototype unit under the most adverse design conditions. This regulatory guide describes a method acceptable to the NRC staff for complying with the Commission's regulations with regard to qualification testing of electric cables, field splices, and connections for service in light-water-cooled nuclear power plants to ensure that the cables, field splices, and connections can perform their safety-related functions. The fire test provisions of this guide do not apply to qualification for an installed configuration

  4. Temperature regulates splicing efficiency of the cold-inducible RNA-binding protein gene Cirbp

    Science.gov (United States)

    Gotic, Ivana; Omidi, Saeed; Fleury-Olela, Fabienne; Molina, Nacho; Naef, Felix; Schibler, Ueli

    2016-01-01

    In mammals, body temperature fluctuates diurnally around a mean value of 36°C–37°C. Despite the small differences between minimal and maximal values, body temperature rhythms can drive robust cycles in gene expression in cultured cells and, likely, animals. Here we studied the mechanisms responsible for the temperature-dependent expression of cold-inducible RNA-binding protein (CIRBP). In NIH3T3 fibroblasts exposed to simulated mouse body temperature cycles, Cirbp mRNA oscillates about threefold in abundance, as it does in mouse livers. This daily mRNA accumulation cycle is directly controlled by temperature oscillations and does not depend on the cells’ circadian clocks. Here we show that the temperature-dependent accumulation of Cirbp mRNA is controlled primarily by the regulation of splicing efficiency, defined as the fraction of Cirbp pre-mRNA processed into mature mRNA. As revealed by genome-wide “approach to steady-state” kinetics, this post-transcriptional mechanism is widespread in the temperature-dependent control of gene expression. PMID:27633015

  5. Genome-wide analysis of SRSF10-regulated alternative splicing by deep sequencing of chicken transcriptome

    Directory of Open Access Journals (Sweden)

    Xuexia Zhou

    2014-12-01

    Full Text Available Splicing factor SRSF10 is known to function as a sequence-specific splicing activator that is capable of regulating alternative splicing both in vitro and in vivo. We recently used an RNA-seq approach coupled with bioinformatics analysis to identify the extensive splicing network regulated by SRSF10 in chicken cells. We found that SRSF10 promoted both exon inclusion and exclusion. Functionally, many of the SRSF10-verified alternative exons are linked to pathways of response to external stimulus. Here we describe in detail the experimental design, bioinformatics analysis and GO/pathway enrichment analysis of SRSF10-regulated genes to correspond with our data in the Gene Expression Omnibus with accession number GSE53354. Our data thus provide a resource for studying regulation of alternative splicing in vivo that underlines biological functions of splicing regulatory proteins in cells.

  6. Dynamic changes in neurexins' alternative splicing: role of Rho-associated protein kinases and relevance to memory formation.

    Directory of Open Access Journals (Sweden)

    Gabriela Rozic

    Full Text Available The three neurexins genes (NRXN1/2/3 encode polymorphic synaptic membrane proteins that are involved in cognitive functioning. Neurexins' selectivity of function is presumably conferred through differential use of 2 promoters and 5 alternative splicing sites (SS#1/2/3/4/5. In day-old rat brain neurons grown in culture, activation (depolarization induces reversible, calcium dependent, repression of NRXN2α SS#3 insert. The effects of depolarization on NRXN1/2/3α splicing and biochemical pathways mediating them were further studied in these neurons. NRXN1/2/3α splicing in the course of memory formation in vivo was also explored, using fear conditioning paradigm in rats in which the animals were trained to associate an aversive stimulus (electrical shock with a neutral context (a tone, resulting in the expression of fear responses to the neutral context.In the cultured neurons depolarization induced, beside NRXN2α SS#3, repression of SS#3 and SS#4 exons in NRXN3α but not NRXN1α. The repressions were mediated by the calcium/protein kinase C/Rho-associated protein kinase (ROCK pathway. Fear conditioning induced significant and transient repressions of the NRXN1/2/3α SS#4 exons in the rat hippocampus. ROCK inhibition prior to training attenuated the behavioral fear response, the NRXN1/2/3α splicing repressions and subsequent recovery and the levels of excitatory (PSD95 and inhibitory (gephyrin synaptic proteins in the hippocampus. No such effects were observed in the prefrontal cortex. Significant correlations existed between the fear response and hippocampal NRXN3α and NRXN2α SS#4 inserts as well as PSD95 protein levels. Hippocampal NRXN1α SS#4 insert and gephyrin levels did not correlate with the behavioral response but were negatively correlated with each other.These results show for the first time dynamic, experience related changes in NRXN1/2/3α alternative splicing in the rat brain and a role for ROCK in them. Specific neurexins

  7. HPV-18 E2circumflexE4 chimera: 2 new spliced transcripts and proteins induced by keratinocyte differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Tan, Chye Ling [Papillomavirus Regulation and Cancer, Institute of Medical Biology, Agency for Science, Technology and Research (A-STAR), Biopolis, 8A Biomedical Grove, Immunos, Singapore 138648 (Singapore); Gunaratne, Jayantha [Mass Spectrometry and Systems Biology Laboratory, Institute of Molecular and Cell Biology, A-STAR, Biopolis, 61 Biopolis Drive, Proteos, Singapore 138673 (Singapore); Lai, Deborah [Papillomavirus Regulation and Cancer, Institute of Medical Biology, Agency for Science, Technology and Research (A-STAR), Biopolis, 8A Biomedical Grove, Immunos, Singapore 138648 (Singapore); Carthagena, Laetitia [UMR-S996, Universite Paris-Sud 11, 32 rue des Carnets, 92140 Clamart (France); Wang, Qian [MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London N10 3UE (United Kingdom); Xue, Yue Zhen; Quek, Ling Shih [Papillomavirus Regulation and Cancer, Institute of Medical Biology, Agency for Science, Technology and Research (A-STAR), Biopolis, 8A Biomedical Grove, Immunos, Singapore 138648 (Singapore); Doorbar, John [MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London N10 3UE (United Kingdom); Bachelerie, Francoise [UMR-S996, Universite Paris-Sud 11, 32 rue des Carnets, 92140 Clamart (France); Thierry, Francoise, E-mail: francoise.thierry@imb.a-star.edu.sg [Papillomavirus Regulation and Cancer, Institute of Medical Biology, Agency for Science, Technology and Research (A-STAR), Biopolis, 8A Biomedical Grove, Immunos, Singapore 138648 (Singapore); Bellanger, Sophie, E-mail: sophie.bellanger@imb.a-star.edu.sg [Papillomavirus Regulation and Cancer, Institute of Medical Biology, Agency for Science, Technology and Research (A-STAR), Biopolis, 8A Biomedical Grove, Immunos, Singapore 138648 (Singapore)

    2012-07-20

    The Human Papillomavirus (HPV) E4 is known to be synthesized as an E1circumflexE4 fusion resulting from splice donor and acceptor sites conserved across HPV types. Here we demonstrate the existence of 2 HPV-18 E2circumflexE4 transcripts resulting from 2 splice donor sites in the 5 Prime part of E2, while the splice acceptor site is the one used for E1circumflexE4. Both E2circumflexE4 transcripts are up-regulated by keratinocyte differentiation in vitro and can be detected in clinical samples containing low-grade HPV-18-positive cells from Pap smears. They give rise to two fusion proteins in vitro, E2circumflexE4-S and E2circumflexE4-L. Whereas we could not differentiate E2circumflexE4-S from E1circumflexE4 in vivo, E2circumflexE4-L could be formally identified as a 23 kDa protein in raft cultures in which the corresponding transcript was also found, and in a biopsy from a patient with cervical intraepithelial neoplasia stage I-II (CINI-II) associated with HPV-18, demonstrating the physiological relevance of E2circumflexE4 products.

  8. Alternative splicing of the human gene SYBL1 modulates protein domain architecture of longin VAMP7/TI-VAMP, showing both non-SNARE and synaptobrevin-like isoforms

    Directory of Open Access Journals (Sweden)

    De Franceschi Nicola

    2011-05-01

    Full Text Available Abstract Background The control of intracellular vesicle trafficking is an ideal target to weigh the role of alternative splicing in shaping genomes to make cells. Alternative splicing has been reported for several Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptors of the vesicle (v-SNAREs or of the target membrane (t-SNARES, which are crucial to intracellular membrane fusion and protein and lipid traffic in Eukaryotes. However, splicing has not yet been investigated in Longins, i.e. the most widespread v-SNAREs. Longins are essential in Eukaryotes and prototyped by VAMP7, Sec22b and Ykt6, sharing a conserved N-terminal Longin domain which regulates membrane fusion and subcellular targeting. Human VAMP7/TI-VAMP, encoded by gene SYBL1, is involved in multiple cell pathways, including control of neurite outgrowth. Results Alternative splicing of SYBL1 by exon skipping events results in the production of a number of VAMP7 isoforms. In-frame or frameshift coding sequence modifications modulate domain architecture of VAMP7 isoforms, which can lack whole domains or domain fragments and show variant or extra domains. Intriguingly, two main types of VAMP7 isoforms either share the inhibitory Longin domain and lack the fusion-promoting SNARE motif, or vice versa. Expression analysis in different tissues and cell lines, quantitative real time RT-PCR and confocal microscopy analysis of fluorescent protein-tagged isoforms demonstrate that VAMP7 variants have different tissue specificities and subcellular localizations. Moreover, design and use of isoform-specific antibodies provided preliminary evidence for the existence of splice variants at the protein level. Conclusions Previous evidence on VAMP7 suggests inhibitory functions for the Longin domain and fusion/growth promoting activity for the Δ-longin molecule. Thus, non-SNARE isoforms with Longin domain and non-longin SNARE isoforms might have somehow opposite regulatory functions

  9. Fox-2 Splicing Factor Binds to a Conserved Intron Motif to PromoteInclusion of Protein 4.1R Alternative Exon 16

    Energy Technology Data Exchange (ETDEWEB)

    Ponthier, Julie L.; Schluepen, Christina; Chen, Weiguo; Lersch,Robert A.; Gee, Sherry L.; Hou, Victor C.; Lo, Annie J.; Short, Sarah A.; Chasis, Joel A.; Winkelmann, John C.; Conboy, John G.

    2006-03-01

    Activation of protein 4.1R exon 16 (E16) inclusion during erythropoiesis represents a physiologically important splicing switch that increases 4.1R affinity for spectrin and actin. Previous studies showed that negative regulation of E16 splicing is mediated by the binding of hnRNP A/B proteins to silencer elements in the exon and that downregulation of hnRNP A/B proteins in erythroblasts leads to activation of E16 inclusion. This paper demonstrates that positive regulation of E16 splicing can be mediated by Fox-2 or Fox-1, two closely related splicing factors that possess identical RNA recognition motifs. SELEX experiments with human Fox-1 revealed highly selective binding to the hexamer UGCAUG. Both Fox-1 and Fox-2 were able to bind the conserved UGCAUG elements in the proximal intron downstream of E16, and both could activate E16 splicing in HeLa cell co-transfection assays in a UGCAUG-dependent manner. Conversely, knockdown of Fox-2 expression, achieved with two different siRNA sequences resulted in decreased E16 splicing. Moreover, immunoblot experiments demonstrate mouse erythroblasts express Fox-2, but not Fox-1. These findings suggest that Fox-2 is a physiological activator of E16 splicing in differentiating erythroid cells in vivo. Recent experiments show that UGCAUG is present in the proximal intron sequence of many tissue-specific alternative exons, and we propose that the Fox family of splicing enhancers plays an important role in alternative splicing switches during differentiation in metazoan organisms.

  10. Organization and alternative splicing of the Caenorhabditis elegans cAMP-dependent protein kinase catalytic-subunit gene (kin-1).

    Science.gov (United States)

    Tabish, M; Clegg, R A; Rees, H H; Fisher, M J

    1999-04-01

    The cAMP-dependent protein kinase (protein kinase A, PK-A) is multifunctional in nature, with key roles in the control of diverse aspects of eukaryotic cellular activity. In the case of the free-living nematode, Caenorhabditis elegans, a gene encoding the PK-A catalytic subunit has been identified and two isoforms of this subunit, arising from a C-terminal alternative-splicing event, have been characterized [Gross, Bagchi, Lu and Rubin (1990) J. Biol. Chem. 265, 6896-6907]. Here we report the occurrence of N-terminal alternative-splicing events that, in addition to generating a multiplicity of non-myristoylatable isoforms, also generate the myristoylated variant(s) of the catalytic subunit that we have recently characterized [Aspbury, Fisher, Rees and Clegg (1997) Biochem. Biophys. Res. Commun. 238, 523-527]. The gene spans more than 36 kb and is divided into a total of 13 exons. Each of the mature transcripts contains only 7 exons. In addition to the already characterized exon 1, the 5'-untranslated region and first intron actually contain 5 other exons, any one of which may be alternatively spliced on to exon 2 at the 5' end of the pre-mRNA. This N-terminal alternative splicing occurs in combination with either of the already characterized C-terminal alternative exons. Thus, C. elegans expresses at least 12 different isoforms of the catalytic subunit of PK-A. The significance of this unprecedented structural diversity in the family of PK-A catalytic subunits is discussed.

  11. Depletion of Arabidopsis SC35 and SC35-like serine/arginine-rich proteins affects the transcription and splicing of a subset of genes.

    Science.gov (United States)

    Yan, Qingqing; Xia, Xi; Sun, Zhenfei; Fang, Yuda

    2017-03-01

    Serine/arginine-rich (SR) proteins are important splicing factors which play significant roles in spliceosome assembly and splicing regulation. However, little is known regarding their biological functions in plants. Here, we analyzed the phenotypes of mutants upon depleting different subfamilies of Arabidopsis SR proteins. We found that loss of the functions of SC35 and SC35-like (SCL) proteins cause pleiotropic changes in plant morphology and development, including serrated leaves, late flowering, shorter roots and abnormal silique phyllotaxy. Using RNA-seq, we found that SC35 and SCL proteins play roles in the pre-mRNA splicing. Motif analysis revealed that SC35 and SCL proteins preferentially bind to a specific RNA sequence containing the AGAAGA motif. In addition, the transcriptions of a subset of genes are affected by the deletion of SC35 and SCL proteins which interact with NRPB4, a specific subunit of RNA polymerase II. The splicing of FLOWERING LOCUS C (FLC) intron1 and transcription of FLC were significantly regulated by SC35 and SCL proteins to control Arabidopsis flowering. Therefore, our findings provide mechanistic insight into the functions of plant SC35 and SCL proteins in the regulation of splicing and transcription in a direct or indirect manner to maintain the proper expression of genes and development.

  12. Unraveling 14-3-3 proteins in C4 panicoids with emphasis on model plant Setaria italica reveals phosphorylation-dependent subcellular localization of RS splicing factor.

    Directory of Open Access Journals (Sweden)

    Karunesh Kumar

    Full Text Available 14-3-3 proteins are a large multigenic family of regulatory proteins ubiquitously found in eukaryotes. In plants, 14-3-3 proteins are reported to play significant role in both development and response to stress stimuli. Therefore, considering their importance, genome-wide analyses have been performed in many plants including Arabidopsis, rice and soybean. But, till date, no comprehensive investigation has been conducted in any C4 panicoid crops. In view of this, the present study was performed to identify 8, 5 and 26 potential 14-3-3 gene family members in foxtail millet (Si14-3-3, sorghum (Sb14-3-3 and maize (Zm14-3-3, respectively. In silico characterization revealed large variations in their gene structures; segmental and tandem duplications have played a major role in expansion of these genes in foxtail millet and maize. Gene ontology annotation showed the participation of 14-3-3 proteins in diverse biological processes and molecular functions, and in silico expression profiling indicated their higher expression in all the investigated tissues. Comparative mapping was performed to derive the orthologous relationships between 14-3-3 genes of foxtail millet and other Poaceae members, which showed a higher, as well as similar percentage of orthology among these crops. Expression profiling of Si14-3-3 genes during different time-points of abiotic stress and hormonal treatments showed a differential expression pattern of these genes, and sub-cellular localization studies revealed the site of action of Si14-3-3 proteins within the cells. Further downstream characterization indicated the interaction of Si14-3-3 with a nucleocytoplasmic shuttling phosphoprotein (SiRSZ21A in a phosphorylation-dependent manner, and this demonstrates that Si14-3-3 might regulate the splicing events by binding with phosphorylated SiRSZ21A. Taken together, the present study is a comprehensive analysis of 14-3-3 gene family members in foxtail millet, sorghum and maize

  13. Unraveling 14-3-3 proteins in C4 panicoids with emphasis on model plant Setaria italica reveals phosphorylation-dependent subcellular localization of RS splicing factor.

    Science.gov (United States)

    Kumar, Karunesh; Muthamilarasan, Mehanathan; Bonthala, Venkata Suresh; Roy, Riti; Prasad, Manoj

    2015-01-01

    14-3-3 proteins are a large multigenic family of regulatory proteins ubiquitously found in eukaryotes. In plants, 14-3-3 proteins are reported to play significant role in both development and response to stress stimuli. Therefore, considering their importance, genome-wide analyses have been performed in many plants including Arabidopsis, rice and soybean. But, till date, no comprehensive investigation has been conducted in any C4 panicoid crops. In view of this, the present study was performed to identify 8, 5 and 26 potential 14-3-3 gene family members in foxtail millet (Si14-3-3), sorghum (Sb14-3-3) and maize (Zm14-3-3), respectively. In silico characterization revealed large variations in their gene structures; segmental and tandem duplications have played a major role in expansion of these genes in foxtail millet and maize. Gene ontology annotation showed the participation of 14-3-3 proteins in diverse biological processes and molecular functions, and in silico expression profiling indicated their higher expression in all the investigated tissues. Comparative mapping was performed to derive the orthologous relationships between 14-3-3 genes of foxtail millet and other Poaceae members, which showed a higher, as well as similar percentage of orthology among these crops. Expression profiling of Si14-3-3 genes during different time-points of abiotic stress and hormonal treatments showed a differential expression pattern of these genes, and sub-cellular localization studies revealed the site of action of Si14-3-3 proteins within the cells. Further downstream characterization indicated the interaction of Si14-3-3 with a nucleocytoplasmic shuttling phosphoprotein (SiRSZ21A) in a phosphorylation-dependent manner, and this demonstrates that Si14-3-3 might regulate the splicing events by binding with phosphorylated SiRSZ21A. Taken together, the present study is a comprehensive analysis of 14-3-3 gene family members in foxtail millet, sorghum and maize, which provides

  14. Aberrant alternative splicing is another hallmark of cancer.

    Science.gov (United States)

    Ladomery, Michael

    2013-01-01

    The vast majority of human genes are alternatively spliced. Not surprisingly, aberrant alternative splicing is increasingly linked to cancer. Splice isoforms often encode proteins that have distinct and even antagonistic properties. The abnormal expression of splice factors and splice factor kinases in cancer changes the alternative splicing of critically important pre-mRNAs. Aberrant alternative splicing should be added to the growing list of cancer hallmarks.

  15. Aberrant Alternative Splicing Is Another Hallmark of Cancer

    OpenAIRE

    Ladomery, Michael

    2013-01-01

    The vast majority of human genes are alternatively spliced. Not surprisingly, aberrant alternative splicing is increasingly linked to cancer. Splice isoforms often encode proteins that have distinct and even antagonistic properties. The abnormal expression of splice factors and splice factor kinases in cancer changes the alternative splicing of critically important pre-mRNAs. Aberrant alternative splicing should be added to the growing list of cancer hallmarks.

  16. Alternative RNA splicing and gastric cancer.

    Science.gov (United States)

    Li, Ying; Yuan, Yuan

    2017-07-01

    Alternative splicing (AS) linked to diseases, especially to tumors. Recently, more and more studies focused on the relationship between AS and gastric cancer (GC). This review surveyed the hot topic from four aspects: First, the common types of AS in cancer, including exon skipping, intron retention, mutually exclusive exon, alternative 5 ' or 3' splice site, alternative first or last exon and alternative 3' untranslated regions. Second, basic mechanisms of AS and its relationship with cancer. RNA splicing in eukaryotes follows the GT-AG rule by both cis-elements and trans-acting factors regulatory. Through RNA splicing, different proteins with different forms and functions can be produced and may be associated with carcinogenesis. Third, AS types of GC-related genes and their splicing variants. In this paper, we listed 10 common genes with AS and illustrated its possible molecular mechanisms owing to genetic variation (mutation and /or polymorphism). Fourth, the splicing variants of GC-associated genes and gastric carcinogenesis, invasion and metastasis. Many studies have found that the different splicing variants of the same gene are differentially expressed in GC and its precancerous diseases, suggesting AS has important implications in GC development. Taking together, this review highlighted the role of AS and splicing variants in the process of GC. We hope that this is not only beneficial to advances in the study field of GC, but also can provide valuable information to other similar tumor research.Although we already know some gene splicing and splicing variants play an important role in the development of GC, but many phenomena and mechanisms are still unknown. For example, how the tumor microenvironment and signal transduction pathway effect the forming and function of AS? Unfortunately, this review did not cover the contents because the current study is limited. It is no doubt that clarifying the phenomena and mechanisms of these unknown may help to reveal

  17. Sex determination in insects: a binary decision based on alternative splicing.

    Science.gov (United States)

    Salz, Helen K

    2011-08-01

    The gene regulatory networks that control sex determination vary between species. Despite these differences, comparative studies in insects have found that alternative splicing is reiteratively used in evolution to control expression of the key sex-determining genes. Sex determination is best understood in Drosophila where activation of the RNA binding protein-encoding gene Sex-lethal is the central female-determining event. Sex-lethal serves as a genetic switch because once activated it controls its own expression by a positive feedback splicing mechanism. Sex fate choice in is also maintained by self-sustaining positive feedback splicing mechanisms in other dipteran and hymenopteran insects, although different RNA binding protein-encoding genes function as the binary switch. Studies exploring the mechanisms of sex-specific splicing have revealed the extent to which sex determination is integrated with other developmental regulatory networks. Copyright © 2011 Elsevier Ltd. All rights reserved.

  18. A conserved RNA structural element within the hepatitis B virus post-transcriptional regulatory element enhance nuclear export of intronless transcripts and repress the splicing mechanism.

    Science.gov (United States)

    Visootsat, Akasit; Payungporn, Sunchai; T-Thienprasert, Nattanan P

    2015-12-01

    Hepatitis B virus (HBV) infection is a primary cause of hepatocellular carcinoma and liver cirrhosis worldwide. To develop novel antiviral drugs, a better understanding of HBV gene expression regulation is vital. One important aspect is to understand how HBV hijacks the cellular machinery to export unspliced RNA from the nucleus. The HBV post-transcriptional regulatory element (HBV PRE) has been proposed to be the HBV RNA nuclear export element. However, the function remains controversial, and the core element is unclear. This study, therefore, aimed to identify functional regulatory elements within the HBV PRE and investigate their functions. Using bioinformatics programs based on sequence conservation and conserved RNA secondary structures, three regulatory elements were predicted, namely PRE 1151-1410, PRE 1520-1620 and PRE 1650-1684. PRE 1151-1410 significantly increased intronless and unspliced luciferase activity in both HepG2 and COS-7 cells. Likewise, PRE 1151-1410 significantly elevated intronless and unspliced HBV surface transcripts in liver cancer cells. Moreover, motif analysis predicted that PRE 1151-1410 contains several regulatory motifs. This study reported the roles of PRE 1151-1410 in intronless transcript nuclear export and the splicing mechanism. Additionally, these results provide knowledge in the field of HBV RNA regulation. Moreover, PRE 1151-1410 may be used to enhance the expression of other mRNAs in intronless reporter plasmids.

  19. Evidence that C9ORF72 Dipeptide Repeat Proteins Associate with U2 snRNP to Cause Mis-splicing in ALS/FTD Patients.

    Science.gov (United States)

    Yin, Shanye; Lopez-Gonzalez, Rodrigo; Kunz, Ryan C; Gangopadhyay, Jaya; Borufka, Carl; Gygi, Steven P; Gao, Fen-Biao; Reed, Robin

    2017-06-13

    Hexanucleotide repeat expansion in the C9ORF72 gene results in production of dipeptide repeat (DPR) proteins that may disrupt pre-mRNA splicing in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) patients. At present, the mechanisms underlying this mis-splicing are not understood. Here, we show that addition of proline-arginine (PR) and glycine-arginine (GR) toxic DPR peptides to nuclear extracts blocks spliceosome assembly and splicing, but not other types of RNA processing. Proteomic and biochemical analyses identified the U2 small nuclear ribonucleoprotein particle (snRNP) as a major interactor of PR and GR peptides. In addition, U2 snRNP, but not other splicing factors, mislocalizes from the nucleus to the cytoplasm both in C9ORF72 patient induced pluripotent stem cell (iPSC)-derived motor neurons and in HeLa cells treated with the toxic peptides. Bioinformatic studies support a specific role for U2-snRNP-dependent mis-splicing in C9ORF72 patient brains. Together, our data indicate that DPR-mediated dysfunction of U2 snRNP could account for as much as ∼44% of the mis-spliced cassette exons in C9ORF72 patient brains. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  20. Evidence that C9ORF72 Dipeptide Repeat Proteins Associate with U2 snRNP to Cause Mis-splicing in ALS/FTD Patients

    Directory of Open Access Journals (Sweden)

    Shanye Yin

    2017-06-01

    Full Text Available Hexanucleotide repeat expansion in the C9ORF72 gene results in production of dipeptide repeat (DPR proteins that may disrupt pre-mRNA splicing in amyotrophic lateral sclerosis (ALS and frontotemporal dementia (FTD patients. At present, the mechanisms underlying this mis-splicing are not understood. Here, we show that addition of proline-arginine (PR and glycine-arginine (GR toxic DPR peptides to nuclear extracts blocks spliceosome assembly and splicing, but not other types of RNA processing. Proteomic and biochemical analyses identified the U2 small nuclear ribonucleoprotein particle (snRNP as a major interactor of PR and GR peptides. In addition, U2 snRNP, but not other splicing factors, mislocalizes from the nucleus to the cytoplasm both in C9ORF72 patient induced pluripotent stem cell (iPSC-derived motor neurons and in HeLa cells treated with the toxic peptides. Bioinformatic studies support a specific role for U2-snRNP-dependent mis-splicing in C9ORF72 patient brains. Together, our data indicate that DPR-mediated dysfunction of U2 snRNP could account for as much as ∼44% of the mis-spliced cassette exons in C9ORF72 patient brains.

  1. A pp32-retinoblastoma protein complex modulates androgen receptor-mediated transcription and associates with components of the splicing machinery

    International Nuclear Information System (INIS)

    Adegbola, Onikepe; Pasternack, Gary R.

    2005-01-01

    We have previously shown pp32 and the retinoblastoma protein interact. pp32 and the retinoblastoma protein are nuclear receptor transcriptional coregulators: the retinoblastoma protein is a coactivator for androgen receptor, the major regulator of prostate cancer growth, while pp32, which is highly expressed in prostate cancer, is a corepressor of the estrogen receptor. We now show pp32 increases androgen receptor-mediated transcription and the retinoblastoma protein modulates this activity. Using affinity purification and mass spectrometry, we identify members of the pp32-retinoblastoma protein complex as PSF and nonO/p54nrb, proteins implicated in coordinate regulation of nuclear receptor-mediated transcription and splicing. We show that the pp32-retinoblastoma protein complex is modulated during TPA-induced K562 differentiation. Present evidence suggests that nuclear receptors assemble multiprotein complexes to coordinately regulate transcription and mRNA processing. Our results suggest that pp32 and the retinoblastoma protein may be part of a multiprotein complex that coordinately regulates nuclear receptor-mediated transcription and mRNA processing

  2. Genetic variations and alternative splicing. The Glioma associated oncogene 1, GLI1.

    Directory of Open Access Journals (Sweden)

    Peter eZaphiropoulos

    2012-07-01

    Full Text Available Alternative splicing is a post-transcriptional regulatory process that is attaining stronger recognition as a modulator of gene expression. Alternative splicing occurs when the primary RNA transcript is differentially processed into more than one mature RNAs. This is the result of a variable definition/inclusion of the exons, the sequences that are excised from the primary RNA to form the mature RNAs. Consequently, RNA expression can generate a collection of differentially spliced RNAs, which may distinctly influence subsequent biological events, such as protein synthesis or other biomolecular interactions. Still the mechanisms that control exon definition and exon inclusion are not fully clarified. This mini-review highlights advances in this field as well as the impact of single nucleotide polymorphisms in affecting splicing decisions. The Glioma associated oncogene 1, GLI1, is taken as an example in addressing the role of nucleotide substitutions for splicing regulation.

  3. Human Splicing Finder: an online bioinformatics tool to predict splicing signals

    OpenAIRE

    Desmet, Francois-Olivier; Hamroun, Dalil; Lalande, Marine; Collod-Beroud, Gwenaelle; Claustres, Mireille; Beroud, Christophe

    2009-01-01

    International audience; Thousands of mutations are identified yearly. Although many directly affect protein expression, an increasing proportion of mutations is now believed to influence mRNA splicing. They mostly affect existing splice sites, but synonymous, non-synonymous or nonsense mutations can also create or disrupt splice sites or auxiliary cis-splicing sequences. To facilitate the analysis of the different mutations, we designed Human Splicing Finder (HSF), a tool to predict the effec...

  4. Spliceosomal protein U1A is involved in alternative splicing and salt stress tolerance in Arabidopsis thaliana

    KAUST Repository

    Gu, Jinbao

    2017-12-01

    Soil salinity is a significant threat to sustainable agricultural production worldwide. Plants must adjust their developmental and physiological processes to cope with salt stress. Although the capacity for adaptation ultimately depends on the genome, the exceptional versatility in gene regulation provided by the spliceosome-mediated alternative splicing (AS) is essential in these adaptive processes. However, the functions of the spliceosome in plant stress responses are poorly understood. Here, we report the in-depth characterization of a U1 spliceosomal protein, AtU1A, in controlling AS of pre-mRNAs under salt stress and salt stress tolerance in Arabidopsis thaliana. The atu1a mutant was hypersensitive to salt stress and accumulated more reactive oxygen species (ROS) than the wild-type under salt stress. RNA-seq analysis revealed that AtU1A regulates AS of many genes, presumably through modulating recognition of 5′ splice sites. We showed that AtU1A is associated with the pre-mRNA of the ROS detoxification-related gene ACO1 and is necessary for the regulation of ACO1 AS. ACO1 is important for salt tolerance because ectopic expression of ACO1 in the atu1a mutant can partially rescue its salt hypersensitive phenotype. Our findings highlight the critical role of AtU1A as a regulator of pre-mRNA processing and salt tolerance in plants.

  5. The fission yeast RNA binding protein Mmi1 regulates meiotic genes by controlling intron specific splicing and polyadenylation coupled RNA turnover.

    Directory of Open Access Journals (Sweden)

    Huei-Mei Chen

    Full Text Available The polyA tails of mRNAs are monitored by the exosome as a quality control mechanism. We find that fission yeast, Schizosaccharomyces pombe, adopts this RNA quality control mechanism to regulate a group of 30 or more meiotic genes at the level of both splicing and RNA turnover. In vegetative cells the RNA binding protein Mmi1 binds to the primary transcripts of these genes. We find the novel motif U(U/C/GAAAC highly over-represented in targets of Mmi1. Mmi1 can specifically regulate the splicing of particular introns in a transcript: it inhibits the splicing of introns that are in the vicinity of putative Mmi1 binding sites, while allowing the splicing of other introns that are far from such sites. In addition, binding of Mmi1, particularly near the 3' end, alters 3' processing to promote extremely long polyA tails of up to a kilobase. The hyperadenylated transcripts are then targeted for degradation by the nuclear exonuclease Rrp6. The nuclear polyA binding protein Pab2 assists this hyperadenylation-mediated RNA decay. Rrp6 also targets other hyperadenylated transcripts, which become hyperadenylated in an unknown, but Mmi1-independent way. Thus, hyperadenylation may be a general signal for RNA degradation. In addition, binding of Mmi1 can affect the efficiency of 3' cleavage. Inactivation of Mmi1 in meiosis allows meiotic expression, through splicing and RNA stabilization, of at least 29 target genes, which are apparently constitutively transcribed.

  6. A synonymous polymorphic variation in ACADM exon 11 affects splicing efficiency and may affect fatty acid oxidation

    DEFF Research Database (Denmark)

    Bruun, Gitte Hoffmann; Doktor, Thomas Koed; Andresen, Brage Storstein

    2013-01-01

    beta-oxidation of medium-chain fatty acids. We examined the functional basis for this association and identified linkage between rs211718 and the intragenic synonymous polymorphic variant c.1161A>G in ACADM exon 11 (rs1061337). Employing minigene studies we show that the c.1161A allele is associated......, perhaps due to improved splicing. This study is a proof of principle that synonymous SNPs are not neutral. By changing the binding sites for splicing regulatory proteins they can have significant effects on pre-mRNA splicing and thus protein function. In addition, this study shows that for a sequence...

  7. An alternatively spliced heat shock transcription factor, OsHSFA2dI, functions in the heat stress-induced unfolded protein response in rice.

    Science.gov (United States)

    Cheng, Q; Zhou, Y; Liu, Z; Zhang, L; Song, G; Guo, Z; Wang, W; Qu, X; Zhu, Y; Yang, D

    2015-03-01

    As sessile organisms, plants have evolved a wide range of defence pathways to cope with environmental stress such as heat shock. However, the molecular mechanism of these defence pathways remains unclear in rice. In this study, we found that OsHSFA2d, a heat shock transcriptional factor, encodes two main splice variant proteins, OsHSFA2dI and OsHSFA2dII in rice. Under normal conditions, OsHSFA2dII is the dominant but transcriptionally inactive spliced form. However, when the plant suffers heat stress, OsHSFA2d is alternatively spliced into a transcriptionally active form, OsHSFA2dI, which participates in the heat stress response (HSR). Further study found that this alternative splicing was induced by heat shock rather than photoperiod. We found that OsHSFA2dI is localised to the nucleus, whereas OsHSFA2dII is localised to the nucleus and cytoplasm. Moreover, expression of the unfolded protein response (UNFOLDED PROTEIN RESPONSE) sensors, OsIRE1, OsbZIP39/OsbZIP60 and the UNFOLDED PROTEIN RESPONSE marker OsBiP1, was up-regulated. Interestingly, OsbZIP50 was also alternatively spliced under heat stress, indicating that UNFOLDED PROTEIN RESPONSE signalling pathways were activated by heat stress to re-establish cellular protein homeostasis. We further demonstrated that OsHSFA2dI participated in the unfolded protein response by regulating expression of OsBiP1. © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands.

  8. Alternative RNA splicing and cancer

    Science.gov (United States)

    Liu, Sali; Cheng, Chonghui

    2015-01-01

    Alternative splicing of pre-messenger RNA (mRNA) is a fundamental mechanism by which a gene can give rise to multiple distinct mRNA transcripts, yielding protein isoforms with different, even opposing, functions. With the recognition that alternative splicing occurs in nearly all human genes, its relationship with cancer-associated pathways has emerged as a rapidly growing field. In this review, we summarize recent findings that have implicated the critical role of alternative splicing in cancer and discuss current understandings of the mechanisms underlying dysregulated alternative splicing in cancer cells. PMID:23765697

  9. Alternative splicing: the pledge, the turn, and the prestige : The key role of alternative splicing in human biological systems.

    Science.gov (United States)

    Gallego-Paez, L M; Bordone, M C; Leote, A C; Saraiva-Agostinho, N; Ascensão-Ferreira, M; Barbosa-Morais, N L

    2017-09-01

    Alternative pre-mRNA splicing is a tightly controlled process conducted by the spliceosome, with the assistance of several regulators, resulting in the expression of different transcript isoforms from the same gene and increasing both transcriptome and proteome complexity. The differences between alternative isoforms may be subtle but enough to change the function or localization of the translated proteins. A fine control of the isoform balance is, therefore, needed throughout developmental stages and adult tissues or physiological conditions and it does not come as a surprise that several diseases are caused by its deregulation. In this review, we aim to bring the splicing machinery on stage and raise the curtain on its mechanisms and regulation throughout several systems and tissues of the human body, from neurodevelopment to the interactions with the human microbiome. We discuss, on one hand, the essential role of alternative splicing in assuring tissue function, diversity, and swiftness of response in these systems or tissues, and on the other hand, what goes wrong when its regulatory mechanisms fail. We also focus on the possibilities that splicing modulation therapies open for the future of personalized medicine, along with the leading techniques in this field. The final act of the spliceosome, however, is yet to be fully revealed, as more knowledge is needed regarding the complex regulatory network that coordinates alternative splicing and how its dysfunction leads to disease.

  10. Complement Factor H-Related Protein 4A Is the Dominant Circulating Splice Variant of CFHR4

    Directory of Open Access Journals (Sweden)

    Richard B. Pouw

    2018-04-01

    Full Text Available Recent research has elucidated circulating levels of almost all factor H-related (FHR proteins. Some of these proteins are hypothesized to act as antagonists of the important complement regulator factor H (FH, fine-tuning complement regulation on human surfaces. For the CFHR4 splice variants FHR-4A and FHR-4B, the individual circulating levels are unknown, with only total levels being described. Specific reagents for FHR-4A or FHR-4B are lacking due to the fact that the unique domains in FHR-4A show high sequence similarity with FHR-4B, making it challenging to distinguish them. We developed an assay that specifically measures FHR-4A using novel, well-characterized monoclonal antibodies (mAbs that target unique domains in FHR-4A only. Using various FHR-4A/FHR-4B-specific mAbs, no FHR-4B was identified in any of the serum samples tested. The results demonstrate that FHR-4A is the dominant splice variant of CFHR4 in the circulation, while casting doubt on the presence of FHR-4B. FHR-4A levels (avg. 2.55 ± 1.46 µg/mL were within the range of most of the previously reported levels for all other FHRs. FHR-4A was found to be highly variable among the population, suggesting a strong genetic regulation. These results shed light on the physiological relevance of the previously proposed role of FHR-4A and FHR-4B as antagonists of FH in the circulation.

  11. TBX3 regulates splicing in vivo: a novel molecular mechanism for Ulnar-mammary syndrome.

    Directory of Open Access Journals (Sweden)

    Pavan Kumar P

    2014-03-01

    Full Text Available TBX3 is a member of the T-box family of transcription factors with critical roles in development, oncogenesis, cell fate, and tissue homeostasis. TBX3 mutations in humans cause complex congenital malformations and Ulnar-mammary syndrome. Previous investigations into TBX3 function focused on its activity as a transcriptional repressor. We used an unbiased proteomic approach to identify TBX3 interacting proteins in vivo and discovered that TBX3 interacts with multiple mRNA splicing factors and RNA metabolic proteins. We discovered that TBX3 regulates alternative splicing in vivo and can promote or inhibit splicing depending on context and transcript. TBX3 associates with alternatively spliced mRNAs and binds RNA directly. TBX3 binds RNAs containing TBX binding motifs, and these motifs are required for regulation of splicing. Our study reveals that TBX3 mutations seen in humans with UMS disrupt its splicing regulatory function. The pleiotropic effects of TBX3 mutations in humans and mice likely result from disrupting at least two molecular functions of this protein: transcriptional regulation and pre-mRNA splicing.

  12. TBX3 regulates splicing in vivo: a novel molecular mechanism for Ulnar-mammary syndrome.

    Science.gov (United States)

    Kumar P, Pavan; Franklin, Sarah; Emechebe, Uchenna; Hu, Hao; Moore, Barry; Lehman, Chris; Yandell, Mark; Moon, Anne M

    2014-03-01

    TBX3 is a member of the T-box family of transcription factors with critical roles in development, oncogenesis, cell fate, and tissue homeostasis. TBX3 mutations in humans cause complex congenital malformations and Ulnar-mammary syndrome. Previous investigations into TBX3 function focused on its activity as a transcriptional repressor. We used an unbiased proteomic approach to identify TBX3 interacting proteins in vivo and discovered that TBX3 interacts with multiple mRNA splicing factors and RNA metabolic proteins. We discovered that TBX3 regulates alternative splicing in vivo and can promote or inhibit splicing depending on context and transcript. TBX3 associates with alternatively spliced mRNAs and binds RNA directly. TBX3 binds RNAs containing TBX binding motifs, and these motifs are required for regulation of splicing. Our study reveals that TBX3 mutations seen in humans with UMS disrupt its splicing regulatory function. The pleiotropic effects of TBX3 mutations in humans and mice likely result from disrupting at least two molecular functions of this protein: transcriptional regulation and pre-mRNA splicing.

  13. Mutations in the human adenosine deaminase gene that affect protein structure and RNA splicing

    International Nuclear Information System (INIS)

    Akeson, A.L.; Wiginton, D.A.; States, C.J.; Perme, C.M.; Dusing, M.R.; Hutton, J.J.

    1987-01-01

    Adenosine deaminase deficiency is one cause of the genetic disease severe combined immunodeficiency. To identify mutations responsible for ADA deficiency, the authors synthesized cDNAs to ADA mRNAs from two cell lines, GM2756 and GM2825A, derived from ADA-deficient immunodeficient patients. Sequence analysis of GM2756 cDNA clones revealed a different point mutation in each allele that causes amino acid changes of alanine to valine and arginine to histidine. One allele of GM2825A also has a point mutation that causes an alanine to valine substitution. The other allele of GM2825A was found to produce an mRNA in which exon 4 had been spliced out but had no other detrimental mutations. S1 nuclease mapping of GM2825A mRNA showed equal abundance of the full-length ADA mRNA and the ADA mRNA that was missing exon 4. Several of the ADA cDNA clones extended 5' of the major initiation start site, indicating multiple start sites for ADA transcription. The point mutations in GM2756 and GM2825A and the absence of exon 4 in GM2825A appear to be directly responsible for the ADA deficiency. Comparison of a number of normal and mutant ADA cDNA sequences showed a number of changes in the third base of codons. These change do not affect the amino acid sequence. Analyses of ADA cDNAs from different cell lines detected aberrant RNA species that either included intron 7 or excluded exon 7. Their presence is a result of aberrant splicing of pre-mRNAs and is not related to mutations that cause ADA deficiency

  14. Protein kinase A regulatory subunit distribution in medulloblastoma

    International Nuclear Information System (INIS)

    Mucignat-Caretta, Carla; Denaro, Luca; Redaelli, Marco; D'Avella, Domenico; Caretta, Antonio

    2010-01-01

    Previous studies showed a differential distribution of the four regulatory subunits of cAMP-dependent protein kinases inside the brain, that changed in rodent gliomas: therefore, the distribution of these proteins inside the brain can give information on the functional state of the cells. Our goal was to examine human brain tumors to provide evidence for a differential distribution of protein kinase A in different tumors. The distribution of detergent insoluble regulatory (R1 and R2) and catalytic subunits of cAMP dependent kinases was examined in pediatric brain tumors by immunohistochemistry and fluorescent cAMP analogues binding. R2 is organized in large single dots in medulloblastomas, while it has a different appearance in other tumors. Fluorescent cAMP labelling was observed only in medulloblastoma. A different distribution of cAMP dependent protein kinases has been observed in medulloblastoma

  15. Control of fibroblast fibronectin expression and alternative splicing via the PI3K/Akt/mTOR pathway

    International Nuclear Information System (INIS)

    White, Eric S.; Sagana, Rommel L.; Booth, Adam J.; Yan, Mei; Cornett, Ashley M.; Bloomheart, Christopher A.; Tsui, Jessica L.; Wilke, Carol A.; Moore, Bethany B.; Ritzenthaler, Jeffrey D.; Roman, Jesse; Muro, Andres F.

    2010-01-01

    Fibronectin (FN), a ubiquitous glycoprotein that plays critical roles in physiologic and pathologic conditions, undergoes alternative splicing which distinguishes plasma FN (pFN) from cellular FN (cFN). Although both pFN and cFN can be incorporated into the extracellular matrix, a distinguishing feature of cFN is the inclusion of an alternatively spliced exon termed EDA (for extra type III domain A). The molecular steps involved in EDA splicing are well-characterized, but pathways influencing EDA splicing are less clear. We have previously found an obligate role for inhibition of the tumor suppressor phosphatase and tensin homologue on chromosome 10 (PTEN), the primary regulator of the PI3K/Akt pathway, in fibroblast activation. Here we show TGF-β, a potent inducer of both EDA splicing and fibroblast activation, inhibits PTEN expression and activity in mesenchymal cells, corresponding with enhanced PI3K/Akt signaling. In pten -/- fibroblasts, which resemble activated fibroblasts, inhibition of Akt attenuated FN production and decreased EDA alternative splicing. Moreover, inhibition of mammalian target of rapamycin (mTOR) in pten -/- cells also blocked FN production and EDA splicing. This effect was due to inhibition of Akt-mediated phosphorylation of the primary EDA splicing regulatory protein SF2/ASF. Importantly, FN silencing in pten -/- cells resulted in attenuated proliferation and migration. Thus, our results demonstrate that the PI3K/Akt/mTOR axis is instrumental in FN transcription and alternative splicing, which regulates cell behavior.

  16. cis-Acting and trans-acting modulation of equine infectious anemia virus alternative RNA splicing

    International Nuclear Information System (INIS)

    Liao, Huey-Jane; Baker, Carl C.; Princler, Gerald L.; Derse, David

    2004-01-01

    Equine infectious anemia virus (EIAV), a lentivirus distantly related to HIV-1, encodes regulatory proteins, EIAV Tat (ETat) and Rev (ERev), from a four-exon mRNA. Exon 3 of the tat/rev mRNA contains a 30-nucleotide purine-rich element (PRE) which binds both ERev and SF2/ASF, a member of the SR family of RNA splicing factors. To better understand the role of this element in the regulation of EIAV pre-mRNA splicing, we quantified the effects of mutation or deletion of the PRE on exon 3 splicing in vitro and on alternative splicing in vivo. We also determined the branch point elements upstream of exons 3 and 4. In vitro splicing of exon 3 to exon 4 was not affected by mutation of the PRE, and addition of purified SR proteins enhanced splicing independently of the PRE. In vitro splicing of exon 2 to exon 3 was dependent on the PRE; under conditions of excess SR proteins, either the PRE or the 5' splice site of exon 3 was sufficient to activate splicing. We applied isoform-specific primers in real-time RT-PCR reactions to quantitatively analyze alternative splicing in cells transfected with rev-minus EIAV provirus constructs. In the context of provirus with wild-type exon 3, greater than 80% of the viral mRNAs were multiply spliced, and of these, less than 1% excluded exon 3. Deletion of the PRE resulted in a decrease in the relative amount of multiply spliced mRNA to about 40% of the total and approximately 39% of the viral mRNA excluded exon 3. Ectopic expression of ERev caused a decrease in the relative amount of multiply spliced mRNA to approximately 50% of the total and increased mRNAs that excluded exon 3 to about 4%. Over-expression of SF2/ASF in cells transfected with wild-type provirus constructs inhibited splicing but did not significantly alter exon 3 skipping

  17. Co-suppression of sterol-regulatory element binding protein ...

    African Journals Online (AJOL)

    Administrator

    2011-06-22

    Jun 22, 2011 ... In Arabidopsis,. At5g35220 gene being sterol regulatory element-binding protein site 2, protease and metalloendopeptidase activity were required for chloroplast development and play a role in regulation of endodermal plastid size and number that are involved in ethylene-dependent gravitropism of light-.

  18. Hijacking Complement Regulatory Proteins for Bacterial Immune Evasion.

    Science.gov (United States)

    Hovingh, Elise S; van den Broek, Bryan; Jongerius, Ilse

    2016-01-01

    The human complement system plays an important role in the defense against invading pathogens, inflammation and homeostasis. Invading microbes, such as bacteria, directly activate the complement system resulting in the formation of chemoattractants and in effective labeling of the bacteria for phagocytosis. In addition, formation of the membrane attack complex is responsible for direct killing of Gram-negative bacteria. In turn, bacteria have evolved several ways to evade complement activation on their surface in order to be able to colonize and invade the human host. One important mechanism of bacterial escape is attraction of complement regulatory proteins to the microbial surface. These molecules are present in the human body for tight regulation of the complement system to prevent damage to host self-surfaces. Therefore, recruitment of complement regulatory proteins to the bacterial surface results in decreased complement activation on the microbial surface which favors bacterial survival. This review will discuss recent advances in understanding the binding of complement regulatory proteins to the bacterial surface at the molecular level. This includes, new insights that have become available concerning specific conserved motives on complement regulatory proteins that are favorable for microbial binding. Finally, complement evasion molecules are of high importance for vaccine development due to their dominant role in bacterial survival, high immunogenicity and homology as well as their presence on the bacterial surface. Here, the use of complement evasion molecules for vaccine development will be discussed.

  19. CONSTRUCTION AND ANALYSIS OF IPBR/XYLS HYBRID REGULATORY PROTEINS

    Science.gov (United States)

    IpbR and XylS are related regulatory proteins (having 56% identity). IpbR responds to isopropylbenzene as well as to a variety of hydrophobic chemicals to activate expression of the isopropylbenzene catabolic pathway operon of pRE4 from ipbOP. XylS responds to substituted benzoic...

  20. Trans-acting translational regulatory RNA binding proteins.

    Science.gov (United States)

    Harvey, Robert F; Smith, Tom S; Mulroney, Thomas; Queiroz, Rayner M L; Pizzinga, Mariavittoria; Dezi, Veronica; Villenueva, Eneko; Ramakrishna, Manasa; Lilley, Kathryn S; Willis, Anne E

    2018-05-01

    The canonical molecular machinery required for global mRNA translation and its control has been well defined, with distinct sets of proteins involved in the processes of translation initiation, elongation and termination. Additionally, noncanonical, trans-acting regulatory RNA-binding proteins (RBPs) are necessary to provide mRNA-specific translation, and these interact with 5' and 3' untranslated regions and coding regions of mRNA to regulate ribosome recruitment and transit. Recently it has also been demonstrated that trans-acting ribosomal proteins direct the translation of specific mRNAs. Importantly, it has been shown that subsets of RBPs often work in concert, forming distinct regulatory complexes upon different cellular perturbation, creating an RBP combinatorial code, which through the translation of specific subsets of mRNAs, dictate cell fate. With the development of new methodologies, a plethora of novel RNA binding proteins have recently been identified, although the function of many of these proteins within mRNA translation is unknown. In this review we will discuss these methodologies and their shortcomings when applied to the study of translation, which need to be addressed to enable a better understanding of trans-acting translational regulatory proteins. Moreover, we discuss the protein domains that are responsible for RNA binding as well as the RNA motifs to which they bind, and the role of trans-acting ribosomal proteins in directing the translation of specific mRNAs. This article is categorized under: RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes Translation > Translation Regulation Translation > Translation Mechanisms. © 2018 Medical Research Council and University of Cambridge. WIREs RNA published by Wiley Periodicals, Inc.

  1. Analysis of MDM2 and MDM4 single nucleotide polymorphisms, mRNA splicing and protein expression in retinoblastoma.

    Directory of Open Access Journals (Sweden)

    Justina McEvoy

    Full Text Available Retinoblastoma is a childhood cancer of the developing retina that begins in utero and is diagnosed in the first years of life. Biallelic RB1 gene inactivation is the initiating genetic lesion in retinoblastoma. The p53 gene is intact in human retinoblastoma but the pathway is believed to be suppressed by increased expression of MDM4 (MDMX and MDM2. Here we quantify the expression of MDM4 and MDM2 mRNA and protein in human fetal retinae, primary retinoblastomas, retinoblastoma cell lines and several independent orthotopic retinoblastoma xenografts. We found that MDM4 is the major p53 antagonist expressed in retinoblastoma and in the developing human retina. We also discovered that MDM4 protein steady state levels are much higher in retinoblastoma than in human fetal retinae. This increase would not have been predicted based on the mRNA levels. We explored several possible post-transcriptional mechanisms that may contribute to the elevated levels of MDM4 protein. A proportion of MDM4 transcripts are alternatively spliced to produce protein products that are reported to be more stable and oncogenic. We also discovered that a microRNA predicted to target MDM4 (miR191 was downregulated in retinoblastoma relative to human fetal retinae and a subset of samples had somatic mutations that eliminated the miR-191 binding site in the MDM4 mRNA. Taken together, these data suggest that post-transcriptional mechanisms may contribute to stabilization of the MDM4 protein in retinoblastoma.

  2. Feline immunodeficiency virus OrfA alters gene expression of splicing factors and proteasome-ubiquitination proteins

    International Nuclear Information System (INIS)

    Sundstrom, Magnus; Chatterji, Udayan; Schaffer, Lana; Rozieres, Sohela de; Elder, John H.

    2008-01-01

    Expression of the feline immunodeficiency virus (FIV) accessory protein OrfA (or Orf2) is critical for efficient viral replication in lymphocytes, both in vitro and in vivo. OrfA has been reported to exhibit functions in common with the human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) accessory proteins Vpr and Tat, although the function of OrfA has not been fully explained. Here, we use microarray analysis to characterize how OrfA modulates the gene expression profile of T-lymphocytes. The primary IL-2-dependent T-cell line 104-C1 was transduced to express OrfA. Functional expression of OrfA was demonstrated by trans complementation of the OrfA-defective clone, FIV-34TF10. OrfA-expressing cells had a slightly reduced cell proliferation rate but did not exhibit any significant alteration in cell cycle distribution. Reverse-transcribed RNA from cells expressing green fluorescent protein (GFP) or GFP + OrfA were hybridized to Affymetrix HU133 Plus 2.0 microarray chips representing more than 47,000 genome-wide transcripts. By using two statistical approaches, 461 (Rank Products) and 277 (ANOVA) genes were identified as modulated by OrfA expression. The functional relevance of the differentially expressed genes was explored by Ingenuity Pathway Analysis. The analyses revealed alterations in genes critical for RNA post-transcriptional modifications and protein ubiquitination as the two most significant functional outcomes of OrfA expression. In these two groups, several subunits of the spliceosome, cellular splicing factors and family members of the proteasome-ubiquitination system were identified. These findings provide novel information on the versatile function of OrfA during FIV infection and indicate a fine-tuning mechanism of the cellular environment by OrfA to facilitate efficient FIV replication

  3. GC content around splice sites affects splicing through pre-mRNA secondary structures

    Directory of Open Access Journals (Sweden)

    Chen Liang

    2011-01-01

    Full Text Available Abstract Background Alternative splicing increases protein diversity by generating multiple transcript isoforms from a single gene through different combinations of exons or through different selections of splice sites. It has been reported that RNA secondary structures are involved in alternative splicing. Here we perform a genomic study of RNA secondary structures around splice sites in humans (Homo sapiens, mice (Mus musculus, fruit flies (Drosophila melanogaster, and nematodes (Caenorhabditis elegans to further investigate this phenomenon. Results We observe that GC content around splice sites is closely associated with the splice site usage in multiple species. RNA secondary structure is the possible explanation, because the structural stability difference among alternative splice sites, constitutive splice sites, and skipped splice sites can be explained by the GC content difference. Alternative splice sites tend to be GC-enriched and exhibit more stable RNA secondary structures in all of the considered species. In humans and mice, splice sites of first exons and long exons tend to be GC-enriched and hence form more stable structures, indicating the special role of RNA secondary structures in promoter proximal splicing events and the splicing of long exons. In addition, GC-enriched exon-intron junctions tend to be overrepresented in tissue-specific alternative splice sites, indicating the functional consequence of the GC effect. Compared with regions far from splice sites and decoy splice sites, real splice sites are GC-enriched. We also found that the GC-content effect is much stronger than the nucleotide-order effect to form stable secondary structures. Conclusion All of these results indicate that GC content is related to splice site usage and it may mediate the splicing process through RNA secondary structures.

  4. An XPA gene splicing mutation resulting in trace protein expression in an elderly patient with xeroderma pigmentosum group A without neurological abnormalities.

    Science.gov (United States)

    Takahashi, Y; Endo, Y; Kusaka-Kikushima, A; Nakamaura, S; Nakazawa, Y; Ogi, T; Uryu, M; Tsuji, G; Furue, M; Moriwaki, S

    2017-07-01

    A certain relationship between XPA gene mutations and the severity of symptoms has been observed in patients with xeroderma pigmentosum group A (XP-A). Patients with mutations within the DNA-binding domain usually exhibit severe symptoms, whereas splicing mutations in the same domain sometimes cause very mild symptoms. This inconsistency can be explained by a small amount of functional XPA protein produced from normally spliced transcripts. We herein report the case of an adult Japanese patient with XP-A with unusually mild symptoms. We identified a homozygous c.529G>A mutation in exon 4 of the XPA gene, which resulted in aberrant splicing with a 29-bp deletion in exon 4 causing a frameshift. Intact mRNA was observable, but a Western blot analysis failed to detect any normal XPA protein. We therefore evaluated the DNA repair capacity in normal cells in which the XPA expression was artificially diminished. The repair capacity was still present in cells with trace levels of the XPA protein. The repair capacity of the cells derived from our patient with mild symptoms was poor by comparison, but still significant compared with that of the cells derived from a patient with XP-A with severe symptoms. These results provide strong evidence that a trace level of XPA protein can still exert a relatively strong repair capacity, resulting in only a mild phenotype. © 2016 British Association of Dermatologists.

  5. Seed Dormancy in Arabidopsis Requires Self-Binding Ability of DOG1 Protein and the Presence of Multiple Isoforms Generated by Alternative Splicing.

    Directory of Open Access Journals (Sweden)

    Kazumi Nakabayashi

    2015-12-01

    Full Text Available The Arabidopsis protein DELAY OF GERMINATION 1 (DOG1 is a key regulator of seed dormancy, which is a life history trait that determines the timing of seedling emergence. The amount of DOG1 protein in freshly harvested seeds determines their dormancy level. DOG1 has been identified as a major dormancy QTL and variation in DOG1 transcript levels between accessions contributes to natural variation for seed dormancy. The DOG1 gene is alternatively spliced. Alternative splicing increases the transcriptome and proteome diversity in higher eukaryotes by producing transcripts that encode for proteins with altered or lost function. It can also generate tissue specific transcripts or affect mRNA stability. Here we suggest a different role for alternative splicing of the DOG1 gene. DOG1 produces five transcript variants encoding three protein isoforms. Transgenic dog1 mutant seeds expressing single DOG1 transcript variants from the endogenous DOG1 promoter did not complement because they were non-dormant and lacked DOG1 protein. However, transgenic plants overexpressing single DOG1 variants from the 35S promoter could accumulate protein and showed complementation. Simultaneous expression of two or more DOG1 transcript variants from the endogenous DOG1 promoter also led to increased dormancy levels and accumulation of DOG1 protein. This suggests that single isoforms are functional, but require the presence of additional isoforms to prevent protein degradation. Subsequently, we found that the DOG1 protein can bind to itself and that this binding is required for DOG1 function but not for protein accumulation. Natural variation for DOG1 binding efficiency was observed among Arabidopsis accessions and contributes to variation in seed dormancy.

  6. Self-hydroxylation of the splicing factor lysyl hydroxylase, JMJD6

    DEFF Research Database (Denmark)

    Mantri, M.; Webby, C.J.; Loik, N.D.

    2012-01-01

    The lysyl 5S-hydroxylase, JMJD6 acts on proteins involved in RNA splicing. We find that in the absence of substrate JMJD6 catalyses turnover of 2OG to succinate. H-NMR analyses demonstrate that consumption of 2OG is coupled to succinate formation. MS analyses reveal that JMJD6 undergoes self......-hydroxylation in the presence of Fe(ii) and 2OG resulting in production of 5S-hydroxylysine residues. JMJD6 in human cells is also found to be hydroxylated. Self-hydroxylation of JMJD6 may play a regulatory role in modulating the hydroxylation status of proteins involved in RNA splicing. This journal is...

  7. Transcriptome and proteome analyses and the role of atypical calpain protein and autophagy in the spliced leader silencing pathway in Trypanosoma brucei.

    Science.gov (United States)

    Hope, Ronen; Egarmina, Katarina; Voloshin, Konstantin; Waldman Ben-Asher, Hiba; Carmi, Shai; Eliaz, Dror; Drori, Yaron; Michaeli, Shulamit

    2016-10-01

    Under persistent ER stress, Trypanosoma brucei parasites induce the spliced leader silencing (SLS) pathway. In SLS, transcription of the SL RNA gene, the SL donor to all mRNAs, is extinguished, arresting trans-splicing and leading to programmed cell death (PCD). In this study, we investigated the transcriptome following silencing of SEC63, a factor essential for protein translocation across the ER membrane, and whose silencing induces SLS. The proteome of SEC63-silenced cells was analyzed with an emphasis on SLS-specific alterations in protein expression, and modifications that do not directly result from perturbations in trans-splicing. One such protein identified is an atypical calpain SKCRP7.1/7.2. Co-silencing of SKCRP7.1/7.2 and SEC63 eliminated SLS induction due its role in translocating the PK3 kinase. This kinase initiates SLS by migrating to the nucleus and phosphorylating TRF4 leading to shut-off of SL RNA transcription. Thus, SKCRP7.1 is involved in SLS signaling and the accompanying PCD. The role of autophagy in SLS was also investigated; eliminating autophagy through VPS34 or ATG7 silencing demonstrated that autophagy is not essential for SLS induction, but is associated with PCD. Thus, this study identified factors that are used by the parasite to cope with ER stress and to induce SLS and PCD. © 2016 John Wiley & Sons Ltd.

  8. Sub-nuclear distribution and mobility of nuclear proteins involved in histone acetylation and pre-mRNA splicing

    International Nuclear Information System (INIS)

    Kruhlak, Michael John

    2001-01-01

    The mitotic relationship between levels of highly acetylated chromatin, chromatin condensation, and HAT/HDAC organization was examined. HATs and HDACs were found to dissociate from chromosomes along with a loss of highly acetylated histones in condensed chromatin in mitosis. We demonstrate that, rather than being enzymatically inactivated, HAT and HDAC activities are decreased in mitosis because the enzymes are sequestered to a non-chromatin domain. Highly acetylated histone species reappear coincident with the reassociation of HATs and HDACs in late telophase/early interphase and before reinitiation of transcription. We propose that HATs and HDACs are spatially regulated through the cell cycle and that this regulation influences which chromatin domains are available for acetylation and deacetylation. We examined the movement of a splicing factor, ASF, green fluorescent fusion protein (ASF:GFP) using timelapse microscopy and the technique fluorescence recovery after photobleaching (FRAP). We found that ASF:GFP moves significantly slower than free diffusion when it is associated with speckles and, surprisingly, also when it is dispersed in the nucleoplasm. The mobility of ASF is consistent with frequent but transient interactions with relatively immobile nuclear binding sites. This mobility is slightly increased in the presence of transcription inhibitors and the ASF molecules further enrich in speckles. We propose that the nonrandom organization of splicing factors reflects spatial differences in the concentration of relatively immobile binding sites. Through a careful analysis of HDAC4 expression we found that HDAC4-containing MAD bodies are not a consistent component of the interphase nucleus. By comparing MAD bodies to PML bodies we found that the assembly, maintenance and distribution of PML bodies is regulated. We investigated the involvement of chromatin condensation in establishing mitotic transcription repression, by analyzing transcriptional activity in

  9. Computational Analysis of an Evolutionarily Conserved VertebrateMuscle Alternative Splicing Program

    Energy Technology Data Exchange (ETDEWEB)

    Das, Debopriya; Clark, Tyson A.; Schweitzer, Anthony; Marr,Henry; Yamamoto, Miki L.; Parra, Marilyn K.; Arribere, Josh; Minovitsky,Simon; Dubchak, Inna; Blume, John E.; Conboy, John G.

    2006-06-15

    A novel exon microarray format that probes gene expression with single exon resolution was employed to elucidate critical features of a vertebrate muscle alternative splicing program. A dataset of 56 microarray-defined, muscle-enriched exons and their flanking introns were examined computationally in order to investigate coordination of the muscle splicing program. Candidate intron regulatory motifs were required to meet several stringent criteria: significant over-representation near muscle-enriched exons, correlation with muscle expression, and phylogenetic conservation among genomes of several vertebrate orders. Three classes of regulatory motifs were identified in the proximal downstream intron, within 200nt of the target exons: UGCAUG, a specific binding site for Fox-1 related splicing factors; ACUAAC, a novel branchpoint-like element; and UG-/UGC-rich elements characteristic of binding sites for CELF splicing factors. UGCAUG was remarkably enriched, being present in nearly one-half of all cases. These studies suggest that Fox and CELF splicing factors play a major role in enforcing the muscle-specific alternative splicing program, facilitating expression of a set of unique isoforms of cytoskeletal proteins that are critical to muscle cell differentiation. Supplementary materials: There are four supplementary tables and one supplementary figure. The tables provide additional detailed information concerning the muscle-enriched datasets, and about over-represented oligonucleotide sequences in the flanking introns. The supplementary figure shows RT-PCR data confirming the muscle-enriched expression of exons predicted from the microarray analysis.

  10. Myristoylated α subunits of guanine nucleotide-binding regulatory proteins

    International Nuclear Information System (INIS)

    Buss, J.E.; Mumby, S.M.; Casey, P.J.; Gilman, A.G.; Sefton, B.M.

    1987-01-01

    Antisera directed against specific subunits of guanine nucleotide-binding regulatory proteins (G proteins) were used to immunoprecipitate these polypeptides from metabolically labeled cells. This technique detects, in extracts of a human astrocytoma cell line, the α subunits of G/sub s/ (stimulatory) (α 45 and α 52 ), a 41-kDa subunit of G/sub i/ (inhibitory) (α 41 ), a 40-kDa protein (α 40 ), and the 36-kDa β subunit. No protein that comigrated with the α subunit of G 0 (unknown function) (α 39 ) was detected. In cells grown in the presence of [ 3 H]myristic acid, α 41 and α 40 contained 3 H label, while the β subunit did not. Chemical analysis of lipids attached covalently to purified α 41 and α 39 from bovine brain also revealed myristic acid. Similar analysis of brain G protein β and γ subunits and of G/sub t/ (Transducin) subunits (α, β, and γ) failed to reveal fatty acids. The fatty acid associated with α 41 , α 40 , and α 39 was stable to treatment with base, suggesting that the lipid is linked to the polypeptide via an amide bond. These GTP binding proteins are thus identified as members of a select group of proteins that contains myristic acid covalently attached to the peptide backbone. Myristate may play an important role in stabilizing interactions of G proteins with phospholipid or with membrane-bound proteins

  11. Characterization of intronic uridine-rich sequence elements acting as possible targets for nuclear proteins during pre-mRNA splicing in Nicotiana plumbaginifolia.

    Science.gov (United States)

    Gniadkowski, M; Hemmings-Mieszczak, M; Klahre, U; Liu, H X; Filipowicz, W

    1996-02-15

    Introns of nuclear pre-mRNAs in dicotyledonous plants, unlike introns in vertebrates or yeast, are distinctly rich in A+U nucleotides and this feature is essential for their processing. In order to define more precisely sequence elements important for intron recognition in plants, we investigated the effects of short insertions, either U-rich or A-rich, on splicing of synthetic introns in transfected protoplast of Nicotiana plumbaginifolia. It was found that insertions of U-rich (sequence UUUUUAU) but not A-rich (AUAAAAA) segments can activate splicing of a GC-rich synthetic infron, and that U-rich segments, or multimers thereof, can function irrespective of the site of insertion within the intron. Insertions of multiple U-rich segments, either at the same or different locations, generally had an additive, stimulatory effect on splicing. Mutational analysis showed that replacement of one or two U residues in the UUUUUAU sequence with A or C residues had only a small effect on splicing, but replacement with G residues was strongly inhibitory. Proteins that interact with fragments of natural and synthetic pre-mRNAs in vitro were identified in nuclear extracts of N.plumbaginifolia by UV cross- linking. The profile of cross-linked plant proteins was considerably less complex than that obtained with a HeLa cell nuclear extract. Two major cross-linkable plant proteins had apparent molecular mass of 50 and 54 kDa and showed affinity for oligouridilates present in synGC introns or for poly(U).

  12. Unexpected role of the steroid-deficiency protein ecdysoneless in pre-mRNA splicing

    Czech Academy of Sciences Publication Activity Database

    Claudius, A.-K.; Romani, P.; Lamkemeyer, T.; Jindra, Marek; Uhlířová, M.

    2014-01-01

    Roč. 10, č. 4 (2014), e1004287 ISSN 1553-7404 Grant - others:Marie Curie Fellowship Award(CZ) GA 276569 Institutional support: RVO:60077344 Keywords : steroid-deficiency protein Subject RIV: ED - Physiology Impact factor: 8.167, year: 2013 http://www.plosgenetics.org/article/info%3Adoi%2F10.1371%2Fjournal.pgen.1004287

  13. Purification of the spliced leader ribonucleoprotein particle from Leptomonas collosoma revealed the existence of an Sm protein in trypanosomes. Cloning the SmE homologue.

    Science.gov (United States)

    Goncharov, I; Palfi, Z; Bindereif, A; Michaeli, S

    1999-04-30

    Trans-splicing in trypanosomes involves the addition of a common spliced leader (SL) sequence, which is derived from a small RNA, the SL RNA, to all mRNA precursors. The SL RNA is present in the cell in the form of a ribonucleoprotein, the SL RNP. Using conventional chromatography and affinity selection with 2'-O-methylated RNA oligonucleotides at high ionic strength, five proteins of 70, 16, 13, 12, and 8 kDa were co-selected with the SL RNA from Leptomonas collosoma, representing the SL RNP core particle. Under conditions of lower ionic strength, additional proteins of 28 and 20 kDa were revealed. On the basis of peptide sequences, the gene coding for a protein with a predicted molecular weight of 11.9 kDa was cloned and identified as homologue of the cis-spliceosomal SmE. The protein carries the Sm motifs 1 and 2 characteristic of Sm antigens that bind to all known cis-spliceosomal uridylic acid-rich small nuclear RNAs (U snRNAs), suggesting the existence of Sm proteins in trypanosomes. This finding is of special interest because trypanosome snRNPs are the only snRNPs examined to date that are not recognized by anti-Sm antibodies. Because of the early divergence of trypanosomes from the eukaryotic lineage, the trypanosome SmE protein represents one of the primordial Sm proteins in nature.

  14. Poliovirus 2A protease triggers a selective nucleo-cytoplasmic redistribution of splicing factors to regulate alternative pre-mRNA splicing.

    Directory of Open Access Journals (Sweden)

    Enrique Álvarez

    Full Text Available Poliovirus protease 2A (2A(pro obstructs host gene expression by reprogramming transcriptional and post-transcriptional regulatory events during infection. Here we demonstrate that expression of 2A(pro induces a selective nucleo-cytoplasm translocation of several important RNA binding proteins and splicing factors. Subcellular fractionation studies, together with immunofluorescence microscopy revealed an asymmetric distribution of HuR and TIA1/TIAR in 2A(pro expressing cells, which modulates splicing of the human Fas exon 6. Consistent with this result, knockdown of HuR or overexpression of TIA1/TIAR, leads to Fas exon 6 inclusion in 2A(pro-expressing cells. Therefore, poliovirus 2A(pro can target alternative pre-mRNA splicing by regulating protein shuttling between the nucleus and the cytoplasm.

  15. Tracing the Evolutionary History of the CAP Superfamily of Proteins Using Amino Acid Sequence Homology and Conservation of Splice Sites.

    Science.gov (United States)

    Abraham, Anup; Chandler, Douglas E

    2017-10-01

    Proteins of the CAP superfamily play numerous roles in reproduction, innate immune responses, cancer biology, and venom toxicology. Here we document the breadth of the CAP (Cysteine-RIch Secretory Protein (CRISP), Antigen 5, and Pathogenesis-Related) protein superfamily and trace the major events in its evolution using amino acid sequence homology and the positions of exon/intron borders within their genes. Seldom acknowledged in the literature, we find that many of the CAP subfamilies present in mammals, where they were originally characterized, have distinct homologues in the invertebrate phyla. Early eukaryotic CAP genes contained only one exon inherited from prokaryotic predecessors and as evolution progressed an increasing number of introns were inserted, reaching 2-5 in the invertebrate world and 5-15 in the vertebrate world. Focusing on the CRISP subfamily, we propose that these proteins evolved in three major steps: (1) origination of the CAP/PR/SCP domain in bacteria, (2) addition of a small Hinge domain to produce the two-domain SCP-like proteins found in roundworms and anthropoids, and (3) addition of an Ion Channel Regulatory domain, borrowed from invertebrate peptide toxins, to produce full length, three-domain CRISP proteins, first seen in insects and later to diversify into multiple subtypes in the vertebrate world.

  16. Protein Kinase A Regulatory Subunits in Human Adipose Tissue

    Science.gov (United States)

    Mantovani, Giovanna; Bondioni, Sara; Alberti, Luisella; Gilardini, Luisa; Invitti, Cecilia; Corbetta, Sabrina; Zappa, Marco A.; Ferrero, Stefano; Lania, Andrea G.; Bosari, Silvano; Beck-Peccoz, Paolo; Spada, Anna

    2009-01-01

    OBJECTIVE—In human adipocytes, the cAMP-dependent pathway mediates signals originating from β-adrenergic activation, thus playing a key role in the regulation of important metabolic processes, i.e., lipolysis and thermogenesis. Cyclic AMP effects are mainly mediated by protein kinase A (PKA), whose R2B regulatory isoform is the most expressed in mouse adipose tissue, where it protects against diet-induced obesity and fatty liver development. The aim of the study was to investigate possible differences in R2B expression, PKA activity, and lipolysis in adipose tissues from obese and nonobese subjects. RESEARCH DESIGN AND METHODS—The expression of the different PKA regulatory subunits was evaluated by immunohistochemistry, Western blot, and real-time PCR in subcutaneous and visceral adipose tissue samples from 20 nonobese and 67 obese patients. PKA activity and glycerol release were evaluated in total protein extract and adipocytes isolated from fresh tissue samples, respectively. RESULTS—Expression techniques showed that R2B was the most abundant regulatory protein, both at mRNA and protein level. Interestingly, R2B mRNA levels were significantly lower in both subcutaneous and visceral adipose tissues from obese than nonobese patients and negatively correlated with BMI, waist circumference, insulin levels, and homeostasis model assessment of insulin resistance. Moreover, both basal and stimulated PKA activity and glycerol release were significantly lower in visceral adipose tissue from obese patients then nonobese subjects. CONCLUSIONS—Our results first indicate that, in human adipose tissue, there are important BMI-related differences in R2B expression and PKA activation, which might be included among the multiple determinants involved in the different lipolytic response to β-adrenergic activation in obesity. PMID:19095761

  17. Differential splicing of the apoptosis-associated speck like protein containing a caspase recruitment domain (ASC regulates inflammasomes

    Directory of Open Access Journals (Sweden)

    Rojanasakul Yon

    2010-05-01

    Full Text Available Abstract Background The apoptotic speck-like protein containing a caspase recruitment domain (ASC is the essential adaptor protein for caspase 1 mediated interleukin (IL-1β and IL-18 processing in inflammasomes. It bridges activated Nod like receptors (NLRs, which are a family of cytosolic pattern recognition receptors of the innate immune system, with caspase 1, resulting in caspase 1 activation and subsequent processing of caspase 1 substrates. Hence, macrophages from ASC deficient mice are impaired in their ability to produce bioactive IL-1β. Furthermore, we recently showed that ASC translocates from the nucleus to the cytosol in response to inflammatory stimulation in order to promote an inflammasome response, which triggers IL-1β processing and secretion. However, the precise regulation of inflammasomes at the level of ASC is still not completely understood. In this study we identified and characterized three novel ASC isoforms for their ability to function as an inflammasome adaptor. Methods To establish the ability of ASC and ASC isoforms as functional inflammasome adaptors, IL-1β processing and secretion was investigated by ELISA in inflammasome reconstitution assays, stable expression in THP-1 and J774A1 cells, and by restoring the lack of endogenous ASC in mouse RAW264.7 macrophages. In addition, the localization of ASC and ASC isoforms was determined by immunofluorescence staining. Results The three novel ASC isoforms, ASC-b, ASC-c and ASC-d display unique and distinct capabilities to each other and to full length ASC in respect to their function as an inflammasome adaptor, with one of the isoforms even showing an inhibitory effect. Consistently, only the activating isoforms of ASC, ASC and ASC-b, co-localized with NLRP3 and caspase 1, while the inhibitory isoform ASC-c, co-localized only with caspase 1, but not with NLRP3. ASC-d did not co-localize with NLRP3 or with caspase 1 and consistently lacked the ability to function as an

  18. Specific CLK inhibitors from a novel chemotype for regulation of alternative splicing

    DEFF Research Database (Denmark)

    Fedorov, Oleg; Huber, Kilian; Eisenreich, Andreas

    2011-01-01

    There is a growing recognition of the importance of protein kinases in the control of alternative splicing. To define the underlying regulatory mechanisms, highly selective inhibitors are needed. Here, we report the discovery and characterization of the dichloroindolyl enaminonitrile KH-CB19......, a potent and highly specific inhibitor of the CDC2-like kinase isoforms 1 and 4 (CLK1/CLK4). Cocrystal structures of KH-CB19 with CLK1 and CLK3 revealed a non-ATP mimetic binding mode, conformational changes in helix aC and the phosphate binding loop and halogen bonding to the kinase hinge region. KH-CB19...... effectively suppressed phosphorylation of SR (serine/arginine) proteins in cells, consistent with its expected mechanism of action. Chemical inhibition of CLK1/CLK4 generated a unique pattern of splicing factor dephosphorylation and had at low nM concentration a profound effect on splicing of the two tissue...

  19. Identification of a novel nuclear localization signal and speckle-targeting sequence of tuftelin-interacting protein 11, a splicing factor involved in spliceosome disassembly

    Energy Technology Data Exchange (ETDEWEB)

    Tannukit, Sissada [Center for Craniofacial Molecular Biology, University of Southern California, 2250 Alcazar Street, CSA Rm103, Los Angeles, CA 90033-1004 (United States); Crabb, Tara L.; Hertel, Klemens J. [Department of Microbiology and Molecular Genetics, University of California Irvine, Irvine, CA 92697-4025 (United States); Wen, Xin [Center for Craniofacial Molecular Biology, University of Southern California, 2250 Alcazar Street, CSA Rm103, Los Angeles, CA 90033-1004 (United States); Jans, David A. [Department of Biochemistry and Molecular Biology, Nuclear Signalling Laboratory, Monash University, Clayton, Victoria 3800 (Australia); Paine, Michael L., E-mail: paine@usc.edu [Center for Craniofacial Molecular Biology, University of Southern California, 2250 Alcazar Street, CSA Rm103, Los Angeles, CA 90033-1004 (United States)

    2009-12-18

    Tuftelin-interacting protein 11 (TFIP11) is a protein component of the spliceosome complex that promotes the release of the lariat-intron during late-stage splicing through a direct recruitment and interaction with DHX15/PRP43. Expression of TFIP11 is essential for cell and organismal survival. TFIP11 contains a G-patch domain, a signature motif of RNA-processing proteins that is responsible for TFIP11-DHX15 interactions. No other functional domains within TFIP11 have been described. TFIP11 is localized to distinct speckled regions within the cell nucleus, although excluded from the nucleolus. In this study sequential C-terminal deletions and mutational analyses have identified two novel protein elements in mouse TFIP11. The first domain covers amino acids 701-706 (VKDKFN) and is an atypical nuclear localization signal (NLS). The second domain is contained within amino acids 711-735 and defines TFIP11's distinct speckled nuclear localization. The identification of a novel TFIP11 nuclear speckle-targeting sequence (TFIP11-STS) suggests that this domain directly interacts with additional spliceosomal components. These data help define the mechanism of nuclear/nuclear speckle localization of the splicing factor TFIP11, with implications for it's function.

  20. Bovine proteins containing poly-glutamine repeats are often polymorphic and enriched for components of transcriptional regulatory complexes

    LENUS (Irish Health Repository)

    Whan, Vicki

    2010-11-23

    Abstract Background About forty human diseases are caused by repeat instability mutations. A distinct subset of these diseases is the result of extreme expansions of polymorphic trinucleotide repeats; typically CAG repeats encoding poly-glutamine (poly-Q) tracts in proteins. Polymorphic repeat length variation is also apparent in human poly-Q encoding genes from normal individuals. As these coding sequence repeats are subject to selection in mammals, it has been suggested that normal variations in some of these typically highly conserved genes are implicated in morphological differences between species and phenotypic variations within species. At present, poly-Q encoding genes in non-human mammalian species are poorly documented, as are their functions and propensities for polymorphic variation. Results The current investigation identified 178 bovine poly-Q encoding genes (Q ≥ 5) and within this group, 26 genes with orthologs in both human and mouse that did not contain poly-Q repeats. The bovine poly-Q encoding genes typically had ubiquitous expression patterns although there was bias towards expression in epithelia, brain and testes. They were also characterised by unusually large sizes. Analysis of gene ontology terms revealed that the encoded proteins were strongly enriched for functions associated with transcriptional regulation and many contributed to physical interaction networks in the nucleus where they presumably act cooperatively in transcriptional regulatory complexes. In addition, the coding sequence CAG repeats in some bovine genes impacted mRNA splicing thereby generating unusual transcriptional diversity, which in at least one instance was tissue-specific. The poly-Q encoding genes were prioritised using multiple criteria for their likelihood of being polymorphic and then the highest ranking group was experimentally tested for polymorphic variation within a cattle diversity panel. Extensive and meiotically stable variation was identified

  1. Enzymatic Mercury Detoxification: The Regulatory Protein MerR

    CERN Multimedia

    Ctortecka, B; Walsh, C T; Comess, K M

    2002-01-01

    Mercury ions and organomercurial reagents are extremely toxic due to their affinity for thiol groups. Many bacteria contain an elaborate detoxification system for a metabolic conversion of toxic Hg$^{2+}$ or organomercurials to less toxic elemental Hg$^0$. The main components of the enzymatic mercury detoxification (see Fig. 1) are the regulatory protein MerR (mercury responsive genetic switch), the organomercurial lyase MerB (cleavage of carbon mercury bonds), and the mercuric ion reductase MerA (reduction of mercuric ions). In these proteins Hg$^{2+}$ is usually coordinated by the thiol groups of cysteines. We utilize the nuclear quadrupole interaction (NQI) of ${\\rm^{199m}}$Hg detected by time differential perturbed angular correlation (TDPAC) to identify the Hg metal site geometries in these proteins in order to elucidate the molecular origin of the ultrasensitivity, selectivity and reaction mechanism of this detoxification system. The short lived TDPAC probe ${\\rm^{199m}}$Hg ($\\tau_{1/2} =$ 43 min) is su...

  2. A conserved regulatory mechanism in bifunctional biotin protein ligases.

    Science.gov (United States)

    Wang, Jingheng; Beckett, Dorothy

    2017-08-01

    Class II bifunctional biotin protein ligases (BirA), which catalyze post-translational biotinylation and repress transcription initiation, are broadly distributed in eubacteria and archaea. However, it is unclear if these proteins all share the same molecular mechanism of transcription regulation. In Escherichia coli the corepressor biotinoyl-5'-AMP (bio-5'-AMP), which is also the intermediate in biotin transfer, promotes operator binding and resulting transcription repression by enhancing BirA dimerization. Like E. coli BirA (EcBirA), Staphylococcus aureus, and Bacillus subtilis BirA (Sa and BsBirA) repress transcription in vivo in a biotin-dependent manner. In this work, sedimentation equilibrium measurements were performed to investigate the molecular basis of this biotin-responsive transcription regulation. The results reveal that, as observed for EcBirA, Sa, and BsBirA dimerization reactions are significantly enhanced by bio-5'-AMP binding. Thus, the molecular mechanism of the Biotin Regulatory System is conserved in the biotin repressors from these three organisms. © 2017 The Protein Society.

  3. Homologous SV40 RNA trans-splicing: Special case or prime example of viral RNA trans-splicing?

    Directory of Open Access Journals (Sweden)

    Sushmita Poddar

    2014-06-01

    Full Text Available To date the Simian Virus 40 (SV40 is the only proven example of a virus that recruits the mechanism of RNA trans-splicing to diversify its sequences and gene products. Thereby, two identical viral transcripts are efficiently joined by homologous trans-splicing triggering the formation of a highly transforming 100 kDa super T antigen. Sequences of other viruses including HIV-1 and the human adenovirus type 5 were reported to be involved in heterologous trans-splicing towards cellular or viral sequences but the meaning of these events remains unclear. We computationally and experimentally investigated molecular features associated with viral RNA trans-splicing and identified a common pattern: Viral RNA trans-splicing occurs between strong cryptic or regular viral splice sites and strong regular or cryptic splice sites of the trans-splice partner sequences. The majority of these splice sites are supported by exonic splice enhancers. Splice sites that could compete with the trans-splicing sites for cis-splice reactions are weaker or inexistent. Finally, all but one of the trans-splice reactions seem to be facilitated by one or more complementary binding domains of 11 to 16 nucleotides in length which, however occur with a statistical probability close to one for the given length of the involved sequences. The chimeric RNAs generated via heterologous viral RNA trans-splicing either did not lead to fusion proteins or led to proteins of unknown function. Our data suggest that distinct viral RNAs are highly susceptible to trans-splicing and that heterologous viral trans-splicing, unlike homologous SV40 trans-splicing, represents a chance event.

  4. The Possible Role of Mena Protein and Its Splicing-Derived Variants in Embryogenesis, Carcinogenesis, and Tumor Invasion: A Systematic Review of the Literature

    Directory of Open Access Journals (Sweden)

    Simona Gurzu

    2013-01-01

    Full Text Available The Ena/VASP (enabled/vasodilator stimulated phosphoprotein family includes the binding actin proteins such as mammalian Ena (Mena, VASP, and Ena-VASP-like. It is known that the perturbation of actin cycle could determine alteration in the mobility of cells and in consequence of organogenesis. Few recent studies have revealed that Mena protein could play a role in breast or pancreatic carcinogenesis. Based on our researches, we observed that the intensity of Mena expression increased from premalignant to malignant lesions in some organs such as large bowel, stomach, cervix, and salivary glands. These findings prove that Mena could be a marker of premalignant epithelial lesions. In premalignant lesions, it could be helpful to define more accurately the risk for malignant transformation. In malignant tumors, correlation of expression of its splice variants could indicate metastatic behavior. In conclusion, we consider that it is necessary to analyze the expression of Mena splice variants in a higher number of cases, in different epithelial lesions, and also in experimental studies to define its exact role in carcinogenesis and also its possible prognostic and predictive values.

  5. The possible role of Mena protein and its splicing-derived variants in embryogenesis, carcinogenesis, and tumor invasion: a systematic review of the literature.

    Science.gov (United States)

    Gurzu, Simona; Ciortea, Diana; Ember, Istvan; Jung, Ioan

    2013-01-01

    The Ena/VASP (enabled/vasodilator stimulated phosphoprotein) family includes the binding actin proteins such as mammalian Ena (Mena), VASP, and Ena-VASP-like. It is known that the perturbation of actin cycle could determine alteration in the mobility of cells and in consequence of organogenesis. Few recent studies have revealed that Mena protein could play a role in breast or pancreatic carcinogenesis. Based on our researches, we observed that the intensity of Mena expression increased from premalignant to malignant lesions in some organs such as large bowel, stomach, cervix, and salivary glands. These findings prove that Mena could be a marker of premalignant epithelial lesions. In premalignant lesions, it could be helpful to define more accurately the risk for malignant transformation. In malignant tumors, correlation of expression of its splice variants could indicate metastatic behavior. In conclusion, we consider that it is necessary to analyze the expression of Mena splice variants in a higher number of cases, in different epithelial lesions, and also in experimental studies to define its exact role in carcinogenesis and also its possible prognostic and predictive values.

  6. A KH-Domain RNA-Binding Protein Interacts with FIERY2/CTD Phosphatase-Like 1 and Splicing Factors and Is Important for Pre-mRNA Splicing in Arabidopsis

    KAUST Repository

    Chen, Tao

    2013-10-17

    Eukaryotic genomes encode hundreds of RNA-binding proteins, yet the functions of most of these proteins are unknown. In a genetic study of stress signal transduction in Arabidopsis, we identified a K homology (KH)-domain RNA-binding protein, HOS5 (High Osmotic Stress Gene Expression 5), as required for stress gene regulation and stress tolerance. HOS5 was found to interact with FIERY2/RNA polymerase II (RNAP II) carboxyl terminal domain (CTD) phosphatase-like 1 (FRY2/CPL1) both in vitro and in vivo. This interaction is mediated by the first double-stranded RNA-binding domain of FRY2/CPL1 and the KH domains of HOS5. Interestingly, both HOS5 and FRY2/CPL1 also interact with two novel serine-arginine (SR)-rich splicing factors, RS40 and RS41, in nuclear speckles. Importantly, FRY2/CPL1 is required for the recruitment of HOS5. In fry2 mutants, HOS5 failed to be localized in nuclear speckles but was found mainly in the nucleoplasm. hos5 mutants were impaired in mRNA export and accumulated a significant amount of mRNA in the nuclei, particularly under salt stress conditions. Arabidopsis mutants of all these genes exhibit similar stress-sensitive phenotypes. RNA-seq analyses of these mutants detected significant intron retention in many stress-related genes under salt stress but not under normal conditions. Our study not only identified several novel regulators of pre-mRNA processing as important for plant stress response but also suggested that, in addition to RNAP II CTD that is a well-recognized platform for the recruitment of mRNA processing factors, FRY2/CPL1 may also recruit specific factors to regulate the co-transcriptional processing of certain transcripts to deal with environmental challenges. © 2013 Chen et al.

  7. A KH-Domain RNA-Binding Protein Interacts with FIERY2/CTD Phosphatase-Like 1 and Splicing Factors and Is Important for Pre-mRNA Splicing in Arabidopsis

    KAUST Repository

    Chen, Tao; Cui, Peng; Chen, Hao; Ali, Shahjahan; Zhang, ShouDong; Xiong, Liming

    2013-01-01

    Eukaryotic genomes encode hundreds of RNA-binding proteins, yet the functions of most of these proteins are unknown. In a genetic study of stress signal transduction in Arabidopsis, we identified a K homology (KH)-domain RNA-binding protein, HOS5 (High Osmotic Stress Gene Expression 5), as required for stress gene regulation and stress tolerance. HOS5 was found to interact with FIERY2/RNA polymerase II (RNAP II) carboxyl terminal domain (CTD) phosphatase-like 1 (FRY2/CPL1) both in vitro and in vivo. This interaction is mediated by the first double-stranded RNA-binding domain of FRY2/CPL1 and the KH domains of HOS5. Interestingly, both HOS5 and FRY2/CPL1 also interact with two novel serine-arginine (SR)-rich splicing factors, RS40 and RS41, in nuclear speckles. Importantly, FRY2/CPL1 is required for the recruitment of HOS5. In fry2 mutants, HOS5 failed to be localized in nuclear speckles but was found mainly in the nucleoplasm. hos5 mutants were impaired in mRNA export and accumulated a significant amount of mRNA in the nuclei, particularly under salt stress conditions. Arabidopsis mutants of all these genes exhibit similar stress-sensitive phenotypes. RNA-seq analyses of these mutants detected significant intron retention in many stress-related genes under salt stress but not under normal conditions. Our study not only identified several novel regulators of pre-mRNA processing as important for plant stress response but also suggested that, in addition to RNAP II CTD that is a well-recognized platform for the recruitment of mRNA processing factors, FRY2/CPL1 may also recruit specific factors to regulate the co-transcriptional processing of certain transcripts to deal with environmental challenges. © 2013 Chen et al.

  8. Dynamic regulation of genome-wide pre-mRNA splicing and stress tolerance by the Sm-like protein LSm5 in Arabidopsis

    KAUST Repository

    Cui, Peng; Zhang, ShouDong; Ding, Feng; Ali, Shahjahan; Xiong, Liming

    2014-01-01

    alternative splicing. Further, SAD1 modulates the splicing of stress-responsive genes, particularly under salt-stress conditions. Finally, we find that overexpression of SAD1 in Arabidopsis improves salt tolerance in transgenic plants, which correlates

  9. Effects of airborne particulate matter on alternative pre-mRNA splicing in colon cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Buggiano, Valeria; Petrillo, Ezequiel; Alló, Mariano; Lafaille, Celina [Laboratorio de Fisiología y Biología Molecular, Departamento de Fisiología, Biología Molecular y Celular, IFIBYNE-CONICET, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pabellón 2, C1428EHA Buenos Aires (Argentina); Redal, María Ana [Instituto de Ciencias Básicas y Medicina Experimental, Hospital Italiano de Buenos Aires (Argentina); Alghamdi, Mansour A. [Department of Environmental Sciences, Faculty of Meteorology, Environment and Arid Land Agriculture, King Abdulaziz University, Jeddah (Saudi Arabia); Khoder, Mamdouh I. [Department of Environmental Sciences, Faculty of Meteorology, Environment and Arid Land Agriculture, King Abdulaziz University, Jeddah (Saudi Arabia); Center of Excellence in Environmental Studies, King Abdulaziz University, Jeddah (Saudi Arabia); Shamy, Magdy [Department of Environmental Sciences, Faculty of Meteorology, Environment and Arid Land Agriculture, King Abdulaziz University, Jeddah (Saudi Arabia); Muñoz, Manuel J., E-mail: mmunoz@fbmc.fcen.uba.ar [Laboratorio de Fisiología y Biología Molecular, Departamento de Fisiología, Biología Molecular y Celular, IFIBYNE-CONICET, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pabellón 2, C1428EHA Buenos Aires (Argentina); and others

    2015-07-15

    Alternative pre-mRNA splicing plays key roles in determining tissue- and species-specific cell differentiation as well as in the onset of hereditary disease and cancer, being controlled by multiple post- and co-transcriptional regulatory mechanisms. We report here that airborne particulate matter, resulting from industrial pollution, inhibits expression and specifically affects alternative splicing at the 5′ untranslated region of the mRNA encoding the bone morphogenetic protein BMP4 in human colon cells in culture. These effects are consistent with a previously reported role for BMP4 in preventing colon cancer development, suggesting that ingestion of particulate matter could contribute to the onset of colon cell proliferation. We also show that the underlying mechanism might involve changes in transcriptional elongation. This is the first study to demonstrate that particulate matter causes non-pleiotropic changes in alternative splicing. - Highlights: • Airborne particulate matter (PM10) affects alternative splicing in colon cells. • PM10 upregulates one of the two mRNA variants of the growth factor BMP-4. • This variant has a longer 5′ unstranslated region and introduces an upstream AUG. • By regulating BMP-4 mRNA splicing PM10 inhibits total expression of BMP-4 protein. • BMP-4 downregulation was previously reported to be associated to colon cancer.

  10. Activation-induced cytidine deaminase (AID)-dependent somatic hypermutation requires a splice isoform of the serine/arginine-rich (SR) protein SRSF1.

    Science.gov (United States)

    Kanehiro, Yuichi; Todo, Kagefumi; Negishi, Misaki; Fukuoka, Junji; Gan, Wenjian; Hikasa, Takuya; Kaga, Yoshiaki; Takemoto, Masayuki; Magari, Masaki; Li, Xialu; Manley, James L; Ohmori, Hitoshi; Kanayama, Naoki

    2012-01-24

    Somatic hypermutation (SHM) of Ig variable region (IgV) genes requires both IgV transcription and the enzyme activation-induced cytidine deaminase (AID). Identification of a cofactor responsible for the fact that IgV genes are much more sensitive to AID-induced mutagenesis than other genes is a key question in immunology. Here, we describe an essential role for a splice isoform of the prototypical serine/arginine-rich (SR) protein SRSF1, termed SRSF1-3, in AID-induced SHM in a DT40 chicken B-cell line. Unexpectedly, we found that SHM does not occur in a DT40 line lacking SRSF1-3 (DT40-ASF), although it is readily detectable in parental DT40 cells. Strikingly, overexpression of AID in DT40-ASF cells led to a large increase in nonspecific (off-target) mutations. In contrast, introduction of SRSF1-3, but not SRSF1, into these cells specifically restored SHM without increasing off-target mutations. Furthermore, we found that SRSF1-3 binds preferentially to the IgV gene and inhibits processing of the Ig transcript, providing a mechanism by which SRSF1-3 makes the IgV gene available for AID-dependent SHM. SRSF1 not only acts as an essential splicing factor but also regulates diverse aspects of mRNA metabolism and maintains genome stability. Our findings, thus, define an unexpected and important role for SRSF1, particularly for its splice variant, in enabling AID to function specifically on its natural substrate during SHM.

  11. Critical protein GAPDH and its regulatory mechanisms in cancer cells

    International Nuclear Information System (INIS)

    Zhang, Jin-Ying; Zhang, Fan; Hong, Chao-Qun; Giuliano, Armando E.; Cui, Xiao-Jiang; Zhou, Guang-Ji; Zhang, Guo-Jun; Cui, Yu-Kun

    2015-01-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), initially identified as a glycolytic enzyme and considered as a housekeeping gene, is widely used as an internal control in experiments on proteins, mRNA, and DNA. However, emerging evidence indicates that GAPDH is implicated in diverse functions independent of its role in energy metabolism; the expression status of GAPDH is also deregulated in various cancer cells. One of the most common effects of GAPDH is its inconsistent role in the determination of cancer cell fate. Furthermore, studies have described GAPDH as a regulator of cell death; other studies have suggested that GAPDH participates in tumor progression and serves as a new therapeutic target. However, related regulatory mechanisms of its numerous cellular functions and deregulated expression levels remain unclear. GAPDH is tightly regulated at transcriptional and posttranscriptional levels, which are involved in the regulation of diverse GAPDH functions. Several cancer-related factors, such as insulin, hypoxia inducible factor-1 (HIF-1), p53, nitric oxide (NO), and acetylated histone, not only modulate GAPDH gene expression but also affect protein functions via common pathways. Moreover, posttranslational modifications (PTMs) occurring in GAPDH in cancer cells result in new activities unrelated to the original glycolytic function of GAPDH. In this review, recent findings related to GAPDH transcriptional regulation and PTMs are summarized. Mechanisms and pathways involved in GAPDH regulation and its different roles in cancer cells are also described

  12. Pleiotropy constrains the evolution of protein but not regulatory sequences in a transcription regulatory network influencing complex social behaviours

    Directory of Open Access Journals (Sweden)

    Daria eMolodtsova

    2014-12-01

    Full Text Available It is increasingly apparent that genes and networks that influence complex behaviour are evolutionary conserved, which is paradoxical considering that behaviour is labile over evolutionary timescales. How does adaptive change in behaviour arise if behaviour is controlled by conserved, pleiotropic, and likely evolutionary constrained genes? Pleiotropy and connectedness are known to constrain the general rate of protein evolution, prompting some to suggest that the evolution of complex traits, including behaviour, is fuelled by regulatory sequence evolution. However, we seldom have data on the strength of selection on mutations in coding and regulatory sequences, and this hinders our ability to study how pleiotropy influences coding and regulatory sequence evolution. Here we use population genomics to estimate the strength of selection on coding and regulatory mutations for a transcriptional regulatory network that influences complex behaviour of honey bees. We found that replacement mutations in highly connected transcription factors and target genes experience significantly stronger negative selection relative to weakly connected transcription factors and targets. Adaptively evolving proteins were significantly more likely to reside at the periphery of the regulatory network, while proteins with signs of negative selection were near the core of the network. Interestingly, connectedness and network structure had minimal influence on the strength of selection on putative regulatory sequences for both transcription factors and their targets. Our study indicates that adaptive evolution of complex behaviour can arise because of positive selection on protein-coding mutations in peripheral genes, and on regulatory sequence mutations in both transcription factors and their targets throughout the network.

  13. Identification of a novel splice acceptor in the HIV-1 genome: independent expression of the cytoplasmic tail of the envelope protein

    NARCIS (Netherlands)

    Berkhout, B.; van Wamel, J. L.

    1996-01-01

    Multiple splicing sites exist in the RNA genome of the human immunodeficiency virus type 1 (HIV-1). In a screen for subgenomic forms of the HIV-1 genome that could be transferred to fresh cells by virus infection, we identified a novel spliced variant of HIV-1 RNA that uses a hitherto unknown splice

  14. Rbfox-regulated alternative splicing is critical for zebrafish cardiac and skeletal muscle function

    Science.gov (United States)

    Gallagher, Thomas L.; Arribere, Joshua A.; Geurts, Paul A.; Exner, Cameron R. T.; McDonald, Kent L.; Dill, Kariena K.; Marr, Henry L.; Adkar, Shaunak S.; Garnett, Aaron T.; Amacher, Sharon L.; Conboy, John G.

    2012-01-01

    Rbfox RNA binding proteins are implicated as regulators of phylogenetically-conserved alternative splicing events important for muscle function. To investigate the function of rbfox genes, we used morpholino-mediated knockdown of muscle-expressed rbfox1l and rbfox2 in zebrafish embryos. Single and double morphant embryos exhibited changes in splicing of overlapping sets of bioinformatically-predicted rbfox target exons, many of which exhibit a muscle-enriched splicing pattern that is conserved in vertebrates. Thus, conservation of intronic Rbfox binding motifs is a good predictor of Rbfox-regulated alternative splicing. Morphology and development of single morphant embryos was strikingly normal; however, muscle development in double morphants was severely disrupted. Defects in cardiac muscle were marked by reduced heart rate and in skeletal muscle by complete paralysis. The predominance of wavy myofibers and abnormal thick and thin filaments in skeletal muscle revealed that myofibril assembly is defective and disorganized in double morphants. Ultra-structural analysis revealed that although sarcomeres with electron dense M- and Z-bands are present in muscle fibers of rbfox1l/rbox2 morphants, they are substantially reduced in number and alignment. Importantly, splicing changes and morphological defects were rescued by expression of morpholino-resistant rbfox cDNA. Additionally, a target-blocking MO complementary to a single UGCAUG motif adjacent to an rbfox target exon of fxr1 inhibited inclusion in a similar manner to rbfox knockdown, providing evidence that Rbfox regulates the splicing of target exons via direct binding to intronic regulatory motifs. We conclude that Rbfox proteins regulate an alternative splicing program essential for vertebrate heart and skeletal muscle function. PMID:21925157

  15. Rbfox-regulated alternative splicing is critical for zebrafish cardiac and skeletal muscle functions.

    Science.gov (United States)

    Gallagher, Thomas L; Arribere, Joshua A; Geurts, Paul A; Exner, Cameron R T; McDonald, Kent L; Dill, Kariena K; Marr, Henry L; Adkar, Shaunak S; Garnett, Aaron T; Amacher, Sharon L; Conboy, John G

    2011-11-15

    Rbfox RNA binding proteins are implicated as regulators of phylogenetically-conserved alternative splicing events important for muscle function. To investigate the function of rbfox genes, we used morpholino-mediated knockdown of muscle-expressed rbfox1l and rbfox2 in zebrafish embryos. Single and double morphant embryos exhibited changes in splicing of overlapping sets of bioinformatically-predicted rbfox target exons, many of which exhibit a muscle-enriched splicing pattern that is conserved in vertebrates. Thus, conservation of intronic Rbfox binding motifs is a good predictor of Rbfox-regulated alternative splicing. Morphology and development of single morphant embryos were strikingly normal; however, muscle development in double morphants was severely disrupted. Defects in cardiac muscle were marked by reduced heart rate and in skeletal muscle by complete paralysis. The predominance of wavy myofibers and abnormal thick and thin filaments in skeletal muscle revealed that myofibril assembly is defective and disorganized in double morphants. Ultra-structural analysis revealed that although sarcomeres with electron dense M- and Z-bands are present in muscle fibers of rbfox1l/rbox2 morphants, they are substantially reduced in number and alignment. Importantly, splicing changes and morphological defects were rescued by expression of morpholino-resistant rbfox cDNA. Additionally, a target-blocking MO complementary to a single UGCAUG motif adjacent to an rbfox target exon of fxr1 inhibited inclusion in a similar manner to rbfox knockdown, providing evidence that Rbfox regulates the splicing of target exons via direct binding to intronic regulatory motifs. We conclude that Rbfox proteins regulate an alternative splicing program essential for vertebrate heart and skeletal muscle functions. Published by Elsevier Inc.

  16. Characterization of a novel splicing variant in the RAPTOR gene

    International Nuclear Information System (INIS)

    Sun Chang; Southard, Catherine; Di Rienzo, Anna

    2009-01-01

    The mammalian target of rapamycin (mTOR) plays an essential role in the regulation of cell growth, proliferation and apoptosis. Raptor, the regulatory associated protein of mTOR, is an important member in this signaling pathway. In the present report, we identified and characterized a novel splicing variant of this gene, RAPTOR v 2, in which exons 14-17, 474 bp in total, are omitted from the mRNA. This deletion does not change the open reading frame, but causes a nearly complete absence of HEAT repeats, which were shown to be involved in the binding of mTOR substrates. Real time PCR performed on 48 different human tissues demonstrated the ubiquitous presence of this splice variant. Quantification of mRNA levels in lymphoblastoid cell lines (LCL) from 56 unrelated HapMap individuals revealed that the expression of this splicing form is quite variable. One synonymous SNP, rs2289759 in exon 14, was predicted by ESEfinder to cause a significant gain/loss of SRp55 and/or SF2/ASF binding sites, and thus potentially influence splicing. This prediction was confirmed by linear regression analysis between the ratio of RAPTOR v 2 to total RAPTOR mRNA levels and the SNP genotype in the above 56 individuals (r = 0.281 and P = 0.036). Moreover, the functional evaluation indicated that this splicing isoform is expected to retain the ability to bind mTOR, but is unlikely to bind mTOR substrates, hence affecting signal transduction and further cell proliferation

  17. Sex Determination in Insects: a binary decision based on alternative splicing

    OpenAIRE

    Salz, Helen K.

    2011-01-01

    The gene regulatory networks that control sex determination vary between species. Despite these differences, comparative studies in insects have found that alternative splicing is reiteratively used in evolution to control expression of the key sex determining genes. Sex determination is best understood in Drosophila where activation of the RNA binding protein encoding gene Sex-lethal is the central female-determining event. Sex-lethal serves as a genetic switch because once activated it cont...

  18. Alternative splicing in cancers: From aberrant regulation to new therapeutics.

    Science.gov (United States)

    Song, Xiaowei; Zeng, Zhenyu; Wei, Huanhuan; Wang, Zefeng

    2018-03-01

    Alternative splicing is one of the most common mechanisms for gene regulation in humans, and plays a vital role to increase the complexity of functional proteins. In this article, we seek to provide a general review on the relationships between alternative splicing and tumorigenesis. We briefly introduce the basic rules for regulation of alternative splicing, and discuss recent advances on dynamic regulation of alternative splicing in cancers by highlighting the roles of a variety of RNA splicing factors in tumorigenesis. We further discuss several important questions regarding the splicing of long noncoding RNAs and back-splicing of circular RNAs in cancers. Finally, we discuss the current technologies that can be used to manipulate alternative splicing and serve as potential cancer treatment. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. The Evolution of the Secreted Regulatory Protein Progranulin.

    Directory of Open Access Journals (Sweden)

    Roger G E Palfree

    Full Text Available Progranulin is a secreted growth factor that is active in tumorigenesis, wound repair, and inflammation. Haploinsufficiency of the human progranulin gene, GRN, causes frontotemporal dementia. Progranulins are composed of chains of cysteine-rich granulin modules. Modules may be released from progranulin by proteolysis as 6kDa granulin polypeptides. Both intact progranulin and some of the granulin polypeptides are biologically active. The granulin module occurs in certain plant proteases and progranulins are present in early diverging metazoan clades such as the sponges, indicating their ancient evolutionary origin. There is only one Grn gene in mammalian genomes. More gene-rich Grn families occur in teleost fish with between 3 and 6 members per species including short-form Grns that have no tetrapod counterparts. Our goals are to elucidate progranulin and granulin module evolution by investigating (i: the origins of metazoan progranulins (ii: the evolutionary relationships between the single Grn of tetrapods and the multiple Grn genes of fish (iii: the evolution of granulin module architectures of vertebrate progranulins (iv: the conservation of mammalian granulin polypeptide sequences and how the conserved granulin amino acid sequences map to the known three dimensional structures of granulin modules. We report that progranulin-like proteins are present in unicellular eukaryotes that are closely related to metazoa suggesting that progranulin is among the earliest extracellular regulatory proteins still employed by multicellular animals. From the genomes of the elephant shark and coelacanth we identified contemporary representatives of a precursor for short-from Grn genes of ray-finned fish that is lost in tetrapods. In vertebrate Grns pathways of exon duplication resulted in a conserved module architecture at the amino-terminus that is frequently accompanied by an unusual pattern of tandem nearly identical module repeats near the carboxyl

  20. The Evolution of the Secreted Regulatory Protein Progranulin.

    Science.gov (United States)

    Palfree, Roger G E; Bennett, Hugh P J; Bateman, Andrew

    2015-01-01

    Progranulin is a secreted growth factor that is active in tumorigenesis, wound repair, and inflammation. Haploinsufficiency of the human progranulin gene, GRN, causes frontotemporal dementia. Progranulins are composed of chains of cysteine-rich granulin modules. Modules may be released from progranulin by proteolysis as 6kDa granulin polypeptides. Both intact progranulin and some of the granulin polypeptides are biologically active. The granulin module occurs in certain plant proteases and progranulins are present in early diverging metazoan clades such as the sponges, indicating their ancient evolutionary origin. There is only one Grn gene in mammalian genomes. More gene-rich Grn families occur in teleost fish with between 3 and 6 members per species including short-form Grns that have no tetrapod counterparts. Our goals are to elucidate progranulin and granulin module evolution by investigating (i): the origins of metazoan progranulins (ii): the evolutionary relationships between the single Grn of tetrapods and the multiple Grn genes of fish (iii): the evolution of granulin module architectures of vertebrate progranulins (iv): the conservation of mammalian granulin polypeptide sequences and how the conserved granulin amino acid sequences map to the known three dimensional structures of granulin modules. We report that progranulin-like proteins are present in unicellular eukaryotes that are closely related to metazoa suggesting that progranulin is among the earliest extracellular regulatory proteins still employed by multicellular animals. From the genomes of the elephant shark and coelacanth we identified contemporary representatives of a precursor for short-from Grn genes of ray-finned fish that is lost in tetrapods. In vertebrate Grns pathways of exon duplication resulted in a conserved module architecture at the amino-terminus that is frequently accompanied by an unusual pattern of tandem nearly identical module repeats near the carboxyl-terminus. Polypeptide

  1. The expression of cytoskeleton regulatory protein Mena in colorectal lesions.

    Science.gov (United States)

    Gurzu, Simona; Jung, I; Prantner, I; Ember, I; Pávai, Z; Mezei, T

    2008-01-01

    The actin regulatory proteins Ena/VASP (Enabled/Vasodilator stimulated phosphoprotein) family is involved in the control of cell motility and adhesion. They are important in the actin-dependent processes where dynamic actin reorganization it is necessary. The deregulation of actin cycle could have an important role in the cells' malignant transformation, tumor invasion or metastasis. Recently studies revealed that the human orthologue of murine Mena is modulated during the breast carcinogenesis. In our study, we tried to observe the immunohistochemical expression of mammalian Ena (Mena) in the colorectal polyps and carcinomas. We analyzed 10 adenomatous polyps (five with dysplasia) and 36 adenocarcinomas. We used the indirect immunoperoxidase staining. BD Biosciences have provided the Mena antibody. We observed that Mena was not expressed in the normal colorectal mucosa neither in polyps without dysplasia, but its expression was very high in polyps with high dysplasia. In colorectal carcinomas, Mena marked the tumoral cells in 80% of cases. In 25% of positive cases, the intensity was 3+, in 60% 2+ and in the other 15% 1+. The Mena intensity was higher in the microsatellite stable tumors (MSS) and was correlated with vascular invasion, with intensity of angiogenesis marked with CD31 and CD105 and with c-erbB-2 and p53 expression. This is the first study in the literature about Mena expression in colorectal lesions.

  2. Human Splicing Finder: an online bioinformatics tool to predict splicing signals.

    Science.gov (United States)

    Desmet, François-Olivier; Hamroun, Dalil; Lalande, Marine; Collod-Béroud, Gwenaëlle; Claustres, Mireille; Béroud, Christophe

    2009-05-01

    Thousands of mutations are identified yearly. Although many directly affect protein expression, an increasing proportion of mutations is now believed to influence mRNA splicing. They mostly affect existing splice sites, but synonymous, non-synonymous or nonsense mutations can also create or disrupt splice sites or auxiliary cis-splicing sequences. To facilitate the analysis of the different mutations, we designed Human Splicing Finder (HSF), a tool to predict the effects of mutations on splicing signals or to identify splicing motifs in any human sequence. It contains all available matrices for auxiliary sequence prediction as well as new ones for binding sites of the 9G8 and Tra2-beta Serine-Arginine proteins and the hnRNP A1 ribonucleoprotein. We also developed new Position Weight Matrices to assess the strength of 5' and 3' splice sites and branch points. We evaluated HSF efficiency using a set of 83 intronic and 35 exonic mutations known to result in splicing defects. We showed that the mutation effect was correctly predicted in almost all cases. HSF could thus represent a valuable resource for research, diagnostic and therapeutic (e.g. therapeutic exon skipping) purposes as well as for global studies, such as the GEN2PHEN European Project or the Human Variome Project.

  3. Systematic Profiling of Poly(A)+ Transcripts Modulated by Core 3’ End Processing and Splicing Factors Reveals Regulatory Rules of Alternative Cleavage and Polyadenylation

    Science.gov (United States)

    Li, Wencheng; You, Bei; Hoque, Mainul; Zheng, Dinghai; Luo, Wenting; Ji, Zhe; Park, Ji Yeon; Gunderson, Samuel I.; Kalsotra, Auinash; Manley, James L.; Tian, Bin

    2015-01-01

    Alternative cleavage and polyadenylation (APA) results in mRNA isoforms containing different 3’ untranslated regions (3’UTRs) and/or coding sequences. How core cleavage/polyadenylation (C/P) factors regulate APA is not well understood. Using siRNA knockdown coupled with deep sequencing, we found that several C/P factors can play significant roles in 3’UTR-APA. Whereas Pcf11 and Fip1 enhance usage of proximal poly(A) sites (pAs), CFI-25/68, PABPN1 and PABPC1 promote usage of distal pAs. Strong cis element biases were found for pAs regulated by CFI-25/68 or Fip1, and the distance between pAs plays an important role in APA regulation. In addition, intronic pAs are substantially regulated by splicing factors, with U1 mostly inhibiting C/P events in introns near the 5’ end of gene and U2 suppressing those in introns with features for efficient splicing. Furthermore, PABPN1 inhibits expression of transcripts with pAs near the transcription start site (TSS), a property possibly related to its role in RNA degradation. Finally, we found that groups of APA events regulated by C/P factors are also modulated in cell differentiation and development with distinct trends. Together, our results support an APA code where an APA event in a given cellular context is regulated by a number of parameters, including relative location to the TSS, splicing context, distance between competing pAs, surrounding cis elements and concentrations of core C/P factors. PMID:25906188

  4. Systematic profiling of poly(A+ transcripts modulated by core 3' end processing and splicing factors reveals regulatory rules of alternative cleavage and polyadenylation.

    Directory of Open Access Journals (Sweden)

    Wencheng Li

    2015-04-01

    Full Text Available Alternative cleavage and polyadenylation (APA results in mRNA isoforms containing different 3' untranslated regions (3'UTRs and/or coding sequences. How core cleavage/polyadenylation (C/P factors regulate APA is not well understood. Using siRNA knockdown coupled with deep sequencing, we found that several C/P factors can play significant roles in 3'UTR-APA. Whereas Pcf11 and Fip1 enhance usage of proximal poly(A sites (pAs, CFI-25/68, PABPN1 and PABPC1 promote usage of distal pAs. Strong cis element biases were found for pAs regulated by CFI-25/68 or Fip1, and the distance between pAs plays an important role in APA regulation. In addition, intronic pAs are substantially regulated by splicing factors, with U1 mostly inhibiting C/P events in introns near the 5' end of gene and U2 suppressing those in introns with features for efficient splicing. Furthermore, PABPN1 inhibits expression of transcripts with pAs near the transcription start site (TSS, a property possibly related to its role in RNA degradation. Finally, we found that groups of APA events regulated by C/P factors are also modulated in cell differentiation and development with distinct trends. Together, our results support an APA code where an APA event in a given cellular context is regulated by a number of parameters, including relative location to the TSS, splicing context, distance between competing pAs, surrounding cis elements and concentrations of core C/P factors.

  5. Depolarization-mediated regulation of alternative splicing

    Directory of Open Access Journals (Sweden)

    Alok eSharma

    2011-12-01

    Full Text Available Alternative splicing in eukaryotes plays an important role in regulating gene expression by selectively including alternative exons. A wealth of information has been accumulated that explains how alternative exons are selected in a developmental stage- or tissue-specific fashion. However, our knowledge of how cells respond to environmental changes to alter alternative splicing is very limited. For example, although a number of alternative exons have been shown to be regulated by calcium level alterations, the underlying mechanisms are not well understood. As calcium signaling in neurons plays a crucial role in essential neuronal functions such as learning and memory formation, it is important to understand how this process is regulated at every level in gene expression. The significance of the dynamic control of alternative splicing in response to changes of calcium levels has been largely unappreciated. In this communication, we will summarize the recent advances in calcium signaling-mediated alternative splicing that have provided some insights into the important regulatory mechanisms. In addition to describing the cis-acting RNA elements on the pre-mRNA molecules that respond to changes of intracellular calcium levels, we will summarize how splicing regulators change and affect alternative splicing in this process. We will also discuss a novel mode of calcium-mediated splicing regulation at the level of chromatin structure and transcription.

  6. Human thyroid peroxidase: complete cDNA and protein sequence, chromosome mapping, and identification of two alternately spliced mRNAs

    International Nuclear Information System (INIS)

    Kimura, S.; Kotani, T.; McBride, O.W.; Umeki, K.; Hirai, K.; Nakayama, T.; Ohtaki, S.

    1987-01-01

    Two forms of human thyroid peroxidase cDNAs were isolated from a λgt11 cDNA library, prepared from Graves disease thyroid tissue mRNA, by use of oligonucleotides. The longest complete cDNA, designated phTPO-1, has 3048 nucleotides and an open reading frame consisting of 933 amino acids, which would encode a protein with a molecular weight of 103,026. Five potential asparagine-linked glycosylation sites are found in the deduced amino acid sequence. The second peroxidase cDNA, designated phTPO-2, is almost identical to phTPO-1 beginning 605 base pairs downstream except that it contains 1-base-pair difference and lacks 171 base pairs in the middle of the sequence. This results in a loss of 57 amino acids corresponding to a molecular weight of 6282. Interestingly, this 171-nucleotide sequence has GT and AG at its 5' and 3' boundaries, respectively, that are in good agreement with donor and acceptor splice site consensus sequences. Using specific oligonucleotide probes for the mRNAs derived from the cDNA sequences hTOP-1 and hTOP-2, the authors show that both are expressed in all thyroid tissues examined and the relative level of two mRNAs is different in each sample. The results suggest that two thyroid peroxidase proteins might be generated through alternate splicing of the same gene. By using somatic cell hybrid lines, the thyroid peroxidase gene was mapped to the short arm of human chromosome 2

  7. An in vivo genetic screen for genes involved in spliced leader trans-splicing indicates a crucial role for continuous de novo spliced leader RNP assembly.

    Science.gov (United States)

    Philippe, Lucas; Pandarakalam, George C; Fasimoye, Rotimi; Harrison, Neale; Connolly, Bernadette; Pettitt, Jonathan; Müller, Berndt

    2017-08-21

    Spliced leader (SL) trans-splicing is a critical element of gene expression in a number of eukaryotic groups. This process is arguably best understood in nematodes, where biochemical and molecular studies in Caenorhabditis elegans and Ascaris suum have identified key steps and factors involved. Despite this, the precise details of SL trans-splicing have yet to be elucidated. In part, this is because the systematic identification of the molecules involved has not previously been possible due to the lack of a specific phenotype associated with defects in this process. We present here a novel GFP-based reporter assay that can monitor SL1 trans-splicing in living C. elegans. Using this assay, we have identified mutants in sna-1 that are defective in SL trans-splicing, and demonstrate that reducing function of SNA-1, SNA-2 and SUT-1, proteins that associate with SL1 RNA and related SmY RNAs, impairs SL trans-splicing. We further demonstrate that the Sm proteins and pICln, SMN and Gemin5, which are involved in small nuclear ribonucleoprotein assembly, have an important role in SL trans-splicing. Taken together these results provide the first in vivo evidence for proteins involved in SL trans-splicing, and indicate that continuous replacement of SL ribonucleoproteins consumed during trans-splicing reactions is essential for effective trans-splicing. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  8. Identification and characterization of novel ERC-55 interacting proteins: evidence for the existence of several ERC-55 splicing variants; including the cytosolic ERC-55-C.

    Science.gov (United States)

    Ludvigsen, Maja; Jacobsen, Christian; Maunsbach, Arvid B; Honoré, Bent

    2009-12-01

    ERC-55, encoded from RCN2, is localized in the ER and belongs to the CREC protein family. ERC-55 is involved in various diseases and abnormal cell behavior, however, the function is not well defined and it has controversially been reported to interact with a cytosolic protein, the vitamin D receptor. We have used a number of proteomic techniques to further our functional understanding of ERC-55. By affinity purification, we observed interaction with a large variety of proteins, including those secreted and localized outside of the secretory pathway, in the cytosol and also in various organelles. We confirm the existence of several ERC-55 splicing variants including ERC-55-C localized in the cytosol in association with the cytoskeleton. Localization was verified by immunoelectron microscopy and sub-cellular fractionation. Interaction of lactoferrin, S100P, calcyclin (S100A6), peroxiredoxin-6, kininogen and lysozyme with ERC-55 was further studied in vitro by SPR experiments. Interaction of S100P requires [Ca(2+)] of approximately 10(-7) M or greater, while calcyclin interaction requires [Ca(2+)] of >10(-5) M. Interaction with peroxiredoxin-6 is independent of Ca(2+). Co-localization of lactoferrin, S100P and calcyclin with ERC-55 in the perinuclear area was analyzed by fluorescence confocal microscopy. The functional variety of the interacting proteins indicates a broad spectrum of ERC-55 activities such as immunity, redox homeostasis, cell cycle regulation and coagulation.

  9. The Human Splicing Factor ASF/SF2 can Specifically Recognize Pre-mRNA 5' Splice Sites

    Science.gov (United States)

    Zuo, Ping; Manley, James L.

    1994-04-01

    ASF/SF2 is a human protein previously shown to function in in vitro pre-mRNA splicing as an essential factor necessary for all splices and also as an alternative splicing factor, capable of switching selection of 5' splice sites. To begin to study the protein's mechanism of action, we have investigated the RNA binding properties of purified recombinant ASF/SF2. Using UV crosslinking and gel shift assays, we demonstrate that the RNA binding region of ASF/SF2 can interact with RNA in a sequence-specific manner, recognizing the 5' splice site in each of two different pre-mRNAs. Point mutations in the 5' splice site consensus can reduce binding by as much as a factor of 100, with the largest effects observed in competition assays. These findings support a model in which ASF/SF2 aids in the recognition of pre-mRNA 5' splice sites.

  10. Antidiabetic effects of glucokinase regulatory protein small-molecule disruptors

    Science.gov (United States)

    Lloyd, David J.; St Jean, David J.; Kurzeja, Robert J. M.; Wahl, Robert C.; Michelsen, Klaus; Cupples, Rod; Chen, Michelle; Wu, John; Sivits, Glenn; Helmering, Joan; Komorowski, Renée; Ashton, Kate S.; Pennington, Lewis D.; Fotsch, Christopher; Vazir, Mukta; Chen, Kui; Chmait, Samer; Zhang, Jiandong; Liu, Longbin; Norman, Mark H.; Andrews, Kristin L.; Bartberger, Michael D.; van, Gwyneth; Galbreath, Elizabeth J.; Vonderfecht, Steven L.; Wang, Minghan; Jordan, Steven R.; Véniant, Murielle M.; Hale, Clarence

    2013-12-01

    Glucose homeostasis is a vital and complex process, and its disruption can cause hyperglycaemia and type II diabetes mellitus. Glucokinase (GK), a key enzyme that regulates glucose homeostasis, converts glucose to glucose-6-phosphate in pancreatic β-cells, liver hepatocytes, specific hypothalamic neurons, and gut enterocytes. In hepatocytes, GK regulates glucose uptake and glycogen synthesis, suppresses glucose production, and is subject to the endogenous inhibitor GK regulatory protein (GKRP). During fasting, GKRP binds, inactivates and sequesters GK in the nucleus, which removes GK from the gluconeogenic process and prevents a futile cycle of glucose phosphorylation. Compounds that directly hyperactivate GK (GK activators) lower blood glucose levels and are being evaluated clinically as potential therapeutics for the treatment of type II diabetes mellitus. However, initial reports indicate that an increased risk of hypoglycaemia is associated with some GK activators. To mitigate the risk of hypoglycaemia, we sought to increase GK activity by blocking GKRP. Here we describe the identification of two potent small-molecule GK-GKRP disruptors (AMG-1694 and AMG-3969) that normalized blood glucose levels in several rodent models of diabetes. These compounds potently reversed the inhibitory effect of GKRP on GK activity and promoted GK translocation both in vitro (isolated hepatocytes) and in vivo (liver). A co-crystal structure of full-length human GKRP in complex with AMG-1694 revealed a previously unknown binding pocket in GKRP distinct from that of the phosphofructose-binding site. Furthermore, with AMG-1694 and AMG-3969 (but not GK activators), blood glucose lowering was restricted to diabetic and not normoglycaemic animals. These findings exploit a new cellular mechanism for lowering blood glucose levels with reduced potential for hypoglycaemic risk in patients with type II diabetes mellitus.

  11. The splicing mutant of the human tumor suppressor protein DFNA5 induces programmed cell death when expressed in the yeast Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Van Rossom, Sofie; Op de Beeck, Ken; Franssens, Vanessa; Swinnen, Erwin; Schepers, Anne; Ghillebert, Ruben; Caldara, Marina; Van Camp, Guy; Winderickx, Joris

    2012-01-01

    DFNA5 was first identified as a gene responsible for autosomal dominant deafness. Different mutations were found, but they all resulted in exon 8 skipping during splicing and premature termination of the protein. Later, it became clear that the protein also has a tumor suppression function and that it can induce apoptosis. Epigenetic silencing of the DFNA5 gene is associated with different types of cancers, including gastric and colorectal cancers as well as breast tumors. We introduced the wild-type and mutant DFNA5 allele in the yeast Saccharomyces cerevisiae. The expression of the wild-type protein was well tolerated by the yeast cells, although the protein was subject of degradation and often deposited in distinct foci when cells entered the diauxic shift. In contrast, cells had problems to cope with mutant DFNA5 and despite an apparent compensatory reduction in expression levels, the mutant protein still triggered a marked growth defect, which in part can be ascribed to its interaction with mitochondria. Consistently, cells with mutant DFNA5 displayed significantly increased levels of ROS and signs of programmed cell death. The latter occurred independently of the yeast caspase, Mca1, but involved the mitochondrial fission protein, Fis1, the voltage-dependent anion channel protein, Por1 and the mitochondrial adenine nucleotide translocators, Aac1 and Aac3. Recent data proposed DFNA5 toxicity to be associated to a globular domain encoded by exon 2–6. We confirmed these data by showing that expression of solely this domain confers a strong growth phenotype. In addition, we identified a point mutant in this domain that completely abrogated its cytotoxicity in yeast as well as human Human Embryonic Kidney 293T cells (HEK293T). Combined, our data underscore that the yeast system offers a valuable tool to further dissect the apoptotic properties of DFNA5.

  12. The splicing mutant of the human tumor suppressor protein DFNA5 induces programmed cell death when expressed in the yeast Saccharomyces cerevisiae

    Energy Technology Data Exchange (ETDEWEB)

    Van Rossom, Sofie [Department of Biology, Functional Biology, KU Leuven, Leuven-Heverlee (Belgium); Department of Biomedical Sciences, Center of Medical Genetics, University of Antwerp, Wilrijk-Antwerp (Belgium); Op de Beeck, Ken [Department of Biomedical Sciences, Center of Medical Genetics, University of Antwerp, Wilrijk-Antwerp (Belgium); Franssens, Vanessa; Swinnen, Erwin [Department of Biology, Functional Biology, KU Leuven, Leuven-Heverlee (Belgium); Schepers, Anne [Department of Biomedical Sciences, Center of Medical Genetics, University of Antwerp, Wilrijk-Antwerp (Belgium); Ghillebert, Ruben; Caldara, Marina [Department of Biology, Functional Biology, KU Leuven, Leuven-Heverlee (Belgium); Van Camp, Guy [Department of Biomedical Sciences, Center of Medical Genetics, University of Antwerp, Wilrijk-Antwerp (Belgium); Winderickx, Joris, E-mail: guy.vancamp@ua.ac.be, E-mail: joris.winderickx@bio.kuleuven.be [Department of Biology, Functional Biology, KU Leuven, Leuven-Heverlee (Belgium)

    2012-07-25

    DFNA5 was first identified as a gene responsible for autosomal dominant deafness. Different mutations were found, but they all resulted in exon 8 skipping during splicing and premature termination of the protein. Later, it became clear that the protein also has a tumor suppression function and that it can induce apoptosis. Epigenetic silencing of the DFNA5 gene is associated with different types of cancers, including gastric and colorectal cancers as well as breast tumors. We introduced the wild-type and mutant DFNA5 allele in the yeast Saccharomyces cerevisiae. The expression of the wild-type protein was well tolerated by the yeast cells, although the protein was subject of degradation and often deposited in distinct foci when cells entered the diauxic shift. In contrast, cells had problems to cope with mutant DFNA5 and despite an apparent compensatory reduction in expression levels, the mutant protein still triggered a marked growth defect, which in part can be ascribed to its interaction with mitochondria. Consistently, cells with mutant DFNA5 displayed significantly increased levels of ROS and signs of programmed cell death. The latter occurred independently of the yeast caspase, Mca1, but involved the mitochondrial fission protein, Fis1, the voltage-dependent anion channel protein, Por1 and the mitochondrial adenine nucleotide translocators, Aac1 and Aac3. Recent data proposed DFNA5 toxicity to be associated to a globular domain encoded by exon 2–6. We confirmed these data by showing that expression of solely this domain confers a strong growth phenotype. In addition, we identified a point mutant in this domain that completely abrogated its cytotoxicity in yeast as well as human Human Embryonic Kidney 293T cells (HEK293T). Combined, our data underscore that the yeast system offers a valuable tool to further dissect the apoptotic properties of DFNA5.

  13. Changes in Cellular mRNA Stability, Splicing, and Polyadenylation through HuR Protein Sequestration by a Cytoplasmic RNA Virus

    Directory of Open Access Journals (Sweden)

    Michael D. Barnhart

    2013-11-01

    Full Text Available The impact of RNA viruses on the posttranscriptional regulation of cellular gene expression is unclear. Sindbis virus causes a dramatic relocalization of the cellular HuR protein from the nucleus to the cytoplasm in infected cells. This is to the result of the expression of large amounts of viral RNAs that contain high-affinity HuR binding sites in their 3′ UTRs effectively serving as a sponge for the HuR protein. Sequestration of HuR by Sindbis virus is associated with destabilization of cellular mRNAs that normally bind HuR and rely on it to regulate their expression. Furthermore, significant changes can be observed in nuclear alternative polyadenylation and splicing events on cellular pre-mRNAs as a result of sequestration of HuR protein by the 3′ UTR of transcripts of this cytoplasmic RNA virus. These studies suggest a molecular mechanism of virus-host interaction that probably has a significant impact on virus replication, cytopathology, and pathogenesis.

  14. Identification of a spliced gene from duck enteritis virus encoding a protein homologous to UL15 of herpes simplex virus 1

    Directory of Open Access Journals (Sweden)

    Wang Yu

    2011-04-01

    Full Text Available Abstract Background In herpesviruses, UL15 homologue is a subunit of terminase complex responsible for cleavage and packaging of the viral genome into pre-assembled capsids. However, for duck enteritis virus (DEV, the causative agent of duck viral enteritis (DVE, the genomic sequence was not completely determined until most recently. There is limited information of this putative spliced gene and its encoding protein. Results DEV UL15 consists of two exons with a 3.5 kilobases (kb inron and transcribes into two transcripts: the full-length UL15 and an N-terminally truncated UL15.5. The 2.9 kb UL15 transcript encodes a protein of 739 amino acids with an approximate molecular mass of 82 kiloDaltons (kDa, whereas the UL15.5 transcript is 1.3 kb in length, containing a putative 888 base pairs (bp ORF that encodes a 32 kDa product. We also demonstrated that UL15 gene belonged to the late kinetic class as its expression was sensitive to cycloheximide and phosphonoacetic acid. UL15 is highly conserved within the Herpesviridae, and contains Walker A and B motifs homologous to the catalytic subunit of the bacteriophage terminase as revealed by sequence analysis. Phylogenetic tree constructed with the amino acid sequences of 23 herpesvirus UL15 homologues suggests a close relationship of DEV to the Mardivirus genus within the Alphaherpesvirinae. Further, the UL15 and UL15.5 proteins can be detected in the infected cell lysate but not in the sucrose density gradient-purified virion when reacting with the antiserum against UL15. Within the CEF cells, the UL15 and/or UL15.5 localize(s in the cytoplasm at 6 h post infection (h p. i. and mainly in the nucleus at 12 h p. i. and at 24 h p. i., while accumulate(s in the cytoplasm in the absence of any other viral protein. Conclusions DEV UL15 is a spliced gene that encodes two products encoded by 2.9 and 1.3 kb transcripts respectively. The UL15 is expressed late during infection. The coding sequences of DEV UL15

  15. Identification of a spliced gene from duck enteritis virus encoding a protein homologous to UL15 of herpes simplex virus 1.

    Science.gov (United States)

    Zhu, Hongwei; Li, Huixin; Han, Zongxi; Shao, Yuhao; Wang, Yu; Kong, Xiangang

    2011-04-06

    In herpesviruses, UL15 homologue is a subunit of terminase complex responsible for cleavage and packaging of the viral genome into pre-assembled capsids. However, for duck enteritis virus (DEV), the causative agent of duck viral enteritis (DVE), the genomic sequence was not completely determined until most recently. There is limited information of this putative spliced gene and its encoding protein. DEV UL15 consists of two exons with a 3.5 kilobases (kb) inron and transcribes into two transcripts: the full-length UL15 and an N-terminally truncated UL15.5. The 2.9 kb UL15 transcript encodes a protein of 739 amino acids with an approximate molecular mass of 82 kiloDaltons (kDa), whereas the UL15.5 transcript is 1.3 kb in length, containing a putative 888 base pairs (bp) ORF that encodes a 32 kDa product. We also demonstrated that UL15 gene belonged to the late kinetic class as its expression was sensitive to cycloheximide and phosphonoacetic acid. UL15 is highly conserved within the Herpesviridae, and contains Walker A and B motifs homologous to the catalytic subunit of the bacteriophage terminase as revealed by sequence analysis. Phylogenetic tree constructed with the amino acid sequences of 23 herpesvirus UL15 homologues suggests a close relationship of DEV to the Mardivirus genus within the Alphaherpesvirinae. Further, the UL15 and UL15.5 proteins can be detected in the infected cell lysate but not in the sucrose density gradient-purified virion when reacting with the antiserum against UL15. Within the CEF cells, the UL15 and/or UL15.5 localize(s) in the cytoplasm at 6 h post infection (h p. i.) and mainly in the nucleus at 12 h p. i. and at 24 h p. i., while accumulate(s) in the cytoplasm in the absence of any other viral protein. DEV UL15 is a spliced gene that encodes two products encoded by 2.9 and 1.3 kb transcripts respectively. The UL15 is expressed late during infection. The coding sequences of DEV UL15 are very similar to those of alphaherpesviruses and

  16. Targeting Splicing in Prostate Cancer

    OpenAIRE

    Effrosyni Antonopoulou; Michael Ladomery

    2018-01-01

    Over 95% of human genes are alternatively spliced, expressing splice isoforms that often exhibit antagonistic functions. We describe genes whose alternative splicing has been linked to prostate cancer; namely VEGFA, KLF6, BCL2L2, ERG, and AR. We discuss opportunities to develop novel therapies that target specific splice isoforms, or that target the machinery of splicing. Therapeutic approaches include the development of small molecule inhibitors of splice factor kinases, splice isoform speci...

  17. Exonic Splicing Mutations Are More Prevalent than Currently Estimated and Can Be Predicted by Using In Silico Tools

    Science.gov (United States)

    Soukarieh, Omar; Gaildrat, Pascaline; Hamieh, Mohamad; Drouet, Aurélie; Baert-Desurmont, Stéphanie; Frébourg, Thierry; Tosi, Mario; Martins, Alexandra

    2016-01-01

    The identification of a causal mutation is essential for molecular diagnosis and clinical management of many genetic disorders. However, even if next-generation exome sequencing has greatly improved the detection of nucleotide changes, the biological interpretation of most exonic variants remains challenging. Moreover, particular attention is typically given to protein-coding changes often neglecting the potential impact of exonic variants on RNA splicing. Here, we used the exon 10 of MLH1, a gene implicated in hereditary cancer, as a model system to assess the prevalence of RNA splicing mutations among all single-nucleotide variants identified in a given exon. We performed comprehensive minigene assays and analyzed patient’s RNA when available. Our study revealed a staggering number of splicing mutations in MLH1 exon 10 (77% of the 22 analyzed variants), including mutations directly affecting splice sites and, particularly, mutations altering potential splicing regulatory elements (ESRs). We then used this thoroughly characterized dataset, together with experimental data derived from previous studies on BRCA1, BRCA2, CFTR and NF1, to evaluate the predictive power of 3 in silico approaches recently described as promising tools for pinpointing ESR-mutations. Our results indicate that ΔtESRseq and ΔHZEI-based approaches not only discriminate which variants affect splicing, but also predict the direction and severity of the induced splicing defects. In contrast, the ΔΨ-based approach did not show a compelling predictive power. Our data indicates that exonic splicing mutations are more prevalent than currently appreciated and that they can now be predicted by using bioinformatics methods. These findings have implications for all genetically-caused diseases. PMID:26761715

  18. Exonic Splicing Mutations Are More Prevalent than Currently Estimated and Can Be Predicted by Using In Silico Tools.

    Directory of Open Access Journals (Sweden)

    Omar Soukarieh

    2016-01-01

    Full Text Available The identification of a causal mutation is essential for molecular diagnosis and clinical management of many genetic disorders. However, even if next-generation exome sequencing has greatly improved the detection of nucleotide changes, the biological interpretation of most exonic variants remains challenging. Moreover, particular attention is typically given to protein-coding changes often neglecting the potential impact of exonic variants on RNA splicing. Here, we used the exon 10 of MLH1, a gene implicated in hereditary cancer, as a model system to assess the prevalence of RNA splicing mutations among all single-nucleotide variants identified in a given exon. We performed comprehensive minigene assays and analyzed patient's RNA when available. Our study revealed a staggering number of splicing mutations in MLH1 exon 10 (77% of the 22 analyzed variants, including mutations directly affecting splice sites and, particularly, mutations altering potential splicing regulatory elements (ESRs. We then used this thoroughly characterized dataset, together with experimental data derived from previous studies on BRCA1, BRCA2, CFTR and NF1, to evaluate the predictive power of 3 in silico approaches recently described as promising tools for pinpointing ESR-mutations. Our results indicate that ΔtESRseq and ΔHZEI-based approaches not only discriminate which variants affect splicing, but also predict the direction and severity of the induced splicing defects. In contrast, the ΔΨ-based approach did not show a compelling predictive power. Our data indicates that exonic splicing mutations are more prevalent than currently appreciated and that they can now be predicted by using bioinformatics methods. These findings have implications for all genetically-caused diseases.

  19. Variation in alternative splicing across human tissues

    OpenAIRE

    Yeo, Gene; Holste, Dirk; Kreiman, Gabriel; Burge, Christopher B

    2004-01-01

    Background: Alternative pre-mRNA splicing (AS) is widely used by higher eukaryotes to generate different protein isoforms in specific cell or tissue types. To compare AS events across human tissues, we analyzed the splicing patterns of genomically aligned expressed sequence tags (ESTs) derived from libraries of cDNAs from different tissues. Results: Controlling for differences in EST coverage among tissues, we found that the brain and testis had the highest levels of exon skipping. The most p...

  20. Landscape of the spliced leader trans-splicing mechanism in Schistosoma mansoni.

    Science.gov (United States)

    Boroni, Mariana; Sammeth, Michael; Gava, Sandra Grossi; Jorge, Natasha Andressa Nogueira; Macedo, Andréa Mara; Machado, Carlos Renato; Mourão, Marina Moraes; Franco, Glória Regina

    2018-03-01

    Spliced leader dependent trans-splicing (SLTS) has been described as an important RNA regulatory process that occurs in different organisms, including the trematode Schistosoma mansoni. We identified more than seven thousand putative SLTS sites in the parasite, comprising genes with a wide spectrum of functional classes, which underlines the SLTS as a ubiquitous mechanism in the parasite. Also, SLTS gene expression levels span several orders of magnitude, showing that SLTS frequency is not determined by the expression level of the target gene, but by the presence of particular gene features facilitating or hindering the trans-splicing mechanism. Our in-depth investigation of SLTS events demonstrates widespread alternative trans-splicing (ATS) acceptor sites occurring in different regions along the entire gene body, highlighting another important role of SLTS generating alternative RNA isoforms in the parasite, besides the polycistron resolution. Particularly for introns where SLTS directly competes for the same acceptor substrate with cis-splicing, we identified for the first time additional and important features that might determine the type of splicing. Our study substantially extends the current knowledge of RNA processing by SLTS in S. mansoni, and provide basis for future studies on the trans-splicing mechanism in other eukaryotes.

  1. Alternative splicing of the porcine glycogen synthase kinase 3β (GSK-3β gene with differential expression patterns and regulatory functions.

    Directory of Open Access Journals (Sweden)

    Linjie Wang

    Full Text Available Glycogen synthase kinase 3 (GSK3α and GSK3β are serine/threonine kinases involved in numerous cellular processes and diverse diseases including mood disorders, Alzheimer's disease, diabetes, and cancer. However, in pigs, the information on GSK3 is very limited. Identification and characterization of pig GSK3 are not only important for pig genetic improvement, but also contribute to the understanding and development of porcine models for human disease prevention and treatment.Five different isoforms of GSK3β were identified in porcine different tissues, in which three isoforms are novel. These isoforms had differential expression patterns in the fetal and adult of the porcine different tissues. The mRNA expression level of GSK3β isoforms was differentially regulated during the course of the insulin treatment, suggesting that different GSK3β isoforms may have different roles in insulin signaling pathway. Moreover, GSK3β5 had a different role on regulating the glycogen synthase activity, phosphorylation and the expression of porcine GYS1 and GYS2 gene compared to other GSK3β isoforms.We are the first to report five different isoforms of GSK3β identified from the porcine different tissues. Splice variants of GSK3β exhibit differential activity towards glycogen synthase. These results provide new insight into roles of the GSK3β on regulating glycogen metabolism.

  2. Deconstructing the pluripotency gene regulatory network

    KAUST Repository

    Li, Mo

    2018-04-04

    Pluripotent stem cells can be isolated from embryos or derived by reprogramming. Pluripotency is stabilized by an interconnected network of pluripotency genes that cooperatively regulate gene expression. Here we describe the molecular principles of pluripotency gene function and highlight post-transcriptional controls, particularly those induced by RNA-binding proteins and alternative splicing, as an important regulatory layer of pluripotency. We also discuss heterogeneity in pluripotency regulation, alternative pluripotency states and future directions of pluripotent stem cell research.

  3. Deconstructing the pluripotency gene regulatory network

    KAUST Repository

    Li, Mo; Belmonte, Juan Carlos Izpisua

    2018-01-01

    Pluripotent stem cells can be isolated from embryos or derived by reprogramming. Pluripotency is stabilized by an interconnected network of pluripotency genes that cooperatively regulate gene expression. Here we describe the molecular principles of pluripotency gene function and highlight post-transcriptional controls, particularly those induced by RNA-binding proteins and alternative splicing, as an important regulatory layer of pluripotency. We also discuss heterogeneity in pluripotency regulation, alternative pluripotency states and future directions of pluripotent stem cell research.

  4. Modulation of 5' splice site selection using tailed oligonucleotides carrying splicing signals

    Directory of Open Access Journals (Sweden)

    Elela Sherif

    2006-01-01

    Full Text Available Abstract Background We previously described the use of tailed oligonucleotides as a means of reprogramming alternative pre-mRNA splicing in vitro and in vivo. The tailed oligonucleotides that were used interfere with splicing because they contain a portion complementary to sequences immediately upstream of the target 5' splice site combined with a non-hybridizing 5' tail carrying binding sites for the hnRNP A1/A2 proteins. In the present study, we have tested the inhibitory activity of RNA oligonucleotides carrying different tail structures. Results We show that an oligonucleotide with a 5' tail containing the human β-globin branch site sequence inhibits the use of the 5' splice site of Bcl-xL, albeit less efficiently than a tail containing binding sites for the hnRNP A1/A2 proteins. A branch site-containing tail positioned at the 3' end of the oligonucleotide also elicited splicing inhibition but not as efficiently as a 5' tail. The interfering activity of a 3' tail was improved by adding a 5' splice site sequence next to the branch site sequence. A 3' tail carrying a Y-shaped branch structure promoted similar splicing interference. The inclusion of branch site or 5' splice site sequences in the Y-shaped 3' tail further improved splicing inhibition. Conclusion Our in vitro results indicate that a variety of tail architectures can be used to elicit splicing interference at low nanomolar concentrations, thereby broadening the scope and the potential impact of this antisense technology.

  5. Approaches to link RNA secondary structures with splicing regulation

    DEFF Research Database (Denmark)

    Plass, Mireya; Eyras, Eduardo

    2014-01-01

    In higher eukaryotes, alternative splicing is usually regulated by protein factors, which bind to the pre-mRNA and affect the recognition of splicing signals. There is recent evidence that the secondary structure of the pre-mRNA may also play an important role in this process, either by facilitat...... describes the steps in the analysis of the secondary structure of the pre-mRNA and its possible relation to splicing. As a working example, we use the case of yeast and the problem of the recognition of the 3' splice site (3'ss).......In higher eukaryotes, alternative splicing is usually regulated by protein factors, which bind to the pre-mRNA and affect the recognition of splicing signals. There is recent evidence that the secondary structure of the pre-mRNA may also play an important role in this process, either...

  6. CDKL5 influences RNA splicing activity by its association to the nuclear speckle molecular machinery.

    Science.gov (United States)

    Ricciardi, Sara; Kilstrup-Nielsen, Charlotte; Bienvenu, Thierry; Jacquette, Aurélia; Landsberger, Nicoletta; Broccoli, Vania

    2009-12-01

    Mutations in the human X-linked cyclin-dependent kinase-like 5 (CDKL5) gene have been shown to cause severe neurodevelopmental disorders including infantile spasms, encephalopathy, West-syndrome and an early-onset variant of Rett syndrome. CDKL5 is a serine/threonine kinase whose involvement in Rett syndrome can be inferred by its ability to directly bind and mediate phosphorylation of MeCP2. However, it remains to be elucidated how CDKL5 exerts its function. Here, we report that CDKL5 localizes to specific nuclear foci referred to as nuclear speckles in both cell lines and tissues. These sub-nuclear structures are traditionally considered as storage/modification sites of pre-mRNA splicing factors. Interestingly, we provide evidence that CDKL5 regulates the dynamic behaviour of nuclear speckles. Indeed, CDKL5 overexpression leads to nuclear speckle disassembly, and this event is strictly dependent on its kinase activity. Conversely, its down-regulation affects nuclear speckle morphology leading to abnormally large and uneven speckles. Similar results were obtained for primary adult fibroblasts isolated from CDKL5-mutated patients. Altogether, these findings indicate that CDKL5 controls nuclear speckle morphology probably by regulating the phosphorylation state of splicing regulatory proteins. Nuclear speckles are dynamic sites that can continuously supply splicing factors to active transcription sites, where splicing occurs. Notably, we proved that CDKL5 influences alternative splicing, at least as proved in heterologous minigene assays. In conclusion, we provide evidence that CDKL5 is involved indirectly in pre-mRNA processing, by controlling splicing factor dynamics. These findings identify a biological process whose disregulation might affect neuronal maturation and activity in CDKL5-related disorders.

  7. Regulatory mechanisms of skeletal muscle protein turnover during exercise

    DEFF Research Database (Denmark)

    Rose, Adam John; Richter, Erik

    2009-01-01

    Skeletal muscle protein turnover is a relatively slow metabolic process that is altered by various physiological stimuli such as feeding/fasting and exercise. During exercise, catabolism of amino acids contributes very little to ATP turnover in working muscle. With regards to protein turnover......, there is now consistent data from tracer studies in rodents and humans showing that global protein synthesis is blunted in working skeletal muscle. Whether there is altered skeletal muscle protein breakdown during exercise remains unclear. The blunting of protein synthesis is believed to be mediated...... downstream of changes in intracellular Ca(2+) and energy turnover. In particular, a signaling cascade involving Ca(2+)-calmodulin-eEF2 kinase-eEF2 is implicated. The possible functional significance of altered protein turnover in working skeletal muscle during exercise is discussed. Further work...

  8. Suppression of HPV-16 late L1 5′-splice site SD3632 by binding of hnRNP D proteins and hnRNP A2/B1 to upstream AUAGUA RNA motifs

    Science.gov (United States)

    Li, Xiaoze; Johansson, Cecilia; Glahder, Jacob; Mossberg, Ann-Kristin; Schwartz, Stefan

    2013-01-01

    Human papillomavirus type 16 (HPV-16) 5′-splice site SD3632 is used exclusively to produce late L1 mRNAs. We identified a 34-nt splicing inhibitory element located immediately upstream of HPV-16 late 5′-splice site SD3632. Two AUAGUA motifs located in these 34 nt inhibited SD3632. Two nucleotide substitutions in each of the HPV-16 specific AUAGUA motifs alleviated splicing inhibition and induced late L1 mRNA production from episomal forms of the HPV-16 genome in primary human keratinocytes. The AUAGUA motifs bind specifically not only to the heterogeneous nuclear RNP (hnRNP) D family of RNA-binding proteins including hnRNP D/AUF, hnRNP DL and hnRNP AB but also to hnRNP A2/B1. Knock-down of these proteins induced HPV-16 late L1 mRNA expression, and overexpression of hnRNP A2/B1, hnRNP AB, hnRNP DL and the two hnRNP D isoforms hnRNP D37 and hnRNP D40 further suppressed L1 mRNA expression. This inhibition may allow HPV-16 to hide from the immune system and establish long-term persistent infections with enhanced risk at progressing to cancer. There is an inverse correlation between expression of hnRNP D proteins and hnRNP A2/B1 and HPV-16 L1 production in the cervical epithelium, as well as in cervical cancer, supporting the conclusion that hnRNP D proteins and A2/B1 inhibit HPV-16 L1 mRNA production. PMID:24013563

  9. Analytical Validation and Clinical Qualification of a New Immunohistochemical Assay for Androgen Receptor Splice Variant-7 Protein Expression in Metastatic Castration-resistant Prostate Cancer.

    Science.gov (United States)

    Welti, Jonathan; Rodrigues, Daniel Nava; Sharp, Adam; Sun, Shihua; Lorente, David; Riisnaes, Ruth; Figueiredo, Ines; Zafeiriou, Zafeiris; Rescigno, Pasquale; de Bono, Johann S; Plymate, Stephen R

    2016-10-01

    The androgen receptor splice variant-7 (AR-V7) has been implicated in the development of castration-resistant prostate cancer (CRPC) and resistance to abiraterone and enzalutamide. To develop a validated assay for detection of AR-V7 protein in tumour tissue and determine its expression and clinical significance as patients progress from hormone-sensitive prostate cancer (HSPC) to CRPC. Following monoclonal antibody generation and validation, we retrospectively identified patients who had HSPC and CRPC tissue available for AR-V7 immunohistochemical (IHC) analysis. Nuclear AR-V7 expression was determined using IHC H score (HS) data. The change in nuclear AR-V7 expression from HSPC to CRPC and the association between nuclear AR-V7 expression and overall survival (OS) was determined. Nuclear AR-V7 expression was significantly lower in HSPC (median HS 50, interquartile range [IQR] 17.5-90) compared to CRPC (HS 135, IQR 80-157.5; pprostate cancer. A higher level of AR-V7 identifies a group of patients who respond less well to certain prostate cancer treatments and live for a shorter period of time. Copyright © 2016 European Association of Urology. Published by Elsevier B.V. All rights reserved.

  10. Dynamic SPR monitoring of yeast nuclear protein binding to a cis-regulatory element

    International Nuclear Information System (INIS)

    Mao, Grace; Brody, James P.

    2007-01-01

    Gene expression is controlled by protein complexes binding to short specific sequences of DNA, called cis-regulatory elements. Expression of most eukaryotic genes is controlled by dozens of these elements. Comprehensive identification and monitoring of these elements is a major goal of genomics. In pursuit of this goal, we are developing a surface plasmon resonance (SPR) based assay to identify and monitor cis-regulatory elements. To test whether we could reliably monitor protein binding to a regulatory element, we immobilized a 16 bp region of Saccharomyces cerevisiae chromosome 5 onto a gold surface. This 16 bp region of DNA is known to bind several proteins and thought to control expression of the gene RNR1, which varies through the cell cycle. We synchronized yeast cell cultures, and then sampled these cultures at a regular interval. These samples were processed to purify nuclear lysate, which was then exposed to the sensor. We found that nuclear protein binds this particular element of DNA at a significantly higher rate (as compared to unsynchronized cells) during G1 phase. Other time points show levels of DNA-nuclear protein binding similar to the unsynchronized control. We also measured the apparent association complex of the binding to be 0.014 s -1 . We conclude that (1) SPR-based assays can monitor DNA-nuclear protein binding and that (2) for this particular cis-regulatory element, maximum DNA-nuclear protein binding occurs during G1 phase

  11. Dynamic Contacts of U2, RES, Cwc25, Prp8 and Prp45 Proteins with the Pre-mRNA Branch-Site and 3' Splice Site during Catalytic Activation and Step 1 Catalysis in Yeast Spliceosomes.

    Directory of Open Access Journals (Sweden)

    Cornelius Schneider

    Full Text Available Little is known about contacts in the spliceosome between proteins and intron nucleotides surrounding the pre-mRNA branch-site and their dynamics during splicing. We investigated protein-pre-mRNA interactions by UV-induced crosslinking of purified yeast B(act spliceosomes formed on site-specifically labeled pre-mRNA, and analyzed their changes after conversion to catalytically-activated B* and step 1 C complexes, using a purified splicing system. Contacts between nucleotides upstream and downstream of the branch-site and the U2 SF3a/b proteins Prp9, Prp11, Hsh49, Cus1 and Hsh155 were detected, demonstrating that these interactions are evolutionarily conserved. The RES proteins Pml1 and Bud13 were shown to contact the intron downstream of the branch-site. A comparison of the B(act crosslinking pattern versus that of B* and C complexes revealed that U2 and RES protein interactions with the intron are dynamic. Upon step 1 catalysis, Cwc25 contacts with the branch-site region, and enhanced crosslinks of Prp8 and Prp45 with nucleotides surrounding the branch-site were observed. Cwc25's step 1 promoting activity was not dependent on its interaction with pre-mRNA, indicating it acts via protein-protein interactions. These studies provide important insights into the spliceosome's protein-pre-mRNA network and reveal novel RNP remodeling events during the catalytic activation of the spliceosome and step 1 of splicing.

  12. The 0.3-kb fragment containing the R-U5-5'leader sequence of Friend murine leukemia virus influences the level of protein expression from spliced mRNA.

    Science.gov (United States)

    Choo, Yeng Cheng; Seki, Yohei; Machinaga, Akihito; Ogita, Nobuo; Takase-Yoden, Sayaka

    2013-04-19

    A neuropathogenic variant of Friend murine leukemia virus (Fr-MLV) clone A8 induces spongiform neurodegeneration when infected into neonatal rats. Studies with chimeras constructed from the A8 virus and the non-neuropathogenic Fr-MLV clone 57 identified a 0.3-kb KpnI-AatII fragment containing a R-U5-5'leader sequence as an important determinant for inducing spongiosis, in addition to the env gene of A8 as the primary determinant. This 0.3-kb fragment contains a 17-nucleotide difference between the A8 and 57 sequences. We previously showed that the 0.3-kb fragment influences expression levels of Env protein in both cultured cells and rat brain, but the corresponding molecular mechanisms are not well understood. Studies with expression vectors constructed from the full-length proviral genome of Fr-MLV that incorporated the luciferase (luc) gene instead of the env gene found that the vector containing the A8-0.3-kb fragment yielded a larger amount of spliced luc-mRNA and showed higher expression of luciferase when compared to the vector containing the 57-0.3-kb fragment. The amount of total transcripts from the vectors, the poly (A) tail length of their mRNAs, and the nuclear-cytoplasm distribution of luc-mRNA in transfected cells were also evaluated. The 0.3-kb fragment did not influence transcription efficiency, mRNA polyadenylation or nuclear export of luc-mRNA. Mutational analyses were carried out to determine the importance of nucleotides that differ between the A8 and 57 sequences within the 0.3-kb fragment. In particular, seven nucleotides upstream of the 5'splice site (5'ss) were found to be important in regulating the level of protein expression from spliced messages. Interestingly, these nucleotides reside within the stem-loop structure that has been speculated to limit the recognition of 5'ss. The 0.3-kb fragment containing the R-U5-5'leader sequence of Fr-MLV influences the level of protein expression from the spliced-mRNA by regulating the splicing

  13. Mechanical rebar splicing

    Directory of Open Access Journals (Sweden)

    Milosavljević Branko

    2014-01-01

    Full Text Available Different mechanical rebar splicing systems are presented, and design situations where mechanical splicing has advantage over reinforcement splicing by overlapping and welding are defined in this paper. New international standards for testing and proof of systems for mechanical rebar splicing quality are considered. Mechanical splicing system for rebar and bolt connection, usable in steel and reinforced concrete structural elements connections, is presented in this paper. There are only few examples of mechanical rebar splicing in our country. The most significant one - the pylon and beam connection at Ada Bridge in Belgrade is presented in the paper. Intensive development of production and use of mechanical rebar splicing systems, research in this area, as well as the publication of international standards prescribing requirements for quality and procedures for proof of quality, represent very good base for development of the corresponding technical norms in Serbia. The legislation in this area would quicken proof of quality procedures, attest and approval issuing for individual products, leading to wider use of this system in all situations where it is in advantage over the classical reinforcement splicing.

  14. spliceR

    DEFF Research Database (Denmark)

    Vitting-Seerup, Kristoffer; Porse, Bo Torben; Sandelin, Albin

    2014-01-01

    RNA-seq data is currently underutilized, in part because it is difficult to predict the functional impact of alternate transcription events. Recent software improvements in full-length transcript deconvolution prompted us to develop spliceR, an R package for classification of alternative splicing...

  15. Interplay between DMD point mutations and splicing signals in Dystrophinopathy phenotypes.

    Directory of Open Access Journals (Sweden)

    Jonàs Juan-Mateu

    Full Text Available DMD nonsense and frameshift mutations lead to severe Duchenne muscular dystrophy while in-frame mutations lead to milder Becker muscular dystrophy. Exceptions are found in 10% of cases and the production of alternatively spliced transcripts is considered a key modifier of disease severity. Several exonic mutations have been shown to induce exon-skipping, while splice site mutations result in exon-skipping or activation of cryptic splice sites. However, factors determining the splicing pathway are still unclear. Point mutations provide valuable information regarding the regulation of pre-mRNA splicing and elements defining exon identity in the DMD gene. Here we provide a comprehensive analysis of 98 point mutations related to clinical phenotype and their effect on muscle mRNA and dystrophin expression. Aberrant splicing was found in 27 mutations due to alteration of splice sites or splicing regulatory elements. Bioinformatics analysis was performed to test the ability of the available algorithms to predict consequences on mRNA and to investigate the major factors that determine the splicing pathway in mutations affecting splicing signals. Our findings suggest that the splicing pathway is highly dependent on the interplay between splice site strength and density of regulatory elements.

  16. Genome-wide survey of allele-specific splicing in humans

    Directory of Open Access Journals (Sweden)

    Scheffler Konrad

    2008-06-01

    Full Text Available Abstract Background Accurate mRNA splicing depends on multiple regulatory signals encoded in the transcribed RNA sequence. Many examples of mutations within human splice regulatory regions that alter splicing qualitatively or quantitatively have been reported and allelic differences in mRNA splicing are likely to be a common and important source of phenotypic diversity at the molecular level, in addition to their contribution to genetic disease susceptibility. However, because the effect of a mutation on the efficiency of mRNA splicing is often difficult to predict, many mutations that cause disease through an effect on splicing are likely to remain undiscovered. Results We have combined a genome-wide scan for sequence polymorphisms likely to affect mRNA splicing with analysis of publicly available Expressed Sequence Tag (EST and exon array data. The genome-wide scan uses published tools and identified 30,977 SNPs located within donor and acceptor splice sites, branch points and exonic splicing enhancer elements. For 1,185 candidate splicing polymorphisms the difference in splicing between alternative alleles was corroborated by publicly available exon array data from 166 lymphoblastoid cell lines. We developed a novel probabilistic method to infer allele-specific splicing from EST data. The method uses SNPs and alternative mRNA isoforms mapped to EST sequences and models both regulated alternative splicing as well as allele-specific splicing. We have also estimated heritability of splicing and report that a greater proportion of genes show evidence of splicing heritability than show heritability of overall gene expression level. Our results provide an extensive resource that can be used to assess the possible effect on splicing of human polymorphisms in putative splice-regulatory sites. Conclusion We report a set of genes showing evidence of allele-specific splicing from an integrated analysis of genomic polymorphisms, EST data and exon array

  17. RNA-Binding Proteins in Trichomonas vaginalis: Atypical Multifunctional Proteins Involved in a Posttranscriptional Iron Regulatory Mechanism

    Science.gov (United States)

    Figueroa-Angulo, Elisa E.; Calla-Choque, Jaeson S.; Mancilla-Olea, Maria Inocente; Arroyo, Rossana

    2015-01-01

    Iron homeostasis is highly regulated in vertebrates through a regulatory system mediated by RNA-protein interactions between the iron regulatory proteins (IRPs) that interact with an iron responsive element (IRE) located in certain mRNAs, dubbed the IRE-IRP regulatory system. Trichomonas vaginalis, the causal agent of trichomoniasis, presents high iron dependency to regulate its growth, metabolism, and virulence properties. Although T. vaginalis lacks IRPs or proteins with aconitase activity, possesses gene expression mechanisms of iron regulation at the transcriptional and posttranscriptional levels. However, only one gene with iron regulation at the transcriptional level has been described. Recently, our research group described an iron posttranscriptional regulatory mechanism in the T. vaginalis tvcp4 and tvcp12 cysteine proteinase mRNAs. The tvcp4 and tvcp12 mRNAs have a stem-loop structure in the 5'-coding region or in the 3'-UTR, respectively that interacts with T. vaginalis multifunctional proteins HSP70, α-Actinin, and Actin under iron starvation condition, causing translation inhibition or mRNA stabilization similar to the previously characterized IRE-IRP system in eukaryotes. Herein, we summarize recent progress and shed some light on atypical RNA-binding proteins that may participate in the iron posttranscriptional regulation in T. vaginalis. PMID:26703754

  18. High-throughput proteomics detection of novel splice isoforms in human platelets.

    LENUS (Irish Health Repository)

    Power, Karen A

    2009-01-01

    Alternative splicing (AS) is an intrinsic regulatory mechanism of all metazoans. Recent findings suggest that 100% of multiexonic human genes give rise to splice isoforms. AS can be specific to tissue type, environment or developmentally regulated. Splice variants have also been implicated in various diseases including cancer. Detection of these variants will enhance our understanding of the complexity of the human genome and provide disease-specific and prognostic biomarkers. We adopted a proteomics approach to identify exon skip events - the most common form of AS. We constructed a database harboring the peptide sequences derived from all hypothetical exon skip junctions in the human genome. Searching tandem mass spectrometry (MS\\/MS) data against the database allows the detection of exon skip events, directly at the protein level. Here we describe the application of this approach to human platelets, including the mRNA-based verification of novel splice isoforms of ITGA2, NPEPPS and FH. This methodology is applicable to all new or existing MS\\/MS datasets.

  19. Regulatory crosstalk by protein kinases on CFTR trafficking and activity

    Science.gov (United States)

    Farinha, Carlos Miguel; Swiatecka-Urban, Agnieszka; Brautigan, David; Jordan, Peter

    2016-01-01

    Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a member of the ATP binding cassette (ABC) transporter superfamily that functions as a cAMP-activated chloride ion channel in fluid-transporting epithelia. There is abundant evidence that CFTR activity (i.e. channel opening and closing) is regulated by protein kinases and phosphatases via phosphorylation and dephosphorylation. Here, we review recent evidence for the role of protein kinases in regulation of CFTR delivery to and retention in the plasma membrane. We review this information in a broader context of regulation of other transporters by protein kinases because the overall functional output of transporters involves the integrated control of both their number at the plasma membrane and their specific activity. While many details of the regulation of intracellular distribution of CFTR and other transporters remain to be elucidated, we hope that this review will motivate research providing new insights into how protein kinases control membrane transport to impact health and disease.

  20. Guanine nucleotide-binding regulatory proteins in retinal pigment epithelial cells.

    OpenAIRE

    Jiang, M; Pandey, S; Tran, V T; Fong, H K

    1991-01-01

    The expression of GTP-binding regulatory proteins (G proteins) in retinal pigment epithelial (RPE) cells was analyzed by RNA blot hybridization and cDNA amplification. Both adult and fetal human RPE cells contain mRNA for multiple G protein alpha subunits (G alpha) including Gs alpha, Gi-1 alpha, Gi-2 alpha, Gi-3 alpha, and Gz alpha (or Gx alpha), where Gs and Gi are proteins that stimulate or inhibit adenylyl cyclase, respectively, and Gz is a protein that may mediate pertussis toxin-insensi...

  1. Evolution of hepatic glucose metabolism: liver-specific glucokinase deficiency explained by parallel loss of the gene for glucokinase regulatory protein (GCKR.

    Directory of Open Access Journals (Sweden)

    Zhao Yang Wang

    Full Text Available Glucokinase (GCK plays an important role in the regulation of carbohydrate metabolism. In the liver, phosphorylation of glucose to glucose-6-phosphate by GCK is the first step for both glycolysis and glycogen synthesis. However, some vertebrate species are deficient in GCK activity in the liver, despite containing GCK genes that appear to be compatible with function in their genomes. Glucokinase regulatory protein (GCKR is the most important post-transcriptional regulator of GCK in the liver; it participates in the modulation of GCK activity and location depending upon changes in glucose levels. In experimental models, loss of GCKR has been shown to associate with reduced hepatic GCK protein levels and activity.GCKR genes and GCKR-like sequences were identified in the genomes of all vertebrate species with available genome sequences. The coding sequences of GCKR and GCKR-like genes were identified and aligned; base changes likely to disrupt coding potential or splicing were also identified.GCKR genes could not be found in the genomes of 9 vertebrate species, including all birds. In addition, in multiple mammalian genomes, whereas GCKR-like gene sequences could be identified, these genes could not predict a functional protein. Vertebrate species that were previously reported to be deficient in hepatic GCK activity were found to have deleted (birds and lizard or mutated (mammals GCKR genes. Our results suggest that mutation of the GCKR gene leads to hepatic GCK deficiency due to the loss of the stabilizing effect of GCKR.

  2. Splicing modulation therapy in the treatment of genetic diseases

    Directory of Open Access Journals (Sweden)

    Arechavala-Gomeza V

    2014-12-01

    Full Text Available Virginia Arechavala-Gomeza,1 Bernard Khoo,2 Annemieke Aartsma-Rus3 1Neuromuscular Disorders Group, BioCruces Health Research Institute, Barakaldo, Bizkaia, Spain; 2Endocrinology, Division of Medicine, University College London, London, UK; 3Department of Human Genetics, Leiden University Medical Center, Leiden, the Netherlands All authors contributed equally to this manuscript Abstract: Antisense-mediated splicing modulation is a tool that can be exploited in several ways to provide a potential therapy for rare genetic diseases. This approach is currently being tested in clinical trials for Duchenne muscular dystrophy and spinal muscular atrophy. The present review outlines the versatility of the approach to correct cryptic splicing, modulate alternative splicing, restore the open reading frame, and induce protein knockdown, providing examples of each. Finally, we outline a possible path forward toward the clinical application of this approach for a wide variety of inherited rare diseases. Keywords: splicing, therapy, antisense oligonucleotides, cryptic splicing, alternative splicing

  3. EWS and FUS bind a subset of transcribed genes encoding proteins enriched in RNA regulatory functions

    DEFF Research Database (Denmark)

    Luo, Yonglun; Friis, Jenny Blechingberg; Fernandes, Ana Miguel

    2015-01-01

    at different levels. Gene Ontology analyses showed that FUS and EWS target genes preferentially encode proteins involved in regulatory processes at the RNA level. Conclusions The presented results yield new insights into gene interactions of EWS and FUS and have identified a set of FUS and EWS target genes...... involved in pathways at the RNA regulatory level with potential to mediate normal and disease-associated functions of the FUS and EWS proteins.......Background FUS (TLS) and EWS (EWSR1) belong to the FET-protein family of RNA and DNA binding proteins. FUS and EWS are structurally and functionally related and participate in transcriptional regulation and RNA processing. FUS and EWS are identified in translocation generated cancer fusion proteins...

  4. Architecture of the 99 bp DNA-six-protein regulatory complex of the lambda att site.

    Science.gov (United States)

    Sun, Xingmin; Mierke, Dale F; Biswas, Tapan; Lee, Sang Yeol; Landy, Arthur; Radman-Livaja, Marta

    2006-11-17

    The highly directional and tightly regulated recombination reaction used to site-specifically excise the bacteriophage lambda chromosome out of its E. coli host chromosome requires the binding of six sequence-specific proteins to a 99 bp segment of the phage att site. To gain structural insights into this recombination pathway, we measured 27 FRET distances between eight points on the 99 bp regulatory DNA bound with all six proteins. Triangulation of these distances using a metric matrix distance-geometry algorithm provided coordinates for these eight points. The resulting path for the protein-bound regulatory DNA, which fits well with the genetics, biochemistry, and X-ray crystal structures describing the individual proteins and their interactions with DNA, provides a new structural perspective into the molecular mechanism and regulation of the recombination reaction and illustrates a design by which different families of higher-order complexes can be assembled from different numbers and combinations of the same few proteins.

  5. Alternative Splicing as a Target for Cancer Treatment.

    Science.gov (United States)

    Martinez-Montiel, Nancy; Rosas-Murrieta, Nora Hilda; Anaya Ruiz, Maricruz; Monjaraz-Guzman, Eduardo; Martinez-Contreras, Rebeca

    2018-02-11

    Alternative splicing is a key mechanism determinant for gene expression in metazoan. During alternative splicing, non-coding sequences are removed to generate different mature messenger RNAs due to a combination of sequence elements and cellular factors that contribute to splicing regulation. A different combination of splicing sites, exonic or intronic sequences, mutually exclusive exons or retained introns could be selected during alternative splicing to generate different mature mRNAs that could in turn produce distinct protein products. Alternative splicing is the main source of protein diversity responsible for 90% of human gene expression, and it has recently become a hallmark for cancer with a full potential as a prognostic and therapeutic tool. Currently, more than 15,000 alternative splicing events have been associated to different aspects of cancer biology, including cell proliferation and invasion, apoptosis resistance and susceptibility to different chemotherapeutic drugs. Here, we present well established and newly discovered splicing events that occur in different cancer-related genes, their modification by several approaches and the current status of key tools developed to target alternative splicing with diagnostic and therapeutic purposes.

  6. Conservation and sex-specific splicing of the doublesex gene

    Indian Academy of Sciences (India)

    Genetic control of sex determination in insects has been best characterized in Drosophila melanogaster, where the master gene Sxl codes for RNA that is sex specifically spliced to produce a functional protein only in females. SXL regulates the sex-specific splicing of transformer (tra) RNA which, in turn, regulates the ...

  7. Co-suppression of sterol-regulatory element binding protein ...

    African Journals Online (AJOL)

    Administrator

    2011-06-22

    Jun 22, 2011 ... protein mediates etiolation in Arabidopsis thaliana .... (A) The scheme of At5g35220 gene and pEGAD-At5g35220; .... stem length of 42-day-old plants; root length of 5-day-old seedlings grown on MS medium; fresh weight of ...

  8. Feast/famine regulatory proteins (FFRPs): Escherichia coli Lrp, AsnC and related archaeal transcription factors.

    Science.gov (United States)

    Yokoyama, Katsushi; Ishijima, Sanae A; Clowney, Lester; Koike, Hideaki; Aramaki, Hironori; Tanaka, Chikako; Makino, Kozo; Suzuki, Masashi

    2006-01-01

    Feast/famine regulatory proteins comprise a diverse family of transcription factors, which have been referred to in various individual identifications, including Escherichia coli leucine-responsive regulatory protein and asparagine synthase C gene product. A full length feast/famine regulatory protein consists of the N-terminal DNA-binding domain and the C-domain, which is involved in dimerization and further assembly, thereby producing, for example, a disc or a chromatin-like cylinder. Various ligands of the size of amino acids bind at the interface between feast/famine regulatory protein dimers, thereby altering their assembly forms. Also, the combination of feast/famine regulatory protein subunits forming the same assembly is altered. In this way, a small number of feast/famine regulatory proteins are able to regulate a large number of genes in response to various environmental changes. Because feast/famine regulatory proteins are shared by archaea and eubacteria, the genome-wide regulation by feast/famine regulatory proteins is traceable back to their common ancestor, being the prototype of highly differentiated transcription regulatory mechanisms found in organisms nowadays.

  9. Influence of endurance training on skeletal muscle mitophagy regulatory proteins in type 2 diabetic men.

    Science.gov (United States)

    Brinkmann, Christian; Przyklenk, Axel; Metten, Alexander; Schiffer, Thorsten; Bloch, Wilhelm; Brixius, Klara; Gehlert, Sebastian

    2017-11-01

    Mitophagy is a form of autophagy for the elimination of mitochondria. Mitochondrial content and function are reduced in the skeletal muscle of patients with type 2 diabetes mellitus (T2DM). Physical training has been shown to restore mitochondrial capacity in T2DM patients, but the role of mitophagy has not been examined in this context. This study analyzes the impact of a 3-month endurance training on important skeletal muscle mitophagy regulatory proteins and oxidative phosphorylation (OXPHOS) complexes in T2DM patients. Muscle biopsies were obtained from eight overweight/obese T2DM men (61±10 years) at T1 (6 weeks pre-training), T2 (1 week pre-training), and T3 (3 to 4 days post-training). Protein contents were determined by Western blotting. The training increased mitochondrial complex II significantly (T2-T3: +29%, p = 0.037). The protein contents of mitophagy regulatory proteins (phosphorylated form of forkhead box O3A (pFOXO3A), mitochondrial E3 ubiquitin protein ligase-1 (MUL1), Bcl-2/adenovirus E1B 19-kD interacting protein-3 (BNIP3), microtubule-associated protein 1 light chain-3B (the ratio LC3B-II/LC3B-I was determined)) did not differ significantly between T1, T2, and T3. The results imply that training-induced changes in OXPHOS subunits (significant increase in complex II) are not accompanied by changes in mitophagy regulatory proteins in T2DM men. Future studies should elucidate whether acute exercise might affect mitophagic processes in T2DM patients (and whether a transient regulation of mitophagy regulatory proteins is evident) to fully clarify the role of physical activity and mitophagy for mitochondrial health in this particular patient group.

  10. Blocking of an intronic splicing silencer completely rescues IKBKAP exon 20 splicing in familial dysautonomia patient cells

    DEFF Research Database (Denmark)

    Bruun, Gitte H; Bang, Jeanne Mv; Christensen, Lise L

    2018-01-01

    designed splice switching oligonucleotides (SSO) that blocks the intronic hnRNP A1 binding site, and demonstrate that this completely rescues splicing of IKBKAP exon 20 in FD patient fibroblasts and increases the amounts of IKAP protein. We propose that this may be developed into a potential new specific...

  11. Hepatitis B spliced protein (HBSP) promotes the carcinogenic effects of benzo [alpha] pyrene by interacting with microsomal epoxide hydrolase and enhancing its hydrolysis activity

    International Nuclear Information System (INIS)

    Chen, Jin-Yan; Chen, Wan-Nan; Jiao, Bo-Yan; Lin, Wan-Song; Wu, Yun-Li; Liu, Ling-Ling; Lin, Xu

    2014-01-01

    The risk of hepatocellular carcinoma (HCC) increases in chronic hepatitis B surface antigen (HBsAg) carriers who often have concomitant increase in the levels of benzo[alpha]pyrene-7,8-diol-9,10-epoxide(±) (BPDE)-DNA adduct in liver tissues, suggesting a possible co-carcinogenesis of Hepatitis B virus (HBV) and benzo[alpha]pyrene in HCC; however the exact mechanisms involved are unclear. The interaction between hepatitis B spliced protein (HBSP) and microsomal epoxide hydrolase (mEH) was confirmed using GST pull-down, co-immunoprecipitation and mammalian two-hybrid assay; the effects of HBSP on mEH-mediated B[alpha]P metabolism was examined by high performance liquid chromatography (HPLC); and the influences of HBSP on B[alpha]P carcinogenicity were evaluated by bromodeoxyuridine cell proliferation, anchorage-independent growth and tumor xenograft. HBSP could interact with mEH in vitro and in vivo, and this interaction was mediated by the N terminal 47 amino acid residues of HBSP. HBSP could greatly enhance the hydrolysis activity of mEH in cell-free mouse liver microsomes, thus accelerating the metabolism of benzo[alpha]pyrene to produce more ultimate carcinnogen, BPDE, and this effect of HBSP requires the intact HBSP molecule. Expression of HBSP significantly increased the formation of BPDE-DNA adduct in benzo[alpha]pyrene-treated Huh-7 hepatoma cells, and this enhancement was blocked by knockdown of mEH. HBSP could enhance the cell proliferation, accelerate the G1/S transition, and promote cell transformation and tumorigenesis of B[alpha]P-treated Huh-7 hepatoma cells. Our results demonstrated that HBSP could promote carcinogenic effects of B[alpha]P by interacting with mEH and enhancing its hydrolysis activity

  12. Optical Fiber Fusion Splicing

    CERN Document Server

    Yablon, Andrew D

    2005-01-01

    This book is an up-to-date treatment of optical fiber fusion splicing incorporating all the recent innovations in the field. It provides a toolbox of general strategies and specific techniques that the reader can apply when optimizing fusion splices between novel fibers. It specifically addresses considerations important for fusion splicing of contemporary specialty fibers including dispersion compensating fiber, erbium-doped gain fiber, polarization maintaining fiber, and microstructured fiber. Finally, it discusses the future of optical fiber fusion splicing including silica and non-silica based optical fibers as well as the trend toward increasing automation. Whilst serving as a self-contained reference work, abundant citations from the technical literature will enable readers to readily locate primary sources.

  13. Novel Alternative Splice Variants of Mouse Cdk5rap2.

    Directory of Open Access Journals (Sweden)

    Nadine Kraemer

    Full Text Available Autosomal recessive primary microcephaly (MCPH is a rare neurodevelopmental disorder characterized by a pronounced reduction of brain volume and intellectual disability. A current model for the microcephaly phenotype invokes a stem cell proliferation and differentiation defect, which has moved the disease into the spotlight of stem cell biology and neurodevelopmental science. Homozygous mutations of the Cyclin-dependent kinase-5 regulatory subunit-associated protein 2 gene CDK5RAP2 are one genetic cause of MCPH. To further characterize the pathomechanism underlying MCPH, we generated a conditional Cdk5rap2 LoxP/hCMV Cre mutant mouse. Further analysis, initiated on account of a lack of a microcephaly phenotype in these mutant mice, revealed the presence of previously unknown splice variants of the Cdk5rap2 gene that are at least in part accountable for the lack of microcephaly in the mice.

  14. Cell-cycle regulatory proteins in human wound healing

    DEFF Research Database (Denmark)

    Bartkova, Jirina; Grøn, Birgitte; Dabelsteen, Erik

    2003-01-01

    Proper healing of mucosal wounds requires careful orchestration of epithelial cell migration and proliferation. To elucidate the molecular basis of the lack of cellular proliferation in the migrating 'epithelial tongue' during the re-epithelialization of oral mucosal wounds, the expression of cell......-cycle regulators critical for G(1)-phase progression and S-phase entry was here analysed immunohistochemically. Compared to normal human mucosa, epithelia migrating to cover 2- or 3-day-old wounds made either in vivo or in an organotypic cell culture all showed loss of the proliferation marker Ki67 and cyclins D(1......) and A, and reduced expression of cyclins D(3) and E, the cyclin D-dependent kinase 4 (CDK4), the MCM7 component of DNA replication origin complexes and the retinoblastoma protein pRb. Among the CDK inhibitors (CKIs), p16ink4a and p21Cip1 were moderately increased and decreased, respectively, whereas...

  15. ISVASE: identification of sequence variant associated with splicing event using RNA-seq data.

    Science.gov (United States)

    Aljohi, Hasan Awad; Liu, Wanfei; Lin, Qiang; Yu, Jun; Hu, Songnian

    2017-06-28

    Exon recognition and splicing precisely and efficiently by spliceosome is the key to generate mature mRNAs. About one third or a half of disease-related mutations affect RNA splicing. Software PVAAS has been developed to identify variants associated with aberrant splicing by directly using RNA-seq data. However, it bases on the assumption that annotated splicing site is normal splicing, which is not true in fact. We develop the ISVASE, a tool for specifically identifying sequence variants associated with splicing events (SVASE) by using RNA-seq data. Comparing with PVAAS, our tool has several advantages, such as multi-pass stringent rule-dependent filters and statistical filters, only using split-reads, independent sequence variant identification in each part of splicing (junction), sequence variant detection for both of known and novel splicing event, additional exon-exon junction shift event detection if known splicing events provided, splicing signal evaluation, known DNA mutation and/or RNA editing data supported, higher precision and consistency, and short running time. Using a realistic RNA-seq dataset, we performed a case study to illustrate the functionality and effectiveness of our method. Moreover, the output of SVASEs can be used for downstream analysis such as splicing regulatory element study and sequence variant functional analysis. ISVASE is useful for researchers interested in sequence variants (DNA mutation and/or RNA editing) associated with splicing events. The package is freely available at https://sourceforge.net/projects/isvase/ .

  16. Vitamin D and alternative splicing of RNA.

    Science.gov (United States)

    Zhou, Rui; Chun, Rene F; Lisse, Thomas S; Garcia, Alejandro J; Xu, Jianzhong; Adams, John S; Hewison, Martin

    2015-04-01

    The active form of vitamin D (1α,25-dihydroxyvitamin D, 1,25(OH)2D) exerts its genomic effects via binding to a nuclear high-affinity vitamin D receptor (VDR). Recent deep sequencing analysis of VDR binding locations across the complete genome has significantly expanded our understanding of the actions of vitamin D and VDR on gene transcription. However, these studies have also promoted appreciation of the extra-transcriptional impact of vitamin D on gene expression. It is now clear that vitamin D interacts with the epigenome via effects on DNA methylation, histone acetylation, and microRNA generation to maintain normal biological functions. There is also increasing evidence that vitamin D can influence pre-mRNA constitutive splicing and alternative splicing, although the mechanism for this remains unclear. Pre-mRNA splicing has long been thought to be a post-transcription RNA processing event, but current data indicate that this occurs co-transcriptionally. Several steroid hormones have been recognized to coordinately control gene transcription and pre-mRNA splicing through the recruitment of nuclear receptor co-regulators that can both control gene transcription and splicing. The current review will discuss this concept with specific reference to vitamin D, and the potential role of heterogeneous nuclear ribonucleoprotein C (hnRNPC), a nuclear factor with an established function in RNA splicing. hnRNPC, has been shown to be involved in the VDR transcriptional complex as a vitamin D-response element-binding protein (VDRE-BP), and may act as a coupling factor linking VDR-directed gene transcription with RNA splicing. In this way hnRNPC may provide an additional mechanism for the fine-tuning of vitamin D-regulated target gene expression. This article is part of a Special Issue entitled '17th Vitamin D Workshop'. Copyright © 2014 Elsevier Ltd. All rights reserved.

  17. EWS and FUS bind a subset of transcribed genes encoding proteins enriched in RNA regulatory functions.

    Science.gov (United States)

    Luo, Yonglun; Blechingberg, Jenny; Fernandes, Ana Miguel; Li, Shengting; Fryland, Tue; Børglum, Anders D; Bolund, Lars; Nielsen, Anders Lade

    2015-11-14

    FUS (TLS) and EWS (EWSR1) belong to the FET-protein family of RNA and DNA binding proteins. FUS and EWS are structurally and functionally related and participate in transcriptional regulation and RNA processing. FUS and EWS are identified in translocation generated cancer fusion proteins and involved in the human neurological diseases amyotrophic lateral sclerosis and fronto-temporal lobar degeneration. To determine the gene regulatory functions of FUS and EWS at the level of chromatin, we have performed chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq). Our results show that FUS and EWS bind to a subset of actively transcribed genes, that binding often is downstream the poly(A)-signal, and that binding overlaps with RNA polymerase II. Functional examinations of selected target genes identified that FUS and EWS can regulate gene expression at different levels. Gene Ontology analyses showed that FUS and EWS target genes preferentially encode proteins involved in regulatory processes at the RNA level. The presented results yield new insights into gene interactions of EWS and FUS and have identified a set of FUS and EWS target genes involved in pathways at the RNA regulatory level with potential to mediate normal and disease-associated functions of the FUS and EWS proteins.

  18. Transcription and splicing regulation in human umbilical vein endothelial cells under hypoxic stress conditions by exon array

    Directory of Open Access Journals (Sweden)

    Wu Yonghong

    2009-03-01

    Full Text Available Abstract Background The balance between endothelial cell survival and apoptosis during stress is an important cellular process for vessel integrity and vascular homeostasis, and it is also pivotal in angiogenesis during the development of many vascular diseases. However, the underlying molecular mechanisms remain largely unknown. Although both transcription and alternative splicing are important in regulating gene expression in endothelial cells under stress, the regulatory mechanisms underlying this state and their interactions have not yet been studied on a genome-wide basis. Results Human umbilical vein endothelial cells (HUVECs were treated with cobalt chloride (CoCl2 both to mimic hypoxia and to induce cell apoptosis and alternative splicing responses. Cell apoptosis rate analysis indicated that HUVECs exposed to 300 μM CoCl2 for 24 hrs were initially counterbalancing apoptosis with cell survival. We therefore used the Affymetrix exon array system to determine genome-wide transcript- and exon-level differential expression. Other than 1583 differentially expressed transcripts, 342 alternatively spliced exons were detected and classified by different splicing types. Sixteen alternatively spliced exons were validated by RT-PCR. Furthermore, direct evidence for the ongoing balance between HUVEC survival and apoptosis was provided by Gene Ontology (GO and protein function, as well as protein domain and pathway enrichment analyses of the differentially expressed transcripts. Importantly, a novel molecular module, in which the heat shock protein (HSP families play a significant role, was found to be activated under mimicked hypoxia conditions. In addition, 46% of the transcripts containing stress-modulated exons were differentially expressed, indicating the possibility of combinatorial regulation of transcription and splicing. Conclusion The exon array system effectively profiles gene expression and splicing on the genome-wide scale. Based on

  19. Discovery of a Splicing Regulator Required for Cell Cycle Progression

    Energy Technology Data Exchange (ETDEWEB)

    Suvorova, Elena S.; Croken, Matthew; Kratzer, Stella; Ting, Li-Min; Conde de Felipe, Magnolia; Balu, Bharath; Markillie, Lye Meng; Weiss, Louis M.; Kim, Kami; White, Michael W.

    2013-02-01

    In the G1 phase of the cell division cycle, eukaryotic cells prepare many of the resources necessary for a new round of growth including renewal of the transcriptional and protein synthetic capacities and building the machinery for chromosome replication. The function of G1 has an early evolutionary origin and is preserved in single and multicellular organisms, although the regulatory mechanisms conducting G1 specific functions are only understood in a few model eukaryotes. Here we describe a new G1 mutant from an ancient family of apicomplexan protozoans. Toxoplasma gondii temperature-sensitive mutant 12-109C6 conditionally arrests in the G1 phase due to a single point mutation in a novel protein containing a single RNA-recognition-motif (TgRRM1). The resulting tyrosine to asparagine amino acid change in TgRRM1 causes severe temperature instability that generates an effective null phenotype for this protein when the mutant is shifted to the restrictive temperature. Orthologs of TgRRM1 are widely conserved in diverse eukaryote lineages, and the human counterpart (RBM42) can functionally replace the missing Toxoplasma factor. Transcriptome studies demonstrate that gene expression is downregulated in the mutant at the restrictive temperature due to a severe defect in splicing that affects both cell cycle and constitutively expressed mRNAs. The interaction of TgRRM1 with factors of the tri-SNP complex (U4/U6 & U5 snRNPs) indicate this factor may be required to assemble an active spliceosome. Thus, the TgRRM1 family of proteins is an unrecognized and evolutionarily conserved class of splicing regulators. This study demonstrates investigations into diverse unicellular eukaryotes, like the Apicomplexa, have the potential to yield new insights into important mechanisms conserved across modern eukaryotic kingdoms.

  20. Role of Bmznf-2, a Bombyx mori CCCH zinc finger gene, in masculinisation and differential splicing of Bmtra-2.

    Science.gov (United States)

    Gopinath, Gajula; Arunkumar, Kallare P; Mita, Kazuei; Nagaraju, Javaregowda

    2016-08-01

    Deciphering the regulatory factors involved in Bombyx mori sex determination has been a puzzle, challenging researchers for nearly a century now. The pre-mRNA of B. mori doublesex (Bmdsx), a master regulator gene of sexual differentiation, is differentially spliced, producing Bmdsxm and Bmdsxf transcripts in males and females respectively. The putative proteins encoded by these differential transcripts orchestrate antagonistic functions, which lead to sexual differentiation. A recent study in B. mori illustrated the role of a W-derived fem piRNA in conferring femaleness. In females, the fem piRNA was shown to suppress the activity of a Z-linked CCCH type zinc finger (znf) gene, Masculiniser (masc), which indirectly promotes the Bmdsxm type of splicing. In this study, we report a novel autosomal (Chr 25) CCCH type znf motif encoding gene Bmznf-2 as one of the potential factors in the Bmdsx sex specific differential splicing, and we also provide insights into its role in the alternative splicing of Bmtra2 by using ovary derived BmN cells. Over-expression of Bmznf-2 induced Bmdsxm type of splicing (masculinisation) with a correspondingly reduced expression of Bmdsxf type isoform in BmN cells. Further, the site-directed mutational studies targeting the tandem CCCH znf motifs revealed their indispensability in the observed phenotype of masculinisation. Additionally, the dual luciferase assays in BmN cells using 5' UTR region of the Bmznf-2 strongly implied the existence of a translational repression over this gene. From these findings, we propose Bmznf-2 to be one of the potential factors of masculinisation similar to Masc. From the growing number of Bmdsx splicing regulators, we assume that the sex determination cascade of B. mori is quite intricate in nature; hence, it has to be further investigated for its comprehensive understanding. Copyright © 2016 Elsevier Ltd. All rights reserved.

  1. DEDB: a database of Drosophila melanogaster exons in splicing graph form

    Directory of Open Access Journals (Sweden)

    Tan Tin

    2004-12-01

    Full Text Available Abstract Background A wealth of quality genomic and mRNA/EST sequences in recent years has provided the data required for large-scale genome-wide analysis of alternative splicing. We have capitalized on this by constructing a database that contains alternative splicing information organized as splicing graphs, where all transcripts arising from a single gene are collected, organized and classified. The splicing graph then serves as the basis for the classification of the various types of alternative splicing events. Description DEDB http://proline.bic.nus.edu.sg/dedb/index.html is a database of Drosophila melanogaster exons obtained from FlyBase arranged in a splicing graph form that permits the creation of simple rules allowing for the classification of alternative splicing events. Pfam domains were also mapped onto the protein sequences allowing users to access the impact of alternative splicing events on domain organization. Conclusions DEDB's catalogue of splicing graphs facilitates genome-wide classification of alternative splicing events for genome analysis. The splicing graph viewer brings together genome, transcript, protein and domain information to facilitate biologists in understanding the implications of alternative splicing.

  2. Phosphorylation of a splice variant of collapsin response mediator protein 2 in the nucleus of tumour cells links cyclin dependent kinase-5 to oncogenesis

    International Nuclear Information System (INIS)

    Grant, Nicola J.; Coates, Philip J.; Woods, Yvonne L.; Bray, Susan E.; Morrice, Nicholas A.; Hastie, C. James; Lamont, Douglas J.; Carey, Francis A.; Sutherland, Calum

    2015-01-01

    Cyclin-dependent protein kinase-5 (CDK5) is an unusual member of the CDK family as it is not cell cycle regulated. However many of its substrates have roles in cell growth and oncogenesis, raising the possibility that CDK5 modulation could have therapeutic benefit. In order to establish whether changes in CDK5 activity are associated with oncogenesis one could quantify phosphorylation of CDK5 targets in disease tissue in comparison to appropriate controls. However the identity of physiological and pathophysiological CDK5 substrates remains the subject of debate, making the choice of CDK5 activity biomarkers difficult. Here we use in vitro and in cell phosphorylation assays to identify novel features of CDK5 target sequence determinants that confer enhanced CDK5 selectivity, providing means to select substrate biomarkers of CDK5 activity with more confidence. We then characterize tools for the best CDK5 substrate we identified to monitor its phosphorylation in human tissue and use these to interrogate human tumour arrays. The close proximity of Arg/Lys amino acids and a proline two residues N-terminal to the phosphorylated residue both improve recognition of the substrate by CDK5. In contrast the presence of a proline two residues C-terminal to the target residue dramatically reduces phosphorylation rate. Serine-522 of Collapsin Response Mediator-2 (CRMP2) is a validated CDK5 substrate with many of these structural criteria. We generate and characterise phosphospecific antibodies to Ser522 and show that phosphorylation appears in human tumours (lung, breast, and lymphoma) in stark contrast to surrounding non-neoplastic tissue. In lung cancer the anti-phospho-Ser522 signal is positive in squamous cell carcinoma more frequently than adenocarcinoma. Finally we demonstrate that it is a specific and unusual splice variant of CRMP2 (CRMP2A) that is phosphorylated in tumour cells. For the first time this data associates altered CDK5 substrate phosphorylation with

  3. Phosphorylation of a splice variant of collapsin response mediator protein 2 in the nucleus of tumour cells links cyclin dependent kinase-5 to oncogenesis.

    Science.gov (United States)

    Grant, Nicola J; Coates, Philip J; Woods, Yvonne L; Bray, Susan E; Morrice, Nicholas A; Hastie, C James; Lamont, Douglas J; Carey, Francis A; Sutherland, Calum

    2015-11-10

    Cyclin-dependent protein kinase-5 (CDK5) is an unusual member of the CDK family as it is not cell cycle regulated. However many of its substrates have roles in cell growth and oncogenesis, raising the possibility that CDK5 modulation could have therapeutic benefit. In order to establish whether changes in CDK5 activity are associated with oncogenesis one could quantify phosphorylation of CDK5 targets in disease tissue in comparison to appropriate controls. However the identity of physiological and pathophysiological CDK5 substrates remains the subject of debate, making the choice of CDK5 activity biomarkers difficult. Here we use in vitro and in cell phosphorylation assays to identify novel features of CDK5 target sequence determinants that confer enhanced CDK5 selectivity, providing means to select substrate biomarkers of CDK5 activity with more confidence. We then characterize tools for the best CDK5 substrate we identified to monitor its phosphorylation in human tissue and use these to interrogate human tumour arrays. The close proximity of Arg/Lys amino acids and a proline two residues N-terminal to the phosphorylated residue both improve recognition of the substrate by CDK5. In contrast the presence of a proline two residues C-terminal to the target residue dramatically reduces phosphorylation rate. Serine-522 of Collapsin Response Mediator-2 (CRMP2) is a validated CDK5 substrate with many of these structural criteria. We generate and characterise phosphospecific antibodies to Ser522 and show that phosphorylation appears in human tumours (lung, breast, and lymphoma) in stark contrast to surrounding non-neoplastic tissue. In lung cancer the anti-phospho-Ser522 signal is positive in squamous cell carcinoma more frequently than adenocarcinoma. Finally we demonstrate that it is a specific and unusual splice variant of CRMP2 (CRMP2A) that is phosphorylated in tumour cells. For the first time this data associates altered CDK5 substrate phosphorylation with

  4. The fitness cost of mis-splicing is the main determinant of alternative splicing patterns.

    Science.gov (United States)

    Saudemont, Baptiste; Popa, Alexandra; Parmley, Joanna L; Rocher, Vincent; Blugeon, Corinne; Necsulea, Anamaria; Meyer, Eric; Duret, Laurent

    2017-10-30

    Most eukaryotic genes are subject to alternative splicing (AS), which may contribute to the production of protein variants or to the regulation of gene expression via nonsense-mediated messenger RNA (mRNA) decay (NMD). However, a fraction of splice variants might correspond to spurious transcripts and the question of the relative proportion of splicing errors to functional splice variants remains highly debated. We propose a test to quantify the fraction of AS events corresponding to errors. This test is based on the fact that the fitness cost of splicing errors increases with the number of introns in a gene and with expression level. We analyzed the transcriptome of the intron-rich eukaryote Paramecium tetraurelia. We show that in both normal and in NMD-deficient cells, AS rates strongly decrease with increasing expression level and with increasing number of introns. This relationship is observed for AS events that are detectable by NMD as well as for those that are not, which invalidates the hypothesis of a link with the regulation of gene expression. Our results show that in genes with a median expression level, 92-98% of observed splice variants correspond to errors. We observed the same patterns in human transcriptomes and we further show that AS rates correlate with the fitness cost of splicing errors. These observations indicate that genes under weaker selective pressure accumulate more maladaptive substitutions and are more prone to splicing errors. Thus, to a large extent, patterns of gene expression variants simply reflect the balance between selection, mutation, and drift.

  5. Diversification of the muscle proteome through alternative splicing.

    Science.gov (United States)

    Nakka, Kiran; Ghigna, Claudia; Gabellini, Davide; Dilworth, F Jeffrey

    2018-03-06

    Skeletal muscles express a highly specialized proteome that allows the metabolism of energy sources to mediate myofiber contraction. This muscle-specific proteome is partially derived through the muscle-specific transcription of a subset of genes. Surprisingly, RNA sequencing technologies have also revealed a significant role for muscle-specific alternative splicing in generating protein isoforms that give specialized function to the muscle proteome. In this review, we discuss the current knowledge with respect to the mechanisms that allow pre-mRNA transcripts to undergo muscle-specific alternative splicing while identifying some of the key trans-acting splicing factors essential to the process. The importance of specific splicing events to specialized muscle function is presented along with examples in which dysregulated splicing contributes to myopathies. Though there is now an appreciation that alternative splicing is a major contributor to proteome diversification, the emergence of improved "targeted" proteomic methodologies for detection of specific protein isoforms will soon allow us to better appreciate the extent to which alternative splicing modifies the activity of proteins (and their ability to interact with other proteins) in the skeletal muscle. In addition, we highlight a continued need to better explore the signaling pathways that contribute to the temporal control of trans-acting splicing factor activity to ensure specific protein isoforms are expressed in the proper cellular context. An understanding of the signal-dependent and signal-independent events driving muscle-specific alternative splicing has the potential to provide us with novel therapeutic strategies to treat different myopathies.

  6. Structural studies of bacterial transcriptional regulatory proteins by multidimensional heteronuclear NMR

    Energy Technology Data Exchange (ETDEWEB)

    Volkman, Brian Finley [Univ. of California, Berkeley, CA (United States)

    1995-02-01

    Nuclear magnetic resonance spectroscopy was used to elucidate detailed structural information for peptide and protein molecules. A small peptide was designed and synthesized, and its three-dimensional structure was calculated using distance information derived from two-dimensional NMR measurements. The peptide was used to induce antibodies in mice, and the cross-reactivity of the antibodies with a related protein was analyzed with enzyme-linked immunosorbent assays. Two proteins which are involved in regulation of transcription in bacteria were also studied. The ferric uptake regulation (Fur) protein is a metal-dependent repressor which controls iron uptake in bacteria. Two- and three-dimensional NMR techniques, coupled with uniform and selective isotope labeling allowed the nearly complete assignment of the resonances of the metal-binding domain of the Fur protein. NTRC is a transcriptional enhancer binding protein whose N-terminal domain is a "receiver domain" in the family of "two-component" regulatory systems. Phosphorylation of the N-terminal domain of NTRC activates the initiation of transcription of aeries encoding proteins involved in nitrogen regulation. Three- and four-dimensional NMR spectroscopy methods have been used to complete the resonance assignments and determine the solution structure of the N-terminal receiver domain of the NTRC protein. Comparison of the solution structure of the NTRC receiver domain with the crystal structures of the homologous protein CheY reveals a very similar fold, with the only significant difference being the position of helix 4 relative to the rest of the protein. The determination of the structure of the NTRC receiver domain is the first step toward understanding a mechanism of signal transduction which is common to many bacterial regulatory systems.

  7. Alternative splicing of mutually exclusive exons--a review.

    Science.gov (United States)

    Pohl, Martin; Bortfeldt, Ralf H; Grützmann, Konrad; Schuster, Stefan

    2013-10-01

    Alternative splicing (AS) of pre-mRNAs in higher eukaryotes and several viruses is one major source of protein diversity. Usually, the following major subtypes of AS are distinguished: exon skipping, intron retention, and alternative 3' and 5' splice sites. Moreover, mutually exclusive exons (MXEs) represent a rare subtype. In the splicing of MXEs, two (or more) splicing events are not independent anymore, but are executed or disabled in a coordinated manner. In this review, several bioinformatics approaches for analyzing MXEs are presented and discussed. In particular, we revisit suitable definitions and nomenclatures, and bioinformatics tools for finding MXEs, adjacent and non-adjacent MXEs, clustered and grouped MXEs. Moreover, the molecular mechanisms for splicing MXEs proposed in the literature are reviewed and discussed. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  8. Identification and functional analysis of two alternatively spliced transcripts of ABSCISIC ACID INSENSITIVE3 (ABI3) in linseed flax (Linum usitatissimum L.).

    Science.gov (United States)

    Wang, Yanyan; Zhang, Tianbao; Song, Xiaxia; Zhang, Jianping; Dang, Zhanhai; Pei, Xinwu; Long, Yan

    2018-01-01

    Alternative splicing is a popular phenomenon in different types of plants. It can produce alternative spliced transcripts that encode proteins with altered functions. Previous studies have shown that one transcription factor, ABSCISIC ACID INSENSITIVE3 (ABI3), which encodes an important component in abscisic acid (ABA) signaling, is subjected to alternative splicing in both mono- and dicotyledons. In the current study, we identified two homologs of ABI3 in the genome of linseed flax. We screened two alternatively spliced flax LuABI3 transcripts, LuABI3-2 and LuABI3-3, and one normal flax LuABI3 transcript, LuABI3-1. Sequence analysis revealed that one of the alternatively spliced transcripts, LuABI3-3, retained a 6 bp intron. RNA accumulation analysis showed that all three transcripts were expressed during seed development, while subcellular localization and transgene experiments showed that LuABI3-3 had no biological function. The two normal transcripts, LuABI3-1 and LuABI3-2, are the important functional isoforms in flax and play significant roles in the ABA regulatory pathway during seed development, germination, and maturation.

  9. Identification and functional analysis of two alternatively spliced transcripts of ABSCISIC ACID INSENSITIVE3 (ABI3 in linseed flax (Linum usitatissimum L..

    Directory of Open Access Journals (Sweden)

    Yanyan Wang

    Full Text Available Alternative splicing is a popular phenomenon in different types of plants. It can produce alternative spliced transcripts that encode proteins with altered functions. Previous studies have shown that one transcription factor, ABSCISIC ACID INSENSITIVE3 (ABI3, which encodes an important component in abscisic acid (ABA signaling, is subjected to alternative splicing in both mono- and dicotyledons. In the current study, we identified two homologs of ABI3 in the genome of linseed flax. We screened two alternatively spliced flax LuABI3 transcripts, LuABI3-2 and LuABI3-3, and one normal flax LuABI3 transcript, LuABI3-1. Sequence analysis revealed that one of the alternatively spliced transcripts, LuABI3-3, retained a 6 bp intron. RNA accumulation analysis showed that all three transcripts were expressed during seed development, while subcellular localization and transgene experiments showed that LuABI3-3 had no biological function. The two normal transcripts, LuABI3-1 and LuABI3-2, are the important functional isoforms in flax and play significant roles in the ABA regulatory pathway during seed development, germination, and maturation.

  10. The Contribution of Serine 194 Phosphorylation to Steroidogenic Acute Regulatory Protein Function

    OpenAIRE

    Sasaki, Goro; Zubair, Mohamad; Ishii, Tomohiro; Mitsui, Toshikatsu; Hasegawa, Tomonobu; Auchus, Richard J.

    2014-01-01

    The steroidogenic acute regulatory protein (StAR) facilitates the delivery of cholesterol to the inner mitochondrial membrane, where the cholesterol side-chain cleavage enzyme catalyzes the initial step of steroid hormone biosynthesis. StAR was initially identified in adrenocortical cells as a phosphoprotein, the expression and phosphorylation of which were stimulated by corticotropin. A number of in vitro studies have implicated cAMP-dependent phosphorylation at serine 194 (S194, S195 in hum...

  11. Connecting the dots: chromatin and alternative splicing in EMT.

    Science.gov (United States)

    Warns, Jessica A; Davie, James R; Dhasarathy, Archana

    2016-02-01

    Nature has devised sophisticated cellular machinery to process mRNA transcripts produced by RNA Polymerase II, removing intronic regions and connecting exons together, to produce mature RNAs. This process, known as splicing, is very closely linked to transcription. Alternative splicing, or the ability to produce different combinations of exons that are spliced together from the same genomic template, is a fundamental means of regulating protein complexity. Similar to transcription, both constitutive and alternative splicing can be regulated by chromatin and its associated factors in response to various signal transduction pathways activated by external stimuli. This regulation can vary between different cell types, and interference with these pathways can lead to changes in splicing, often resulting in aberrant cellular states and disease. The epithelial to mesenchymal transition (EMT), which leads to cancer metastasis, is influenced by alternative splicing events of chromatin remodelers and epigenetic factors such as DNA methylation and non-coding RNAs. In this review, we will discuss the role of epigenetic factors including chromatin, chromatin remodelers, DNA methyltransferases, and microRNAs in the context of alternative splicing, and discuss their potential involvement in alternative splicing during the EMT process.

  12. CELF1 preferentially binds to exon-intron boundary and regulates alternative splicing in HeLa cells.

    Science.gov (United States)

    Xia, Heng; Chen, Dong; Wu, Qijia; Wu, Gang; Zhou, Yanhong; Zhang, Yi; Zhang, Libin

    2017-09-01

    The current RIP-seq approach has been developed for the identification of genome-wide interaction between RNA binding protein (RBP) and the bound RNA transcripts, but still rarely for identifying its binding sites. In this study, we performed RIP-seq experiments in HeLa cells using a monoclonal antibody against CELF1. Mapping of the RIP-seq reads showed a biased distribution at the 3'UTR and intronic regions. A total of 15,285 and 1384 CELF1-specific sense and antisense peaks were identified using the ABLIRC software tool. Our bioinformatics analyses revealed that 5' and 3' splice site motifs and GU-rich motifs were highly enriched in the CELF1-bound peaks. Furthermore, transcriptome analyses revealed that alternative splicing was globally regulated by CELF1 in HeLa cells. For example, the inclusion of exon 16 of LMO7 gene, a marker gene of breast cancer, is positively regulated by CELF1. Taken together, we have shown that RIP-seq data can be used to decipher RBP binding sites and reveal an unexpected landscape of the genome-wide CELF1-RNA interactions in HeLa cells. In addition, we found that CELF1 globally regulates the alternative splicing by binding the exon-intron boundary in HeLa cells, which will deepen our understanding of the regulatory roles of CELF1 in the pre-mRNA splicing process. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Effects of sterol regulatory element-binding protein (SREBP in chickens

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    Alipour Fahimeh

    2012-02-01

    Full Text Available Abstract Sterol regulatory element binding protein- 1 and -2 (SREBP-1 and -2 are key transcription factors involved in the biosynthesis of cholesterol and fatty acids. The SREBP have mostly been studied in rodents in which lipogenesis is regulated in both liver and adipose tissue. There is, though, a paucity of information on birds, in which lipogenesis occurs essentially in the liver as in humans. Since a prelude to the investigation of the role of SREBP in lipid metabolism regulation in chicken, we review Size and Tissue expression Pattern of SREBP and role of this protein in chickens.

  14. The Trypanosoma brucei La protein is a candidate poly(U) shield that impacts spliced leader RNA maturation and tRNA intron removal

    Czech Academy of Sciences Publication Activity Database

    Trantírková, Silvie; Paris, Zdeněk; Sturm, N. R.; Campbell, D. A.; Lukeš, Julius

    2005-01-01

    Roč. 35, č. 4 (2005), s. 359-366 ISSN 0020-7519 R&D Projects: GA AV ČR IAA5022302 Grant - others:NIH(US) AI34536; NIH(US) AI056034 Institutional research plan: CEZ:AV0Z60220518 Keywords : splicing * Trypanosoma * RNA interference Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.346, year: 2005

  15. Chemical correction of pre-mRNA splicing defects associated with sequestration of muscleblind-like 1 protein by expanded r(CAG) transcripts

    Science.gov (United States)

    Kumar, Amit; Parkesh, Raman; Sznajder, Lukasz J.; Childs-Disney, Jessica; Sobczak, Krzysztof; Disney, Matthew D.

    2012-01-01

    Recently, it was reported that expanded r(CAG) triplet repeats (r(CAG)exp) associated with untreatable neurological diseases cause pre-mRNA mis-splicing likely due to sequestration of muscleblind-like 1 (MBNL1) splicing factor. Bioactive small molecules that bind the 5’CAG/3’GAC motif found in r(CAG)exp hairpin structure were identified by using RNA binding studies and virtual screening/chemical similarity searching. Specifically, a benzylguanidine-containing small molecule was found to improve pre-mRNA alternative splicing of MBNL1-sensitive exons in cells expressing the toxic r(CAG)exp. The compound was identified by first studying the binding of RNA 1×1 nucleotide internal loops to small molecules known to have affinity for nucleic acids. Those studies identified 4',6-diamidino-2-phenylindole (DAPI) as a specific binder to RNAs with the 5’CAG/3’GAC motif. DAPI was then used as a query molecule in a shape- and chemistry alignment-based virtual screen to identify compounds with improved properties, which identified 4-guanidinophenyl 4-guanidinobenzoate as small molecule capable of improving pre-mRNA splicing defects associated with the r(CAG)exp-MBNL1 complex. This compound may facilitate the development of therapeutics to treat diseases caused by r(CAG)exp and could serve as a useful chemical tool to dissect the mechanisms of r(CAG)exp toxicity. The approach used in these studies, defining the small RNA motifs that bind known nucleic acid binders and then using virtual screening to optimize them for bioactivity, may be generally applicable for designing small molecules that target other RNAs in human genomic sequence. PMID:22252896

  16. Solubilization and reconstitution of the formylmethionylleucylphenylalanine receptor coupled to guanine nucleotide regulatory protein

    International Nuclear Information System (INIS)

    Williamson, K.; Dickey, B.F.; Pyun, H.Y.; Navarro, J.

    1988-01-01

    The authors describe the solubilization, resolution, and reconstitution of the formylmethionylleucylphenylalanine (fMet-Leu-Phe) receptor and guanine nucleotide regulatory proteins (G-proteins). The receptor was solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Guanine nucleotides decreased the number of high-affinity binding sites and accelerated the rate of dissociation of the receptor-ligand complex, suggesting that the solubilized receptor remained coupled to endogenous G-proteins. The solubilized receptor was resolved from endogenous G-proteins by fractionation on a wheat germ agglutinin (WGA)-Sepharose 4B column. High-affinity [ 3 H]fMet-Leu-Phe binding to the WGA-purified receptor was diminished and exhibited reduced guanine nucleotide sensitivity. High-affinity [ 3 H]fMET-Leu-Phe binding and guanine nucleotide sensitivity were reconstituted upon the addition of purified brain G-proteins. Similar results were obtained when the receptor was reconstituted with brain G-proteins into phospholipid vesicles by gel filtration chromatography. In addition, they also demonstrated fMET-Leu-Phe-dependent GTP hydrolysis in the reconstituted vesicles. The results of this work indicate that coupling of the fMet-Leu-Phe receptor to G-proteins converts the receptor to a high-affinity binding state and that agonist produces activation of G-proteins. The resolution and functional reconstitution of this receptor should provide an important step toward the elucidation of the molecular mechanism of the fMet-Leu-Phe transduction system in neutrophils

  17. Aberrant and alternative splicing in skeletal system disease.

    Science.gov (United States)

    Fan, Xin; Tang, Liling

    2013-10-01

    The main function of skeletal system is to support the body and help movement. A variety of factors can lead to skeletal system disease, including age, exercise, and of course genetic makeup and expression. Pre-mRNA splicing plays a crucial role in gene expression, by creating multiple protein variants with different biological functions. The recent studies show that several skeletal system diseases are related to pre-mRNA splicing. This review focuses on the relationship between pre-mRNA splicing and skeletal system disease. On the one hand, splice site mutation that leads to aberrant splicing often causes genetic skeletal system disease, like COL1A1, SEDL and LRP5. On the other hand, alternative splicing without genomic mutation may generate some marker protein isoforms, for example, FN, VEGF and CD44. Therefore, understanding the relationship between pre-mRNA splicing and skeletal system disease will aid in uncovering the mechanism of disease and contribute to the future development of gene therapy. © 2013 Elsevier B.V. All rights reserved.

  18. RAGE splicing variants in mammals.

    Science.gov (United States)

    Sterenczak, Katharina Anna; Nolte, Ingo; Murua Escobar, Hugo

    2013-01-01

    The receptor for advanced glycation end products (RAGE) is a multiligand receptor of environmental stressors which plays key roles in pathophysiological processes, including immune/inflammatory disorders, Alzheimer's disease, diabetic arteriosclerosis, tumorigenesis, and metastasis. Besides the full-length RAGE protein in humans nearly 20 natural occurring RAGE splicing variants were described on mRNA and protein level. These naturally occurring isoforms are characterized by either N-terminally or C-terminally truncations and are discussed as possible regulators of the full-length RAGE receptor either by competitive ligand binding or by displacing the full-length protein in the membrane. Accordingly, expression deregulations of the naturally occurring isoforms were supposed to have significant effect on RAGE-mediated disorders. Thereby the soluble C-truncated RAGE isoforms present in plasma and tissues are the mostly focused isoforms in research and clinics. Deregulations of the circulating levels of soluble RAGE forms were reported in several RAGE-associated pathological disorders including for example atherosclerosis, diabetes, renal failure, Alzheimer's disease, and several cancer types. Regarding other mammalian species, the canine RAGE gene showed high similarities to the corresponding human structures indicating RAGE to be evolutionary highly conserved between both species. Similar to humans the canine RAGE showed a complex and extensive splicing activity leading to a manifold pattern of RAGE isoforms. Due to the similarities seen in several canine and human diseases-including cancer-comparative structural and functional analyses allow the development of RAGE and ligand-specific therapeutic approaches beneficial for human and veterinary medicine.

  19. The conserved splicing factor SUA controls alternative splicing of the developmental regulator ABI3 in Arabidopsis.

    NARCIS (Netherlands)

    Sugliani, M.; Brambilla, V.; Clerkx, E.J.M.; Koornneef, M.; Soppe, W.J.J.

    2010-01-01

    ABSCISIC ACID INSENSITIVE3 (ABI3) is a major regulator of seed maturation in Arabidopsis thaliana. We detected two ABI3 transcripts, ABI3- and ABI3-ß, which encode full-length and truncated proteins, respectively. Alternative splicing of ABI3 is developmentally regulated, and the ABI3-ß transcript

  20. Ca2+-regulatory proteins in cardiomyocytes from the right ventricle in children with congenital heart disease

    Directory of Open Access Journals (Sweden)

    Wu Yihe

    2012-04-01

    Full Text Available Abstract Background Hypoxia and hypertrophy are the most frequent pathophysiological consequence of congenital heart disease (CHD which can induce the alteration of Ca2+-regulatory proteins and inhibit cardiac contractility. Few studies have been performed to examine Ca2+-regulatory proteins in human cardiomyocytes from the hypertrophic right ventricle with or without hypoxia. Methods Right ventricle tissues were collected from children with tetralogy of Fallot [n = 25, hypoxia and hypertrophy group (HH group], pulmonary stenosis [n = 25, hypertrophy group (H group], or small isolated ventricular septal defect [n = 25, control group (C group] during open-heart surgery. Paraffin sections of tissues were stained with 3,3′-dioctadecyloxacarbocyanine perchlorate to measure cardiomyocyte size. Expression levels of Ca2+-regulatory proteins [sarcoplasmic reticulum Ca2+-ATPase (SERCA2a, ryanodine receptor (RyR2, sodiumcalcium exchanger (NCX, sarcolipin (SLN and phospholamban (PLN] were analysed by means of real-time PCR, western blot, or immunofluorescence. Additionally, phosphorylation level of RyR and PLN and activity of protein phosphatase (PP1 were evaluated using western blot. Results Mild cardiomyocyte hypertrophy of the right ventricle in H and HH groups was confirmed by comparing cardiomyocyte size. A significant reduction of SERCA2a in mRNA (P16-phosphorylated PLN was down-regulated (PP Conclusions The decreased SERCA2a mRNA may be a biomarker of the pathological process in the early stage of cyanotic CHD with the hypertrophic right ventricle. A combination of hypoxia and hypertrophy can induce the adverse effect of PLN-Ser16 dephosphorylation. Increased PP1 could result in the decreased PLN-Ser16 and inhibition of PP1 is a potential therapeutic target for heart dysfunction in pediatrics.

  1. Conditional Toxin Splicing Using a Split Intein System.

    Science.gov (United States)

    Alford, Spencer C; O'Sullivan, Connor; Howard, Perry L

    2017-01-01

    Protein toxin splicing mediated by split inteins can be used as a strategy for conditional cell ablation. The approach requires artificial fragmentation of a potent protein toxin and tethering each toxin fragment to a split intein fragment. The toxin-intein fragments are, in turn, fused to dimerization domains, such that addition of a dimerizing agent reconstitutes the split intein. These chimeric toxin-intein fusions remain nontoxic until the dimerizer is added, resulting in activation of intein splicing and ligation of toxin fragments to form an active toxin. Considerations for the engineering and implementation of conditional toxin splicing (CTS) systems include: choice of toxin split site, split site (extein) chemistry, and temperature sensitivity. The following method outlines design criteria and implementation notes for CTS using a previously engineered system for splicing a toxin called sarcin, as well as for developing alternative CTS systems.

  2. Muscle-Specific Mis-Splicing and Heart Disease Exemplified by RBM20.

    Science.gov (United States)

    Rexiati, Maimaiti; Sun, Mingming; Guo, Wei

    2018-01-05

    Alternative splicing is an essential post-transcriptional process to generate multiple functional RNAs or proteins from a single transcript. Progress in RNA biology has led to a better understanding of muscle-specific RNA splicing in heart disease. The recent discovery of the muscle-specific splicing factor RNA-binding motif 20 (RBM20) not only provided great insights into the general alternative splicing mechanism but also demonstrated molecular mechanism of how this splicing factor is associated with dilated cardiomyopathy. Here, we review our current knowledge of muscle-specific splicing factors and heart disease, with an emphasis on RBM20 and its targets, RBM20-dependent alternative splicing mechanism, RBM20 disease origin in induced Pluripotent Stem Cells (iPSCs), and RBM20 mutations in dilated cardiomyopathy. In the end, we will discuss the multifunctional role of RBM20 and manipulation of RBM20 as a potential therapeutic target for heart disease.

  3. Intravitreal Injection of Splice-switching Oligonucleotides to Manipulate Splicing in Retinal Cells

    Directory of Open Access Journals (Sweden)

    Xavier Gérard

    2015-01-01

    Full Text Available Leber congenital amaurosis is a severe hereditary retinal dystrophy responsible for neonatal blindness. The most common disease-causing mutation (c.2991+1655A>G; 10–15% creates a strong splice donor site that leads to insertion of a cryptic exon encoding a premature stop codon. Recently, we reported that splice-switching oligonucleotides (SSO allow skipping of the mutant cryptic exon and the restoration of ciliation in fibroblasts of affected patients, supporting the feasibility of a SSO-mediated exon skipping strategy to correct the aberrant splicing. Here, we present data in the wild-type mouse, which demonstrate that intravitreal administration of 2’-OMePS-SSO allows selective alteration of Cep290 splicing in retinal cells, including photoreceptors as shown by successful alteration of Abca4 splicing using the same approach. We show that both SSOs and Cep290 skipped mRNA were detectable for at least 1 month and that intravitreal administration of oligonucleotides did not provoke any serious adverse event. These data suggest that intravitreal injections of SSO should be considered to bypass protein truncation resulting from the c.2991+1655A>G mutation as well as other truncating mutations in genes which like CEP290 or ABCA4 have a mRNA size that exceed cargo capacities of US Food and Drug Administration (FDA-approved adeno-associated virus (AAV-vectors, thus hampering gene augmentation therapy.

  4. Features generated for computational splice-site prediction correspond to functional elements

    Directory of Open Access Journals (Sweden)

    Wilbur W John

    2007-10-01

    Full Text Available Abstract Background Accurate selection of splice sites during the splicing of precursors to messenger RNA requires both relatively well-characterized signals at the splice sites and auxiliary signals in the adjacent exons and introns. We previously described a feature generation algorithm (FGA that is capable of achieving high classification accuracy on human 3' splice sites. In this paper, we extend the splice-site prediction to 5' splice sites and explore the generated features for biologically meaningful splicing signals. Results We present examples from the observed features that correspond to known signals, both core signals (including the branch site and pyrimidine tract and auxiliary signals (including GGG triplets and exon splicing enhancers. We present evidence that features identified by FGA include splicing signals not found by other methods. Conclusion Our generated features capture known biological signals in the expected sequence interval flanking splice sites. The method can be easily applied to other species and to similar classification problems, such as tissue-specific regulatory elements, polyadenylation sites, promoters, etc.

  5. Functions for fission yeast splicing factors SpSlu7 and SpPrp18 in alternative splice-site choice and stress-specific regulated splicing.

    Directory of Open Access Journals (Sweden)

    Geetha Melangath

    Full Text Available Budding yeast spliceosomal factors ScSlu7 and ScPrp18 interact and mediate intron 3'ss choice during second step pre-mRNA splicing. The fission yeast genome with abundant multi-intronic transcripts, degenerate splice signals and SR proteins is an apt unicellular fungal model to deduce roles for core spliceosomal factors in alternative splice-site choice, intron retention and to study the cellular implications of regulated splicing. From our custom microarray data we deduce a stringent reproducible subset of S. pombe alternative events. We examined the role of factors SpSlu7 or SpPrp18 for these splice events and investigated the relationship to growth phase and stress. Wild-type log and stationary phase cells showed ats1+ exon 3 skipped and intron 3 retained transcripts. Interestingly the non-consensus 5'ss in ats1+ intron 3 caused SpSlu7 and SpPrp18 dependent intron retention. We validated the use of an alternative 5'ss in dtd1+ intron 1 and of an upstream alternative 3'ss in DUF3074 intron 1. The dtd1+ intron 1 non-canonical 5'ss yielded an alternative mRNA whose levels increased in stationary phase. Utilization of dtd1+ intron 1 sub-optimal 5' ss required functional SpPrp18 and SpSlu7 while compromise in SpSlu7 function alone hampered the selection of the DUF3074 intron 1 non canonical 3'ss. We analysed the relative abundance of these splice isoforms during mild thermal, oxidative and heavy metal stress and found stress-specific splice patterns for ats1+ and DUF3074 intron 1 some of which were SpSlu7 and SpPrp18 dependent. By studying ats1+ splice isoforms during compromised transcription elongation rates in wild-type, spslu7-2 and spprp18-5 mutant cells we found dynamic and intron context-specific effects in splice-site choice. Our work thus shows the combinatorial effects of splice site strength, core splicing factor functions and transcription elongation kinetics to dictate alternative splice patterns which in turn serve as an additional

  6. Abnormalities in Alternative Splicing of Apoptotic Genes and Cardiovascular Diseases

    Directory of Open Access Journals (Sweden)

    Zodwa Dlamini

    2015-11-01

    Full Text Available Apoptosis is required for normal heart development in the embryo, but has also been shown to be an important factor in the occurrence of heart disease. Alternative splicing of apoptotic genes is currently emerging as a diagnostic and therapeutic target for heart disease. This review addresses the involvement of abnormalities in alternative splicing of apoptotic genes in cardiac disorders including cardiomyopathy, myocardial ischemia and heart failure. Many pro-apoptotic members of the Bcl-2 family have alternatively spliced isoforms that lack important active domains. These isoforms can play a negative regulatory role by binding to and inhibiting the pro-apoptotic forms. Alternative splicing is observed to be increased in various cardiovascular diseases with the level of alternate transcripts increasing elevated in diseased hearts compared to healthy subjects. In many cases these isoforms appear to be the underlying cause of the disease, while in others they may be induced in response to cardiovascular pathologies. Regardless of this, the detection of alternate splicing events in the heart can serve as useful diagnostic or prognostic tools, while those splicing events that seem to play a causative role in cardiovascular disease make attractive future drug targets.

  7. The Functional Impact of Alternative Splicing in Cancer.

    Science.gov (United States)

    Climente-González, Héctor; Porta-Pardo, Eduard; Godzik, Adam; Eyras, Eduardo

    2017-08-29

    Alternative splicing changes are frequently observed in cancer and are starting to be recognized as important signatures for tumor progression and therapy. However, their functional impact and relevance to tumorigenesis remain mostly unknown. We carried out a systematic analysis to characterize the potential functional consequences of alternative splicing changes in thousands of tumor samples. This analysis revealed that a subset of alternative splicing changes affect protein domain families that are frequently mutated in tumors and potentially disrupt protein-protein interactions in cancer-related pathways. Moreover, there was a negative correlation between the number of these alternative splicing changes in a sample and the number of somatic mutations in drivers. We propose that a subset of the alternative splicing changes observed in tumors may represent independent oncogenic processes that could be relevant to explain the functional transformations in cancer, and some of them could potentially be considered alternative splicing drivers (AS drivers). Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  8. Alternative REST Splicing Underappreciated

    OpenAIRE

    Chen, Guo-Lin; Miller, Gregory

    2017-01-01

    As a major orchestrator of the cellular epigenome, the repressor element-1 silencing transcription factor (REST) can either repress or activate thousands of genes depending on cellular context, suggesting a highly context-dependent REST function tuned by environmental cues. While REST shows cell-type non-selective active transcription, an N-terminal REST4 isoform caused by alternative splicing - inclusion of an extra exon (N3c) which introduces a pre-mature stop codon - has been implicated in...

  9. A splice site mutation in a gene encoding for PDK4, a mitochondrial protein, is associated with the development of dilated cardiomyopathy in the Doberman pinscher.

    Science.gov (United States)

    Meurs, Kathryn M; Lahmers, Sunshine; Keene, Bruce W; White, Stephen N; Oyama, Mark A; Mauceli, Evan; Lindblad-Toh, Kerstin

    2012-08-01

    Familial dilated cardiomyopathy is a primary myocardial disease that can result in the development of congestive heart failure and sudden cardiac death. Spontaneous animal models of familial dilated cardiomyopathy exist and the Doberman pinscher dog is one of the most commonly reported canine breeds. The objective of this study was to evaluate familial dilated cardiomyopathy in the Doberman pinscher dog using a genome-wide association study for a genetic alteration(s) associated with the development of this disease in this canine model. Genome-wide association analysis identified an area of statistical significance on canine chromosome 14 (p(raw) = 9.999e-05 corrected for genome-wide significance), fine-mapping of additional SNPs flanking this region localized a signal to 23,774,190-23,781,919 (p = 0.001) and DNA sequencing identified a 16-base pair deletion in the 5' donor splice site of intron 10 of the pyruvate dehydrogenase kinase 4 gene in affected dogs (p dilation, marked pleomorphic mitochondrial alterations with megamitochondria, scattered mitochondria with whorling and vacuolization and mild aggregates of lipofuscin granules. In conclusion, we report the identification of a splice site deletion in the PDK4 gene that is associated with the development of familial dilated cardiomyopathy in the Doberman pinscher dog.

  10. Pro-protein convertases control the maturation and processing of the iron-regulatory protein, RGMc/hemojuvelin

    Directory of Open Access Journals (Sweden)

    Rotwein Peter

    2008-04-01

    Full Text Available Abstract Background Repulsive guidance molecule c (RGMc or hemojuvelin, a glycosylphosphatidylinositol-linked glycoprotein expressed in liver and striated muscle, plays a central role in systemic iron balance. Inactivating mutations in the RGMc gene cause juvenile hemochromatosis (JH, a rapidly progressing iron storage disorder with severe systemic manifestations. RGMc undergoes complex biosynthetic steps leading to membrane-bound and soluble forms of the protein, including both 50 and 40 kDa single-chain species. Results We now show that pro-protein convertases (PC are responsible for conversion of 50 kDa RGMc to a 40 kDa protein with a truncated COOH-terminus. Unlike related molecules RGMa and RGMb, RGMc encodes a conserved PC recognition and cleavage site, and JH-associated RGMc frame-shift mutants undergo COOH-terminal cleavage only if this site is present. A cell-impermeable peptide PC inhibitor blocks the appearance of 40 kDa RGMc in extra-cellular fluid, as does an engineered mutation in the conserved PC recognition sequence, while the PC furin cleaves 50 kDa RGMc in vitro into a 40 kDa molecule with an intact NH2-terminus. Iron loading reduces release of RGMc from the cell membrane, and diminishes accumulation of the 40 kDa species in cell culture medium. Conclusion Our results define a role for PCs in the maturation of RGMc that may have implications for the physiological actions of this critical iron-regulatory protein.

  11. A functional screen reveals an extensive layer of transcriptional and splicing control underlying RAS/MAPK signaling in Drosophila.

    Directory of Open Access Journals (Sweden)

    Dariel Ashton-Beaucage

    2014-03-01

    Full Text Available The small GTPase RAS is among the most prevalent oncogenes. The evolutionarily conserved RAF-MEK-MAPK module that lies downstream of RAS is one of the main conduits through which RAS transmits proliferative signals in normal and cancer cells. Genetic and biochemical studies conducted over the last two decades uncovered a small set of factors regulating RAS/MAPK signaling. Interestingly, most of these were found to control RAF activation, thus suggesting a central regulatory role for this event. Whether additional factors are required at this level or further downstream remains an open question. To obtain a comprehensive view of the elements functionally linked to the RAS/MAPK cascade, we used a quantitative assay in Drosophila S2 cells to conduct a genome-wide RNAi screen for factors impacting RAS-mediated MAPK activation. The screen led to the identification of 101 validated hits, including most of the previously known factors associated to this pathway. Epistasis experiments were then carried out on individual candidates to determine their position relative to core pathway components. While this revealed several new factors acting at different steps along the pathway--including a new protein complex modulating RAF activation--we found that most hits unexpectedly work downstream of MEK and specifically influence MAPK expression. These hits mainly consist of constitutive splicing factors and thereby suggest that splicing plays a specific role in establishing MAPK levels. We further characterized two representative members of this group and surprisingly found that they act by regulating mapk alternative splicing. This study provides an unprecedented assessment of the factors modulating RAS/MAPK signaling in Drosophila. In addition, it suggests that pathway output does not solely rely on classical signaling events, such as those controlling RAF activation, but also on the regulation of MAPK levels. Finally, it indicates that core splicing

  12. iTRAQ-Based Quantitative Proteomics Identifies Potential Regulatory Proteins Involved in Chicken Eggshell Brownness.

    Directory of Open Access Journals (Sweden)

    Guangqi Li

    Full Text Available Brown eggs are popular in many countries and consumers regard eggshell brownness as an important indicator of egg quality. However, the potential regulatory proteins and detailed molecular mechanisms regulating eggshell brownness have yet to be clearly defined. In the present study, we performed quantitative proteomics analysis with iTRAQ technology in the shell gland epithelium of hens laying dark and light brown eggs to investigate the candidate proteins and molecular mechanisms underlying variation in chicken eggshell brownness. The results indicated 147 differentially expressed proteins between these two groups, among which 65 and 82 proteins were significantly up-regulated in the light and dark groups, respectively. Functional analysis indicated that in the light group, the down-regulated iron-sulfur cluster assembly protein (Iba57 would decrease the synthesis of protoporphyrin IX; furthermore, the up-regulated protein solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator, member 5 (SLC25A5 and down-regulated translocator protein (TSPO would lead to increased amounts of protoporphyrin IX transported into the mitochondria matrix to form heme with iron, which is supplied by ovotransferrin protein (TF. In other words, chickens from the light group produce less protoporphyrin IX, which is mainly used for heme synthesis. Therefore, the exported protoporphyrin IX available for eggshell deposition and brownness is reduced in the light group. The current study provides valuable information to elucidate variation of chicken eggshell brownness, and demonstrates the feasibility and sensitivity of iTRAQ-based quantitative proteomics analysis in providing useful insights into the molecular mechanisms underlying brown eggshell pigmentation.

  13. Guanine nucleotide-binding regulatory proteins in retinal pigment epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Jiang, Meisheng; Tran, V.T.; Fong, H.K.W. (Univ. of Southern California, Los Angeles (United States)); Pandey, S. (Doheny Eye Inst., Los Angeles, CA (United States))

    1991-05-01

    The expression of GTP-binding regulatory proteins (G proteins) in retinal pigment epithelial (RPE) cells was analyzed by RNA blot hybridization and cDNA amplification. Both adult and fetal human RPE cells contain mRNA for multiple G protein {alpha} subunits (G{alpha}) including G{sub s}{alpha}, G{sub i-1}{alpha}, G{sub i-2}{alpha}, G{sub i-3}{alpha}, and G{sub z}{alpha} (or G{sub x}{alpha}), where G{sub s} and G{sub i} are proteins that stimulate or inhibit adenylyl cyclase, respectively, and G{sub z} is a protein that may mediate pertussis toxin-insensitive events. Other G{alpha}-related mRNA transcripts were detected in fetal RPE cells by low-stringency hybridization to G{sub i-2}{alpha} and G{sub s}{alpha} protein-coding cDNA probes. The diversity of G proteins in RPE cells was further studied by cDNA amplification with reverse transcriptase and the polymerase chain reaction. This approach revealed that, besides the above mentioned members of the G{alpha} gene family, at least two other G{alpha} subunits are expressed in RPE cells. Human retinal cDNA clones that encode one of the additional G{alpha} subunits were isolated and characterized. The results indicate that this G{alpha} subunit belongs to a separate subfamily of G proteins that may be insensitive to inhibition by pertussis toxin.

  14. The selective phosphorylation of a guanine nucleotide-binding regulatory protein

    International Nuclear Information System (INIS)

    Carlson, K.E.

    1989-01-01

    Receptor-activated signal transduction pathways regulate the responsiveness of cells to external stimuli. These transduction pathways themselves are subject to regulation, most commonly by phosphorylation. Guanine nucleotide-binding regulatory proteins (G Proteins), as requisite signal transducing elements for many plasma membrane receptors, are considered likely targets for regulation by phosphorylation. Protein kinase C (PKC) has been shown to phosphorylate the α subunit of G i and other G proteins in solution. However, the occurrence of the phosphorylation of G 1 within intact cells in response to activation of PKC has not been rigorously demonstrated. In this thesis, the extent to which the α subunits of G i undergo phosphorylation within human platelets in response to activation of PKC was examined by means of radiolabeling and immunoprecipitation. Incubation of platelets with phorbol-12-myristate-13-acetate (PMA), a potent activator of PKC, promoted the phosphorylation of several proteins within saponin-permeabilized and intact platelets incubated with [γ 32 P]ATP and [ 32 P]H 3 PO 4 , respectively. None of the phosphoproteins, however, were precipitated by either of two antisera containing antibodies differing in specificities for epitopes within G iα -despite precipitation of a substantial fraction of the subunit itself. In contrast, other antisera, containing antibodies specific for the recently describe G zα , or antibodies for both G zα and G iα , precipitated a 40-kDa phosphoprotein

  15. Specific interactions between DNA and regulatory protein controlled by ligand-binding: Ab initio molecular simulation

    International Nuclear Information System (INIS)

    Matsushita, Y.; Murakawa, T.; Shimamura, K.; Oishi, M.; Ohyama, T.; Kurita, N.

    2015-01-01

    The catabolite activator protein (CAP) is one of the regulatory proteins controlling the transcription mechanism of gene. Biochemical experiments elucidated that the complex of CAP with cyclic AMP (cAMP) is indispensable for controlling the mechanism, while previous molecular simulations for the monomer of CAP+cAMP complex revealed the specific interactions between CAP and cAMP. However, the effect of cAMP-binding to CAP on the specific interactions between CAP and DNA is not elucidated at atomic and electronic levels. We here considered the ternary complex of CAP, cAMP and DNA in solvating water molecules and investigated the specific interactions between them at atomic and electronic levels using ab initio molecular simulations based on classical molecular dynamics and ab initio fragment molecular orbital methods. The results highlight the important amino acid residues of CAP for the interactions between CAP and cAMP and between CAP and DNA

  16. Specific interactions between DNA and regulatory protein controlled by ligand-binding: Ab initio molecular simulation

    Energy Technology Data Exchange (ETDEWEB)

    Matsushita, Y., E-mail: kurita@cs.tut.ac.jp; Murakawa, T., E-mail: kurita@cs.tut.ac.jp; Shimamura, K., E-mail: kurita@cs.tut.ac.jp; Oishi, M., E-mail: kurita@cs.tut.ac.jp; Ohyama, T., E-mail: kurita@cs.tut.ac.jp; Kurita, N., E-mail: kurita@cs.tut.ac.jp [Department of Computer Science and Engineering, Toyohashi University of Technology, Tempaku-cho, Toyohashi, Aichi, 441-8580 (Japan)

    2015-02-27

    The catabolite activator protein (CAP) is one of the regulatory proteins controlling the transcription mechanism of gene. Biochemical experiments elucidated that the complex of CAP with cyclic AMP (cAMP) is indispensable for controlling the mechanism, while previous molecular simulations for the monomer of CAP+cAMP complex revealed the specific interactions between CAP and cAMP. However, the effect of cAMP-binding to CAP on the specific interactions between CAP and DNA is not elucidated at atomic and electronic levels. We here considered the ternary complex of CAP, cAMP and DNA in solvating water molecules and investigated the specific interactions between them at atomic and electronic levels using ab initio molecular simulations based on classical molecular dynamics and ab initio fragment molecular orbital methods. The results highlight the important amino acid residues of CAP for the interactions between CAP and cAMP and between CAP and DNA.

  17. Discovery of a mammalian splice variant of myostatin that stimulates myogenesis.

    Directory of Open Access Journals (Sweden)

    Ferenc Jeanplong

    Full Text Available Myostatin plays a fundamental role in regulating the size of skeletal muscles. To date, only a single myostatin gene and no splice variants have been identified in mammals. Here we describe the splicing of a cryptic intron that removes the coding sequence for the receptor binding moiety of sheep myostatin. The deduced polypeptide sequence of the myostatin splice variant (MSV contains a 256 amino acid N-terminal domain, which is common to myostatin, and a unique C-terminus of 65 amino acids. Western immunoblotting demonstrated that MSV mRNA is translated into protein, which is present in skeletal muscles. To determine the biological role of MSV, we developed an MSV over-expressing C2C12 myoblast line and showed that it proliferated faster than that of the control line in association with an increased abundance of the CDK2/Cyclin E complex in the nucleus. Recombinant protein made for the novel C-terminus of MSV also stimulated myoblast proliferation and bound to myostatin with high affinity as determined by surface plasmon resonance assay. Therefore, we postulated that MSV functions as a binding protein and antagonist of myostatin. Consistent with our postulate, myostatin protein was co-immunoprecipitated from skeletal muscle extracts with an MSV-specific antibody. MSV over-expression in C2C12 myoblasts blocked myostatin-induced Smad2/3-dependent signaling, thereby confirming that MSV antagonizes the canonical myostatin pathway. Furthermore, MSV over-expression increased the abundance of MyoD, Myogenin and MRF4 proteins (P<0.05, which indicates that MSV stimulates myogenesis through the induction of myogenic regulatory factors. To help elucidate a possible role in vivo, we observed that MSV protein was more abundant during early post-natal muscle development, while myostatin remained unchanged, which suggests that MSV may promote the growth of skeletal muscles. We conclude that MSV represents a unique example of intra-genic regulation in which a

  18. RRM domain of Arabidopsis splicing factor SF1 is important for pre-mRNA splicing of a specific set of genes

    KAUST Repository

    Lee, Keh Chien; Jang, Yun Hee; Kim, SoonKap; Park, Hyo-Young; Thu, May Phyo; Lee, Jeong Hwan; Kim, Jeong-Kook

    2017-01-01

    , but not the abscisic acid sensitivity response during seed germination. The alternative splicing of FLOWERING LOCUS M (FLM) pre-mRNA is involved in flowering time control. We found that the RRM domain of AtSF1 protein alters the production of alternatively spliced FLM

  19. Evolution of context dependent regulation by expansion of feast/famine regulatory proteins.

    Science.gov (United States)

    Plaisier, Christopher L; Lo, Fang-Yin; Ashworth, Justin; Brooks, Aaron N; Beer, Karlyn D; Kaur, Amardeep; Pan, Min; Reiss, David J; Facciotti, Marc T; Baliga, Nitin S

    2014-11-14

    Expansion of transcription factors is believed to have played a crucial role in evolution of all organisms by enabling them to deal with dynamic environments and colonize new environments. We investigated how the expansion of the Feast/Famine Regulatory Protein (FFRP) or Lrp-like proteins into an eight-member family in Halobacterium salinarum NRC-1 has aided in niche-adaptation of this archaeon to a complex and dynamically changing hypersaline environment. We mapped genome-wide binding locations for all eight FFRPs, investigated their preference for binding different effector molecules, and identified the contexts in which they act by analyzing transcriptional responses across 35 growth conditions that mimic different environmental and nutritional conditions this organism is likely to encounter in the wild. Integrative analysis of these data constructed an FFRP regulatory network with conditionally active states that reveal how interrelated variations in DNA-binding domains, effector-molecule preferences, and binding sites in target gene promoters have tuned the functions of each FFRP to the environments in which they act. We demonstrate how conditional regulation of similar genes by two FFRPs, AsnC (an activator) and VNG1237C (a repressor), have striking environment-specific fitness consequences for oxidative stress management and growth, respectively. This study provides a systems perspective into the evolutionary process by which gene duplication within a transcription factor family contributes to environment-specific adaptation of an organism.

  20. CCAAT/enhancer-binding proteins regulate expression of the human steroidogenic acute regulatory protein (StAR) gene.

    Science.gov (United States)

    Christenson, L K; Johnson, P F; McAllister, J M; Strauss, J F

    1999-09-10

    Two putative CCAAT/enhancer-binding protein (C/EBP) response elements were identified in the proximal promoter of the human steroidogenic acute regulatory protein (StAR) gene, which encodes a key protein-regulating steroid hormone synthesis. Expression of C/EBPalpha and -beta increased StAR promoter activity in COS-1 and HepG2 cells. Cotransfection of C/EBPalpha or -beta and steroidogenic factor 1, a transcription factor required for cAMP regulation of StAR expression, into COS-1 augmented 8-bromoadenosine 3':5'-cyclic monophosphate (8-Br-cAMP)-stimulated promoter activity. When the putative C/EBP response elements were mutated, individually or together, a pronounced decline in basal StAR promoter activity in human granulosa-lutein cells resulted, but the fold stimulation of promoter activity by 8-Br-cAMP was unaffected. Recombinant C/EBPalpha and -beta bound to the two identified sequences but not the mutated elements. Human granulosa-lutein cell nuclear extracts also bound these elements but not the mutated sequences. An antibody to C/EBPbeta, but not C/EBPalpha, supershifted the nuclear protein complex associated with the more distal element. The complex formed by nuclear extracts with the proximal element was not supershifted by either antibody. Western blot analysis revealed the presence of C/EBPalpha and C/EBPbeta in human granulosa-lutein cell nuclear extracts. C/EBPbeta levels were up-regulated 3-fold by 8-Br-cAMP treatment. Our studies demonstrate a role for C/EBPbeta as well as yet to be identified proteins, which can bind to C/EBP response elements, in the regulation of StAR gene expression and suggest a mechanism by which C/EBPbeta participates in the cAMP regulation of StAR gene transcription.

  1. Investigation of tissue-specific human orthologous alternative splice events in pig

    DEFF Research Database (Denmark)

    Hillig, Ann-Britt Nygaard; Jørgensen, Claus Bøttcher; Salicio, Susanna Cirera

    2010-01-01

    Alternative splicing of pre-mRNA can contribute to differences between tissues or cells either by regulating gene expression or creating proteins with various functions encoded by one gene. The number of investigated alternative splice events in pig has so far been limited. In this study we have ...... in preservation of open reading frame are indicative of a functional significance of the splice variants of the gene....

  2. Mutual interdependence of splicing and transcription elongation.

    Science.gov (United States)

    Brzyżek, Grzegorz; Świeżewski, Szymon

    2015-01-01

    Transcription and splicing are intrinsically linked, as splicing needs a pre-mRNA substrate to commence. The more nuanced view is that the rate of transcription contributes to splicing regulation. On the other hand there is accumulating evidence that splicing has an active role in controlling transcription elongation by DNA-dependent RNA polymerase II (RNAP II). We briefly review those mechanisms and propose a unifying model where splicing controls transcription elongation to provide an optimal timing for successive rounds of splicing.

  3. Nuclear Magnetic Resonance structural studies of peptides and proteins from the vaso-regulatory System

    International Nuclear Information System (INIS)

    Sizun, Philippe

    1991-01-01

    The aim of the present work is to show how Nuclear Magnetic Resonance (NMR) allows to determine the 3D structure of peptides and proteins in solution. A comparative study of peptides involved in the vaso-regulatory System (form small hormonal peptide to the 65 amido-acid protein hirudin) has allowed to design most efficient NMR 1D and 2D strategies. It rapidly appeared that the size of the peptide plays a key role in the structuration of the molecule, smallest peptides being weakly structured owing to the lack of cooperative effects. As the molecular size increases or if conformational locks are present (disulfide bridges) the probability of stable secondary structure increases. For the protein hirudin, a combination of ail available NMR parameters deduced form dedicated experiments (chemical shifts, coupling constants, overhauser effects, accessibility of amide protons) and molecular modelling under constraints allows a clear 3D structure to be proposed for this protein in solution. Finally, a comparative study of the experimental structures and of those deduced form prediction rules has shed light on the concept of structural predisposition, the latter being of high value for a better understanding of structure-activity relationships. (author) [fr

  4. Mutations in complement regulatory proteins predispose to preeclampsia: a genetic analysis of the PROMISSE cohort.

    Directory of Open Access Journals (Sweden)

    Jane E Salmon

    2011-03-01

    Full Text Available Pregnancy in women with systemic lupus erythematosus (SLE or antiphospholipid antibodies (APL Ab--autoimmune conditions characterized by complement-mediated injury--is associated with increased risk of preeclampsia and miscarriage. Our previous studies in mice indicate that complement activation targeted to the placenta drives angiogenic imbalance and placental insufficiency.We use PROMISSE, a prospective study of 250 pregnant patients with SLE and/or APL Ab, to test the hypothesis in humans that impaired capacity to limit complement activation predisposes to preeclampsia. We sequenced genes encoding three complement regulatory proteins--membrane cofactor protein (MCP, complement factor I (CFI, and complement factor H (CFH--in 40 patients who had preeclampsia and found heterozygous mutations in seven (18%. Five of these patients had risk variants in MCP or CFI that were previously identified in atypical hemolytic uremic syndrome, a disease characterized by endothelial damage. One had a novel mutation in MCP that impairs regulation of C4b. These findings constitute, to our knowledge, the first genetic defects associated with preeclampsia in SLE and/or APL Ab. We confirmed the association of hypomorphic variants of MCP and CFI in a cohort of non-autoimmune preeclampsia patients in which five of 59 were heterozygous for mutations.The presence of risk variants in complement regulatory proteins in patients with SLE and/or APL Ab who develop preeclampsia, as well as in preeclampsia patients lacking autoimmune disease, links complement activation to disease pathogenesis and suggests new targets for treatment of this important public health problem.ClinicalTrials.gov NCT00198068.

  5. DNA-binding site of major regulatory protein alpha 4 specifically associated with promoter-regulatory domains of alpha genes of herpes simplex virus type 1.

    OpenAIRE

    Kristie, T M; Roizman, B

    1986-01-01

    Herpes simplex virus type 1 genes form at least five groups (alpha, beta 1, beta 2, gamma 1, and gamma 2) whose expression is coordinately regulated and sequentially ordered in a cascade fashion. Previous studies have shown that functional alpha 4 gene product is essential for the transition from alpha to beta protein synthesis and have suggested that alpha 4 gene expression is autoregulatory. We have previously reported that labeled DNA fragments containing promoter-regulatory domains of thr...

  6. RRM domain of Arabidopsis splicing factor SF1 is important for pre-mRNA splicing of a specific set of genes

    KAUST Repository

    Lee, Keh Chien

    2017-04-11

    The RNA recognition motif of Arabidopsis splicing factor SF1 affects the alternative splicing of FLOWERING LOCUS M pre-mRNA and a heat shock transcription factor HsfA2 pre-mRNA. Splicing factor 1 (SF1) plays a crucial role in 3\\' splice site recognition by binding directly to the intron branch point. Although plant SF1 proteins possess an RNA recognition motif (RRM) domain that is absent in its fungal and metazoan counterparts, the role of the RRM domain in SF1 function has not been characterized. Here, we show that the RRM domain differentially affects the full function of the Arabidopsis thaliana AtSF1 protein under different experimental conditions. For example, the deletion of RRM domain influences AtSF1-mediated control of flowering time, but not the abscisic acid sensitivity response during seed germination. The alternative splicing of FLOWERING LOCUS M (FLM) pre-mRNA is involved in flowering time control. We found that the RRM domain of AtSF1 protein alters the production of alternatively spliced FLM-β transcripts. We also found that the RRM domain affects the alternative splicing of a heat shock transcription factor HsfA2 pre-mRNA, thereby mediating the heat stress response. Taken together, our results suggest the importance of RRM domain for AtSF1-mediated alternative splicing of a subset of genes involved in the regulation of flowering and adaptation to heat stress.

  7. Two new splice variants in porcine PPARGC1A

    Directory of Open Access Journals (Sweden)

    Peelman Luc J

    2008-12-01

    Full Text Available Abstract Background Peroxisome proliferator-activated receptor γ coactivator 1α (PPARGC1A is a coactivator with a vital and central role in fat and energy metabolism. It is considered to be a candidate gene for meat quality in pigs and is involved in the development of obesity and diabetes in humans. How its many functions are regulated, is however still largely unclear. Therefore a transcription profile of PPARGC1A in 32 tissues and 4 embryonic developmental stages in the pig was constructed by screening its cDNA for possible splice variants with exon-spanning primers. Findings This led to the discovery of 2 new splice variants in the pig, which were subsequently also detected in human tissues. In these variants, exon 8 was either completely or partly (the last 66 bp were conserved spliced out, potentially coding for a much shorter protein of respectively 337 and 359 amino acids (aa, of which the first 291 aa would be the same compared to the complete protein (796 aa. Conclusion Considering the functional domains of the PPARGC1A protein, it is very likely these splice variants considerably affect the function of the protein and alternative splicing could be one of the mechanisms by which the diverse functions of PPARGC1A are regulated.

  8. A method of predicting changes in human gene splicing induced by genetic variants in context of cis-acting elements

    Directory of Open Access Journals (Sweden)

    Hicks Chindo

    2010-01-01

    Full Text Available Abstract Background Polymorphic variants and mutations disrupting canonical splicing isoforms are among the leading causes of human hereditary disorders. While there is a substantial evidence of aberrant splicing causing Mendelian diseases, the implication of such events in multi-genic disorders is yet to be well understood. We have developed a new tool (SpliceScan II for predicting the effects of genetic variants on splicing and cis-regulatory elements. The novel Bayesian non-canonical 5'GC splice site (SS sensor used in our tool allows inference on non-canonical exons. Results Our tool performed favorably when compared with the existing methods in the context of genes linked to the Autism Spectrum Disorder (ASD. SpliceScan II was able to predict more aberrant splicing isoforms triggered by the mutations, as documented in DBASS5 and DBASS3 aberrant splicing databases, than other existing methods. Detrimental effects behind some of the polymorphic variations previously associated with Alzheimer's and breast cancer could be explained by changes in predicted splicing patterns. Conclusions We have developed SpliceScan II, an effective and sensitive tool for predicting the detrimental effects of genomic variants on splicing leading to Mendelian and complex hereditary disorders. The method could potentially be used to screen resequenced patient DNA to identify de novo mutations and polymorphic variants that could contribute to a genetic disorder.

  9. Body Temperature Cycles Control Rhythmic Alternative Splicing in Mammals.

    Science.gov (United States)

    Preußner, Marco; Goldammer, Gesine; Neumann, Alexander; Haltenhof, Tom; Rautenstrauch, Pia; Müller-McNicoll, Michaela; Heyd, Florian

    2017-08-03

    The core body temperature of all mammals oscillates with the time of the day. However, direct molecular consequences of small, physiological changes in body temperature remain largely elusive. Here we show that body temperature cycles drive rhythmic SR protein phosphorylation to control an alternative splicing (AS) program. A temperature change of 1°C is sufficient to induce a concerted splicing switch in a large group of functionally related genes, rendering this splicing-based thermometer much more sensitive than previously described temperature-sensing mechanisms. AS of two exons in the 5' UTR of the TATA-box binding protein (Tbp) highlights the general impact of this mechanism, as it results in rhythmic TBP protein levels with implications for global gene expression in vivo. Together our data establish body temperature-driven AS as a core clock-independent oscillator in mammalian peripheral clocks. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Cell-cycle-dependent three-dimensional redistribution of nuclear proteins, P 120, pKi-67, and SC 35 splicing factor, in the presence of the topoisomerase I inhibitor camptothecin.

    Science.gov (United States)

    Elias, Emmanuel; Lalun, Nathalie; Lorenzato, Marianne; Blache, Laurent; Chelidze, Pavel; O'Donohue, Marie-Françoise; Ploton, Dominique; Bobichon, Hélène

    2003-11-15

    Topoisomerase I (Topo I) is mostly known for its role in DNA relaxation, which is required for duplication and transcription. Topo I acts as a protein kinase mainly directed to the mRNA splicing factor SC35. Camptothecin is one of the specific Topo I inhibitors and is effective on the two functions of the enzyme. In this study we demonstrated that treatment of KB cells with camptothecin for only 30 min induced the 3D reorganization and redistribution of three proteins involved in the nucleus machinery, P 120, pKi-67, and SC 35, and this occurred in a cell cycle-dependent manner. Our data were obtained from confocal microscopic studies after immunolabeling, 3D reconstruction, and measurement of the nuclear components volumes. In the presence of camptothecin, P 120, which occupied the nucleolar volume, lost its reticulation and pKi-67 was redistributed within the nucleoplasm and even into the cytoplasm. Finally, for SC 35 the fusion of its dots into bigger volumes was observed specifically during the G1 phase. Variations of volumes were also observed for the nucleolus and for the nucleus. These results pointed out that, depending on the cell cycle phase, Topo I functions were selective toward the three different proteins.

  11. Evidence for roles of the Escherichia coli Hda protein beyond regulatory inactivation of DnaA.

    Science.gov (United States)

    Baxter, Jamie C; Sutton, Mark D

    2012-08-01

    The ATP-bound form of the Escherichia coli DnaA protein binds 'DnaA boxes' present in the origin of replication (oriC) and operator sites of several genes, including dnaA, to co-ordinate their transcription with initiation of replication. The Hda protein, together with the β sliding clamp, stimulates the ATPase activity of DnaA via a process termed regulatory inactivation of DnaA (RIDA), to regulate the activity of DnaA in DNA replication. Here, we used the mutant dnaN159 strain, which expresses the β159 clamp protein, to gain insight into how the actions of Hda are co-ordinated with replication. Elevated expression of Hda impeded growth of the dnaN159 strain in a Pol II- and Pol IV-dependent manner, suggesting a role for Hda managing the actions of these Pols. In a wild-type strain, elevated levels of Hda conferred sensitivity to nitrofurazone, and suppressed the frequency of -1 frameshift mutations characteristic of Pol IV, while loss of hda conferred cold sensitivity. Using the dnaN159 strain, we identified 24 novel hda alleles, four of which supported E. coli viability despite their RIDA defect. Taken together, these findings suggest that although one or more Hda functions are essential for cell viability, RIDA may be dispensable. © 2012 Blackwell Publishing Ltd.

  12. Systematic comparison of the response properties of protein and RNA mediated gene regulatory motifs.

    Science.gov (United States)

    Iyengar, Bharat Ravi; Pillai, Beena; Venkatesh, K V; Gadgil, Chetan J

    2017-05-30

    We present a framework enabling the dissection of the effects of motif structure (feedback or feedforward), the nature of the controller (RNA or protein), and the regulation mode (transcriptional, post-transcriptional or translational) on the response to a step change in the input. We have used a common model framework for gene expression where both motif structures have an activating input and repressing regulator, with the same set of parameters, to enable a comparison of the responses. We studied the global sensitivity of the system properties, such as steady-state gain, overshoot, peak time, and peak duration, to parameters. We find that, in all motifs, overshoot correlated negatively whereas peak duration varied concavely with peak time. Differences in the other system properties were found to be mainly dependent on the nature of the controller rather than the motif structure. Protein mediated motifs showed a higher degree of adaptation i.e. a tendency to return to baseline levels; in particular, feedforward motifs exhibited perfect adaptation. RNA mediated motifs had a mild regulatory effect; they also exhibited a lower peaking tendency and mean overshoot. Protein mediated feedforward motifs showed higher overshoot and lower peak time compared to the corresponding feedback motifs.

  13. Expression of the cytoskeleton regulatory protein Mena in human gastric carcinoma and its prognostic significance.

    Science.gov (United States)

    Xu, Lihua; Tan, Huo; Liu, Ruiming; Huang, Qungai; Zhang, Nana; Li, Xi; Wang, Jiani

    2017-11-01

    The cytoskeleton regulatory protein Mena is reportedly overexpressed in breast cancer; however, data regarding its expression level and clinical significance in gastric carcinoma (GC) is limited. The aim of the present study was to investigate Mena expression levels and prognostic significance in GC. Mena mRNA expression level was determined by reverse transcription-quantitative polymerase chain reaction in 10 paired GC and adjacent normal tissues. The Mena protein expression level was analyzed in paraffin-embedded GC samples and adjacent normal tissues by immunohistochemistry. Statistical analyses were also performed to evaluate the clinicopathological significance of Mena. The results revealed that the mRNA expression level of Mena was significantly higher in G Ct issues compared with in adjacent normal tissues from10 paired samples. In the paraffin-embedded tissue samples, the protein expression level of Mena was higher in G Ct issues compared with in adjacent normal tissues. Compared with adjacent normal tissues, Mena overexpression was observed in 52.83% (56/106) of patients. The overexpression of Mena was significantly associated with the T stage (P=0.033), tumor-node-metastasis (TNM) stage (PMena expression level was an independent prognostic factor for overall survival time. In conclusion, Mena wasoverexpressed in G C tissues and significantly associated with the T stage, TNM stage and overall survival time. Mena may therefore be suitable as a prognostic indicator for patients with GC.

  14. Properties of Sequence Conservation in Upstream Regulatory and Protein Coding Sequences among Paralogs in Arabidopsis thaliana

    Science.gov (United States)

    Richardson, Dale N.; Wiehe, Thomas

    Whole genome duplication (WGD) has catalyzed the formation of new species, genes with novel functions, altered expression patterns, complexified signaling pathways and has provided organisms a level of genetic robustness. We studied the long-term evolution and interrelationships of 5’ upstream regulatory sequences (URSs), protein coding sequences (CDSs) and expression correlations (EC) of duplicated gene pairs in Arabidopsis. Three distinct methods revealed significant evolutionary conservation between paralogous URSs and were highly correlated with microarray-based expression correlation of the respective gene pairs. Positional information on exact matches between sequences unveiled the contribution of micro-chromosomal rearrangements on expression divergence. A three-way rank analysis of URS similarity, CDS divergence and EC uncovered specific gene functional biases. Transcription factor activity was associated with gene pairs exhibiting conserved URSs and divergent CDSs, whereas a broad array of metabolic enzymes was found to be associated with gene pairs showing diverged URSs but conserved CDSs.

  15. Expression profiling of cell cycle regulatory proteins in oropharyngeal carcinomas using tissue microarrays.

    Science.gov (United States)

    Ribeiro, Daniel A; Nascimento, Fabio D; Fracalossi, Ana Carolina C; Gomes, Thiago S; Oshima, Celina T F; Franco, Marcello F

    2010-01-01

    The aim of this study was to investigate the expressions of cell cycle regulatory proteins such as p53, p16, p21, and Rb in squamous cell carcinoma of the oropharynx and their relation to histological differentiation, staging of disease, and prognosis. Paraffin blocks from 21 primary tumors were obtained from archives of the Department of Pathology, Paulista Medical School, Federal University of Sao Paulo, UNIFESP/EPM. Immunohistochemistry was used to detect the expression of p53, p16, p21, and Rb by means of tissue microarrays. Expression of p53, p21, p16 and Rb was not correlated with the stage of disease, histopathological grading or recurrence in squamous cell carcinoma of the oropharynx. Taken together, our results suggest that p53, p16, p21 and Rb are not reliable biomarkers for prognosis of the tumor severity or recurrence in squamous cell carcinoma of the oropharynx as depicted by tissue microarrays and immunohistochemistry.

  16. The Role of Alternative Splicing in the Control of Immune Homeostasis and Cellular Differentiation.

    Science.gov (United States)

    Yabas, Mehmet; Elliott, Hannah; Hoyne, Gerard F

    2015-12-22

    Alternative splicing of pre-mRNA helps to enhance the genetic diversity within mammalian cells by increasing the number of protein isoforms that can be generated from one gene product. This provides a great deal of flexibility to the host cell to alter protein function, but when dysregulation in splicing occurs this can have important impact on health and disease. Alternative splicing is widely used in the mammalian immune system to control the development and function of antigen specific lymphocytes. In this review we will examine the splicing of pre-mRNAs yielding key proteins in the immune system that regulate apoptosis, lymphocyte differentiation, activation and homeostasis, and discuss how defects in splicing can contribute to diseases. We will describe how disruption to trans-acting factors, such as heterogeneous nuclear ribonucleoproteins (hnRNPs), can impact on cell survival and differentiation in the immune system.

  17. Differentially regulated splice variants and systems biology analysis of Kaposi's sarcoma-associated herpesvirus-infected lymphatic endothelial cells.

    Science.gov (United States)

    Chang, Ting-Yu; Wu, Yu-Hsuan; Cheng, Cheng-Chung; Wang, Hsei-Wei

    2011-09-01

    Alternative RNA splicing greatly increases proteome diversity, and the possibility of studying genome-wide alternative splicing (AS) events becomes available with the advent of high-throughput genomics tools devoted to this issue. Kaposi's sarcoma associated herpesvirus (KSHV) is the etiological agent of KS, a tumor of lymphatic endothelial cell (LEC) lineage, but little is known about the AS variations induced by KSHV. We analyzed KSHV-controlled AS using high-density microarrays capable of detecting all exons in the human genome. Splicing variants and altered exon-intron usage in infected LEC were found, and these correlated with protein domain modification. The different 3'-UTR used in new transcripts also help isoforms to escape microRNA-mediated surveillance. Exome-level analysis further revealed information that cannot be disclosed using classical gene-level profiling: a significant exon usage difference existed between LEC and CD34(+) precursor cells, and KSHV infection resulted in LEC-to-precursor, dedifferentiation-like exon level reprogramming. Our results demonstrate the application of exon arrays in systems biology research, and suggest the regulatory effects of AS in endothelial cells are far more complex than previously observed. This extra layer of molecular diversity helps to account for various aspects of endothelial biology, KSHV life cycle and disease pathogenesis that until now have been unexplored.

  18. The multifaceted activity of the VirF regulatory protein in the Shigella lifestyle

    Directory of Open Access Journals (Sweden)

    Maria Letizia Di Martino

    2016-09-01

    Full Text Available Shigella is a highly adapted human pathogen, mainly found in the developing world and causing a severe enteric syndrome. The highly sophisticated infectious strategy of Shigella banks on the capacity to invade the intestinal epithelial barrier and cause its inflammatory destruction. The cellular pathogenesis and clinical presentation of shigellosis are the sum of the complex action of a large number of bacterial virulence factors mainly located on a large virulence plasmid (pINV. The expression of pINV genes is controlled by multiple environmental stimuli through a regulatory cascade involving proteins and sRNAs encoded by both the pINV and the chromosome. The primary regulator of the virulence phenotype is VirF, a DNA-binding protein belonging to the AraC family of transcriptional regulators. The virF gene, located on the pINV, is expressed only within the host, mainly in response to the temperature transition occurring when the bacterium transits from the outer environment to the intestinal milieu. VirF then acts as anti-H-NS protein and directly activates the icsA and virB genes, triggering the full expression of the invasion program of Shigella. In this review we will focus on the structure of VirF, on its sophisticated regulation, and on its role as major player in the path leading from the non invasive to the invasive phenotype of Shigella. We will address also the involvement of VirF in mechanisms aimed at withstanding adverse conditions inside the host, indicating that this protein is emerging as a global regulator whose action is not limited to virulence systems. Finally, we will discuss recent observations conferring VirF the potential of a novel antibacterial target for shigellosis.

  19. Sterol regulatory element binding protein-1 (SREBP1) gene expression is similarly increased in polycystic ovary syndrome and endometrial cancer.

    Science.gov (United States)

    Shafiee, Mohamad N; Mongan, Nigel; Seedhouse, Claire; Chapman, Caroline; Deen, Suha; Abu, Jafaru; Atiomo, William

    2017-05-01

    Women with polycystic ovary syndrome have a three-fold higher risk of endometrial cancer. Insulin resistance and hyperlipidemia may be pertinent factors in the pathogenesis of both conditions. The aim of this study was to investigate endometrial sterol regulatory element binding protein-1 gene expression in polycystic ovary syndrome and endometrial cancer endometrium, and to correlate endometrial sterol regulatory element binding protein-1 gene expression with serum lipid profiles. A cross-sectional study was performed at Nottingham University Hospital, UK. A total of 102 women (polycystic ovary syndrome, endometrial cancer and controls; 34 participants in each group) were recruited. Clinical and biochemical assessments were performed before endometrial biopsies were obtained from all participants. Taqman real-time polymerase chain reaction for endometrial sterol regulatory element binding protein-1 gene and its systemic protein expression were analyzed. The body mass indices of women with polycystic ovary syndrome (29.28 ± 2.91 kg/m 2 ) and controls (28.58 ± 2.62 kg/m 2 ) were not significantly different. Women with endometrial cancer had a higher mean body mass index (32.22 ± 5.70 kg/m 2 ). Sterol regulatory element binding protein-1 gene expression was significantly increased in polycystic ovary syndrome and endometrial cancer endometrium compared with controls (p ovary syndrome, but this was not statistically significant. Similarly, statistically insignificant positive correlations were found between endometrial sterol regulatory element binding protein-1 gene expression and body mass index in endometrial cancer (r = 0.643, p = 0.06) and waist-hip ratio (r = 0.096, p = 0.073). Sterol regulatory element binding protein-1 gene expression was significantly positively correlated with triglyceride in both polycystic ovary syndrome and endometrial cancer (p = 0.028 and p = 0.027, respectively). Quantitative serum sterol regulatory element

  20. Identification and analysis of Eimeria nieschulzi gametocyte genes reveal splicing events of gam genes and conserved motifs in the wall-forming proteins within the genus Eimeria (Coccidia, Apicomplexa

    Directory of Open Access Journals (Sweden)

    Wiedmer Stefanie

    2017-01-01

    Full Text Available The genus Eimeria (Apicomplexa, Coccidia provides a wide range of different species with different hosts to study common and variable features within the genus and its species. A common characteristic of all known Eimeria species is the oocyst, the infectious stage where its life cycle starts and ends. In our study, we utilized Eimeria nieschulzi as a model organism. This rat-specific parasite has complex oocyst morphology and can be transfected and even cultivated in vitro up to the oocyst stage. We wanted to elucidate how the known oocyst wall-forming proteins are preserved in this rodent Eimeria species compared to other Eimeria. In newly obtained genomics data, we were able to identify different gametocyte genes that are orthologous to already known gam genes involved in the oocyst wall formation of avian Eimeria species. These genes appeared putatively as single exon genes, but cDNA analysis showed alternative splicing events in the transcripts. The analysis of the translated sequence revealed different conserved motifs but also dissimilar regions in GAM proteins, as well as polymorphic regions. The occurrence of an underrepresented gam56 gene version suggests the existence of a second distinct E. nieschulzi genotype within the E. nieschulzi Landers isolate that we maintain.

  1. Identification and analysis of Eimeria nieschulzi gametocyte genes reveal splicing events of gam genes and conserved motifs in the wall-forming proteins within the genus Eimeria (Coccidia, Apicomplexa)

    Science.gov (United States)

    Wiedmer, Stefanie; Erdbeer, Alexander; Volke, Beate; Randel, Stephanie; Kapplusch, Franz; Hanig, Sacha; Kurth, Michael

    2017-01-01

    The genus Eimeria (Apicomplexa, Coccidia) provides a wide range of different species with different hosts to study common and variable features within the genus and its species. A common characteristic of all known Eimeria species is the oocyst, the infectious stage where its life cycle starts and ends. In our study, we utilized Eimeria nieschulzi as a model organism. This rat-specific parasite has complex oocyst morphology and can be transfected and even cultivated in vitro up to the oocyst stage. We wanted to elucidate how the known oocyst wall-forming proteins are preserved in this rodent Eimeria species compared to other Eimeria. In newly obtained genomics data, we were able to identify different gametocyte genes that are orthologous to already known gam genes involved in the oocyst wall formation of avian Eimeria species. These genes appeared putatively as single exon genes, but cDNA analysis showed alternative splicing events in the transcripts. The analysis of the translated sequence revealed different conserved motifs but also dissimilar regions in GAM proteins, as well as polymorphic regions. The occurrence of an underrepresented gam56 gene version suggests the existence of a second distinct E. nieschulzi genotype within the E. nieschulzi Landers isolate that we maintain. PMID:29210668

  2. Distinct forms of the β subunit of GTP-binding regulatory proteins identified by molecular cloning

    International Nuclear Information System (INIS)

    Fong, H.K.W.; Amatruda, T.T. III; Birren, B.W.; Simon, M.I.

    1987-01-01

    Two distinct β subunits of guanine nucleotide-binding regulatory proteins have been identified by cDNA cloning and are referred to as β 1 and β 1 subunits. The bovine transducin β subunit (β 1 ) has been cloned previously. The author now isolated and analyzed cDNA clones that encode the β 2 subunit from bovine adrenal, bovine brain, and a human myeloid leukemia cell line, HL-60. The 340-residue M/sub r/ 37,329 Β 2 protein is 90% identical with β 1 in predicted amino acid sequence, and it is also organized as a series of repetitive homologous segments. The major mRNA that encodes the bovine β 2 subunit is 1.7 kilobases in length. It is expressed at lower levels than β 1 subunit mRNA in all tissues examined. The β 1 and β 2 messages are expressed in cloned human cell lines. Hybridization of cDNA probes to bovine DNA showed that β 1 and β 2 are encoded by separate genes. The amino acid sequences for the bovine and human β 2 subunit are identical, as are the amino acid sequences for the bovine and human β 1 subunit. This evolutionary conservation suggests that the two β subunits have different roles in the signal transduction process

  3. Casein kinase 1 regulates sterol regulatory element-binding protein (SREBP) to control sterol homeostasis.

    Science.gov (United States)

    Brookheart, Rita T; Lee, Chih-Yung S; Espenshade, Peter J

    2014-01-31

    Sterol homeostasis is tightly controlled by the sterol regulatory element-binding protein (SREBP) transcription factor that is highly conserved from fungi to mammals. In fission yeast, SREBP functions in an oxygen-sensing pathway to promote adaptation to decreased oxygen supply that limits oxygen-dependent sterol synthesis. Low oxygen stimulates proteolytic cleavage of the SREBP homolog Sre1, generating the active transcription factor Sre1N that drives expression of sterol biosynthetic enzymes. In addition, low oxygen increases the stability and DNA binding activity of Sre1N. To identify additional signals controlling Sre1 activity, we conducted a genetic overexpression screen. Here, we describe our isolation and characterization of the casein kinase 1 family member Hhp2 as a novel regulator of Sre1N. Deletion of Hhp2 increases Sre1N protein stability and ergosterol levels in the presence of oxygen. Hhp2-dependent Sre1N degradation by the proteasome requires Hhp2 kinase activity, and Hhp2 binds and phosphorylates Sre1N at specific residues. Our results describe a role for casein kinase 1 as a direct regulator of sterol homeostasis. Given the role of mammalian Hhp2 homologs, casein kinase 1δ and 1ε, in regulation of the circadian clock, these findings may provide a mechanism for coordinating circadian rhythm and lipid metabolism.

  4. COMK ENCODES THE COMPETENCE TRANSCRIPTION FACTOR, THE KEY REGULATORY PROTEIN FOR COMPETENCE DEVELOPMENT IN BACILLUS-SUBTILIS

    NARCIS (Netherlands)

    VANSINDEREN, D; LUTTINGER, A; KONG, LY; DUBNAU, D; VENEMA, G; HAMOEN, L

    comK is a positive autoregulatory gene occupying a central position in the com petence-signal-transduction network. All regulatory routes identified in this network converge at the level of comK expression. The ComK protein is required for the transcriptional induction of comK and the late

  5. Crystallization and quaternary structure analysis of an Lrp-like regulatory protein from the hyperthermophile Pyrococcus furiosus

    NARCIS (Netherlands)

    Sedelnikova, S.E.; Smits, S.H.J.; Leonard, P.M.; Brinkman, A.B.; Oost, van der J.; Rafferty, J.B.

    2001-01-01

    The LrpA transcriptional regulator from Pyrococcus furiosus, a member of the leucine-responsive regulatory protein (Lrp) family, has been crystallized by the hanging-drop method of vapour diffusion using ammonium sulfate as the precipitant. The crystals belong to the tetragonal system and are in

  6. The regulatory beta-subunit of protein kinase CK2 regulates cell-cycle progression at the onset of mitosis

    DEFF Research Database (Denmark)

    Yde, C W; Olsen, B B; Meek, D

    2008-01-01

    25 dual-specificity phosphatase family members. In somatic cells, Wee1 is downregulated by phosphorylation and ubiquitin-mediated degradation to ensure rapid activation of CDK1 at the beginning of M phase. Here, we show that downregulation of the regulatory beta-subunit of protein kinase CK2 by RNA...

  7. Epitopes of human immunodeficiency virus regulatory proteins tat, nef, and rev are expressed in normal human tissue

    NARCIS (Netherlands)

    Parmentier, H. K.; van Wichen, D. F.; Meyling, F. H.; Goudsmit, J.; Schuurman, H. J.

    1992-01-01

    The expression of regulatory proteins tat, rev, and nef of human immunodeficiency virus type-1 (HIV-1) and tat of HIV-2 was studied in frozen sections of lymph nodes from HIV-1-infected individuals, and various tissues from uninfected persons. In HIV-1-positive lymph nodes, monoclonal antibodies to

  8. Adaptation of Tri-molecular fluorescence complementation allows assaying of regulatory Csr RNA-protein interactions in bacteria.

    Science.gov (United States)

    Gelderman, Grant; Sivakumar, Anusha; Lipp, Sarah; Contreras, Lydia

    2015-02-01

    sRNAs play a significant role in controlling and regulating cellular metabolism. One of the more interesting aspects of certain sRNAs is their ability to make global changes in the cell by interacting with regulatory proteins. In this work, we demonstrate the use of an in vivo Tri-molecular Fluorescence Complementation assay to detect and visualize the central regulatory sRNA-protein interaction of the Carbon Storage Regulatory system in E. coli. The Carbon Storage Regulator consists primarily of an RNA binding protein, CsrA, that alters the activity of mRNA targets and of an sRNA, CsrB, that modulates the activity of CsrA. We describe the construction of a fluorescence complementation system that detects the interactions between CsrB and CsrA. Additionally, we demonstrate that the intensity of the fluorescence of this system is able to detect changes in the affinity of the CsrB-CsrA interaction, as caused by mutations in the protein sequence of CsrA. While previous methods have adopted this technique to study mRNA or RNA localization, this is the first attempt to use this technique to study the sRNA-protein interaction directly in bacteria. This method presents a potentially powerful tool to study complex bacterial RNA protein interactions in vivo. © 2014 Wiley Periodicals, Inc.

  9. Nitric oxide-mediated modulation of iron regulatory proteins: implication for cellular iron homeostasis.

    Science.gov (United States)

    Kim, Sangwon; Ponka, Prem

    2002-01-01

    Iron regulatory proteins (IRP1 and IRP2) control the synthesis of transferrin receptors (TfR) and ferritin by binding to iron-responsive elements (IREs) that are located in the 3' untranslated region (UTR) and the 5' UTR of their respective mRNAs. Cellular iron levels affect binding of IRPs to IREs and consequently expression of TfR and ferritin. Moreover, NO(.), a redox species of nitric oxide that interacts primarily with iron, can activate IRP1 RNA-binding activity resulting in an increase in TfR mRNA levels and a decrease in ferritin synthesis. We have shown that treatment of RAW 264.7 cells (a murine macrophage cell line) with NO(+) (nitrosonium ion, which causes S-nitrosylation of thiol groups) resulted in a rapid decrease in RNA-binding of IRP2, followed by IRP2 degradation, and these changes were associated with a decrease in TfR mRNA levels and a dramatic increase in ferritin synthesis. Moreover, we demonstrated that stimulation of RAW 264.7 cells with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) increased IRP1 binding activity, whereas RNA-binding of IRP2 decreased and was followed by a degradation of this protein. Furthermore, the decrease of IRP2 binding/protein levels was associated with a decrease in TfR mRNA levels and an increase in ferritin synthesis in LPS/IFN-gamma-treated cells, and these changes were prevented by inhibitors of inducible nitric oxide synthase. These results suggest that NO(+)-mediated degradation of IRP2 plays a major role in iron metabolism during inflammation.

  10. Changes in regulatory molecules for lymphangiogenesis in intestinal lymphangiectasia with enteric protein loss.

    Science.gov (United States)

    Hokari, Ryota; Kitagawa, Noritake; Watanabe, Chikako; Komoto, Shunsuke; Kurihara, Chie; Okada, Yoshikiyo; Kawaguchi, Atsushi; Nagao, Shigeaki; Hibi, Toshifumi; Miura, Soichiro

    2008-07-01

    Vascular endothelial growth factor receptor 3 (VEGFR3) and LYVE-1 are specifically expressed in the endothelium of the lymphatic systems. VEGF-C, D, FOXC2, Prox 1, and SOX18 are known to play central roles in lymphatic development. We investigated the expression of regulatory molecules for lymphangiogenesis in the duodenal mucosa of idiopathic intestinal lymphangiectasia. Biopsy samples were obtained from duodenal biopsies in patients with intestinal lymphangiectasia complicated with protein-losing from white spot lesions in which lymphangiectasia was histologically confirmed. Immunohistochemical analysis for VEGFR3 and LYVE-1 was performed. mRNA expression of VEGF-C, VEGF-D, VEGFR3, and transcription factors was determined by the quantitative reverse transcription-polymerase chain reaction method. In the control mucosa, VEGFR3 was weakly expressed on the central lymphatic vessels in the lamina propria and LYVE-1 was expressed mainly on the lymphatic vessels in the submucosa. In intestinal lymphangiectasia, VEGFR3 and LYVE-1 expression levels were increased on the mucosal surface corresponding to widely dilated lymphatic vessels, while they were decreased in the deeper mucosa. mRNA expression study showed a significant increase in the expression level of VEGFR3 in lymphangiectasia, but the expression of VEGF-C and -D mRNA was significantly suppressed compared with that in controls despite the presence of lymphangiectasia. The mRNA expression levels of FOXC2 and SOX18 were also decreased, whereas Prox 1 was not altered. There is an altered expression of regulatory molecules for lymphangiogenesis in the duodenal mucosa in these patients.

  11. Overproduction of lactimidomycin by cross-overexpression of genes encoding Streptomyces antibiotic regulatory proteins.

    Science.gov (United States)

    Zhang, Bo; Yang, Dong; Yan, Yijun; Pan, Guohui; Xiang, Wensheng; Shen, Ben

    2016-03-01

    The glutarimide-containing polyketides represent a fascinating class of natural products that exhibit a multitude of biological activities. We have recently cloned and sequenced the biosynthetic gene clusters for three members of the glutarimide-containing polyketides-iso-migrastatin (iso-MGS) from Streptomyces platensis NRRL 18993, lactimidomycin (LTM) from Streptomyces amphibiosporus ATCC 53964, and cycloheximide (CHX) from Streptomyces sp. YIM56141. Comparative analysis of the three clusters identified mgsA and chxA, from the mgs and chx gene clusters, respectively, that were predicted to encode the PimR-like Streptomyces antibiotic regulatory proteins (SARPs) but failed to reveal any regulatory gene from the ltm gene cluster. Overexpression of mgsA or chxA in S. platensis NRRL 18993, Streptomyces sp. YIM56141 or SB11024, and a recombinant strain of Streptomyces coelicolor M145 carrying the intact mgs gene cluster has no significant effect on iso-MGS or CHX production, suggesting that MgsA or ChxA regulation may not be rate-limiting for iso-MGS and CHX production in these producers. In contrast, overexpression of mgsA or chxA in S. amphibiosporus ATCC 53964 resulted in a significant increase in LTM production, with LTM titer reaching 106 mg/L, which is five-fold higher than that of the wild-type strain. These results support MgsA and ChxA as members of the SARP family of positive regulators for the iso-MGS and CHX biosynthetic machinery and demonstrate the feasibility to improve glutarimide-containing polyketide production in Streptomyces strains by exploiting common regulators.

  12. TGFβ1-mediated expression and alternative splicing of Fibronectin Extra Domain A in human podocyte culture.

    Science.gov (United States)

    Madne, Tarunkumar Hemraj; Dockrell, Mark Edward Carl

    2018-02-28

    Alternative splicing is a fundamental phenomenon to build protein diversity in health and diseases. Extra Domain A+ Fibronectin (EDA+Fn) is an alternatively spliced form of fibronectin protein present in the extra cellular matrix (ECM) in renal fibrosis. Podocytes are spectacular cell type and play a key role in filtration and synthesise ECM proteins in renal physiology and pathology. TGFβ1 is a strong stimulator of ECM proteins in renal injury. In this study, we have investigated alternative splicing of EDA+ Fn in human podocytes in response to TGFβ1. We have performed western blotting and immunofluorescence to characterise the expression of the EDA+Fn protein, real-time PCR for RNA expression and RT-PCR to look for alternative splicing of EDA+Fn in conditionally immortalised human podocytes culture.We used TGFβ1 as a stimulator and SB431542 and SRPIN340 for inhibitory studies. In this work, for the first time we have demonstrated in human podocytes culture EDA+Fn is expressed in the basal condition and TGFβ1 2.5ng/ml induced the Fn mRNA and EDA+Fn protein expression demonstrated by real-time PCR, western blotting and immunofluorescence. TGFβ1 2.5ng/ml induced the alternative splicing of EDA+Fn shown by conventional RT-PCR. Studies with ALK5 inhibitor SB431542 and SRPIN340 show that TGFβ1 induced alternative splicing of EDA+Fn was by the ALK5 receptor and the SR proteins.  In human podocytes culture, alternative splicing of EDA+Fn occurs at basal conditions and TGFβ1 further induced the alternative splicing of EDA+Fn via ALK5 receptor activation and SR proteins. This is the first evidence of basal and TGFβ1 mediated alternative splicing of EDA+Fn in human podocytes culture.

  13. FOX-2 Dependent Splicing of Ataxin-2 Transcript Is Affected by Ataxin-1 Overexpression

    Science.gov (United States)

    Welzel, Franziska; Kaehler, Christian; Isau, Melanie; Hallen, Linda; Lehrach, Hans; Krobitsch, Sylvia

    2012-01-01

    Alternative splicing is a fundamental posttranscriptional mechanism for controlling gene expression, and splicing defects have been linked to various human disorders. The splicing factor FOX-2 is part of a main protein interaction hub in a network related to human inherited ataxias, however, its impact remains to be elucidated. Here, we focused on the reported interaction between FOX-2 and ataxin-1, the disease-causing protein in spinocerebellar ataxia type 1. In this line, we further evaluated this interaction by yeast-2-hybrid analyses and co-immunoprecipitation experiments in mammalian cells. Interestingly, we discovered that FOX-2 localization and splicing activity is affected in the presence of nuclear ataxin-1 inclusions. Moreover, we observed that FOX-2 directly interacts with ataxin-2, a protein modulating spinocerebellar ataxia type 1 pathogenesis. Finally, we provide evidence that splicing of pre-mRNA of ataxin-2 depends on FOX-2 activity, since reduction of FOX-2 levels led to increased skipping of exon 18 in ataxin-2 transcripts. Most striking, we observed that ataxin-1 overexpression has an effect on this splicing event as well. Thus, our results demonstrate that FOX-2 is involved in splicing of ataxin-2 transcripts and that this splicing event is altered by overexpression of ataxin-1. PMID:22666429

  14. Widespread Inhibition of Posttranscriptional Splicing Shapes the Cellular Transcriptome following Heat Shock

    Directory of Open Access Journals (Sweden)

    Reut Shalgi

    2014-06-01

    Full Text Available During heat shock and other proteotoxic stresses, cells regulate multiple steps in gene expression in order to globally repress protein synthesis and selectively upregulate stress response proteins. Splicing of several mRNAs is known to be inhibited during heat stress, often meditated by SRp38, but the extent and specificity of this effect have remained unclear. Here, we examined splicing regulation genome-wide during heat shock in mouse fibroblasts. We observed widespread retention of introns in transcripts from ∼1,700 genes, which were enriched for tRNA synthetase, nuclear pore, and spliceosome functions. Transcripts with retained introns were largely nuclear and untranslated. However, a group of 580+ genes biased for oxidation reduction and protein folding functions continued to be efficiently spliced. Interestingly, these unaffected transcripts are mostly cotranscriptionally spliced under both normal and stress conditions, whereas splicing-inhibited transcripts are mostly spliced posttranscriptionally. Altogether, our data demonstrate widespread repression of splicing in the mammalian heat stress response, disproportionately affecting posttranscriptionally spliced genes.

  15. Handbook of knotting and splicing

    CERN Document Server

    Hasluck, Paul N

    2005-01-01

    Clearly written and amply illustrated with 208 figures, this classic guide ranges from simple and useful knots to complex varieties. Additional topics include rope splicing, working cordage, hammock making, more.

  16. SmD1 Modulates the miRNA Pathway Independently of Its Pre-mRNA Splicing Function.

    Directory of Open Access Journals (Sweden)

    Xiao-Peng Xiong

    2015-08-01

    Full Text Available microRNAs (miRNAs are a class of endogenous regulatory RNAs that play a key role in myriad biological processes. Upon transcription, primary miRNA transcripts are sequentially processed by Drosha and Dicer ribonucleases into ~22-24 nt miRNAs. Subsequently, miRNAs are incorporated into the RNA-induced silencing complexes (RISCs that contain Argonaute (AGO family proteins and guide RISC to target RNAs via complementary base pairing, leading to post-transcriptional gene silencing by a combination of translation inhibition and mRNA destabilization. Select pre-mRNA splicing factors have been implicated in small RNA-mediated gene silencing pathways in fission yeast, worms, flies and mammals, but the underlying molecular mechanisms are not well understood. Here, we show that SmD1, a core component of the Drosophila small nuclear ribonucleoprotein particle (snRNP implicated in splicing, is required for miRNA biogenesis and function. SmD1 interacts with both the microprocessor component Pasha and pri-miRNAs, and is indispensable for optimal miRNA biogenesis. Depletion of SmD1 impairs the assembly and function of the miRISC without significantly affecting the expression of major canonical miRNA pathway components. Moreover, SmD1 physically and functionally associates with components of the miRISC, including AGO1 and GW182. Notably, miRNA defects resulting from SmD1 silencing can be uncoupled from defects in pre-mRNA splicing, and the miRNA and splicing machineries are physically and functionally distinct entities. Finally, photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP analysis identifies numerous SmD1-binding events across the transcriptome and reveals direct SmD1-miRNA interactions. Our study suggests that SmD1 plays a direct role in miRNA-mediated gene silencing independently of its pre-mRNA splicing activity and indicates that the dual roles of splicing factors in post-transcriptional gene regulation may be

  17. Insights into alternative splicing of sarcomeric genes in the heart

    NARCIS (Netherlands)

    Weeland, Cornelis J.; van den Hoogenhof, Maarten M.; Beqqali, Abdelaziz; Creemers, Esther E.

    2015-01-01

    Driven by rapidly evolving technologies in next-generation sequencing, alternative splicing has emerged as a crucial layer in gene expression, greatly expanding protein diversity and governing complex biological processes in the cardiomyocyte. At the core of cardiac contraction, the physical

  18. fruitless alternative splicing and sex behaviour in insects

    Indian Academy of Sciences (India)

    In Drosophila melanogaster, male courtship requires proteins encoded by the fruitless (fru) gene that are produced in different sex-specific isoforms via alternative splicing. Drosophila mutant flies with loss-of-function alleles of the fru gene exhibit blocked male courtship behaviour. However, various individual steps in the ...

  19. The physiological functions of iron regulatory proteins in iron homeostasis - an update

    Directory of Open Access Journals (Sweden)

    De-Liang eZhang

    2014-06-01

    Full Text Available Iron regulatory proteins (IRPs regulate the expression of genes involved in iron metabolism by binding to RNA stem-loop structures known as iron responsive elements (IREs in target mRNAs. IRP binding inhibits the translation of mRNAs that contain an IRE in the 5’untranslated region of the transcripts, and increases the stability of mRNAs that contain IREs in the 3'untranslated region of transcripts. By these mechanisms, IRPs increase cellular iron absorption and decrease storage and export of iron to maintain an optimal intracellular iron balance. There are two members of the mammalian IRP protein family, IRP1 and IRP2, and they have redundant functions as evidenced by the embryonic lethality of the mice that completely lack IRP expression (Irp1-/-/Irp2-/- mice, which contrasts with the fact that Irp1-/- and Irp2-/- mice are viable. In addition, Irp2-/- mice also display neurodegenerative symptoms and microcytic hypochromic anemia, suggesting that IRP2 function predominates in the nervous system and erythropoietic homeostasis. Though the physiological significance of IRP1 had been unclear since Irp1-/- animals were first assessed in the early 1990’s, recent studies indicate that IRP1 plays an essential function in orchestrating the balance between erythropoiesis and bodily iron homeostasis. Additionally, Irp1-/- mice develop pulmonary hypertension, and they experience sudden death when maintained on an iron-deficient diet, indicating that IRP1 has a critical role in the pulmonary and cardiovascular systems. This review summarizes recent progress that has been made in understanding the physiological roles of IRP1 and IRP2, and further discusses the implications for clinical research on patients with idiopathic polycythemia, pulmonary hypertension and neurodegeneration.

  20. Espins are multifunctional actin cytoskeletal regulatory proteins in the microvilli of chemosensory and mechanosensory cells

    Science.gov (United States)

    Sekerková, Gabriella; Zheng, Lili; Loomis, Patricia A.; Changyaleket, Benjarat; Whitlon, Donna S.; Mugnaini, Enrico; Bartles, James R.

    2010-01-01

    Espins are associated with the parallel actin bundles of hair cell stereocilia and are the target of mutations that cause deafness and vestibular dysfunction in mice and humans. Here, we report that espins are also concentrated in the microvilli of a number of other sensory cells: vomeronasal organ sensory neurons, solitary chemoreceptor cells, taste cells and Merkel cells. Moreover, we show that hair cells and these other sensory cells contain novel espin isoforms that arise from a different transcriptional start site and differ significantly from other espin isoforms in their complement of ligand-binding activities and their effects on actin polymerization. The novel espin isoforms of sensory cells bundled actin filaments with high affinity in a Ca2+-resistant fashion, bound actin monomer via a WASP homology 2 domain, bound profilin via a single proline-rich peptide, and caused a dramatic elongation of microvillus-type parallel actin bundles in transfected epithelial cells. In addition, the novel espin isoforms of sensory cells differed from other espin isoforms in that they potently inhibited actin polymerization in vitro, did not bind the Src homology 3 domain of the adapter protein insulin receptor substrate p53 and did not bind the acidic, signaling phospholipid phosphatidylinositol 4,5- bisphosphate. Thus, the espins constitute a family of multifunctional actin cytoskeletal regulatory proteins with the potential to differentially influence the organization, dimensions, dynamics and signaling capabilities of the actin filament-rich, microvillus-type specializations that mediate sensory transduction in a variety of mechanosensory and chemosensory cells. PMID:15190118

  1. Human Sterol Regulatory Element-Binding Protein 1a Contributes Significantly to Hepatic Lipogenic Gene Expression

    Directory of Open Access Journals (Sweden)

    Andreas Bitter

    2015-01-01

    Full Text Available Background/Aims: Sterol regulatory element-binding protein (SREBP 1, the master regulator of lipogenesis, was shown to be associated with non-alcoholic fatty liver disease, which is attributed to its major isoform SREBP1c. Based on studies in mice, the minor isoform SREBP1a is regarded as negligible for hepatic lipogenesis. This study aims to elucidate the expression and functional role of SREBP1a in human liver. Methods: mRNA expression of both isoforms was quantified in cohorts of human livers and primary human hepatocytes. Hepatocytes were treated with PF-429242 to inhibit the proteolytic activation of SREBP precursor protein. SREBP1a-specifc and pan-SREBP1 knock-down were performed by transfection of respective siRNAs. Lipogenic SREBP-target gene expression was analyzed by real-time RT-PCR. Results: In human liver, SREBP1a accounts for up to half of the total SREBP1 pool. Treatment with PF-429242 indicated SREBP-dependent auto-regulation of SREBP1a, which however was much weaker than of SREBP1c. SREBP1a-specifc knock-down also reduced significantly the expression of SREBP1c and of SREBP-target genes. Regarding most SREBP-target genes, simultaneous knock-down of both isoforms resulted in effects of only similar extent as SREBP1a-specific knock-down. Conclusion: We here showed that SREBP1a is significantly contributing to the human hepatic SREBP1 pool and has a share in human hepatic lipogenic gene expression.

  2. Characterization of the regulatory subunit from brain cyclic AMP-dependent protein kinase II

    International Nuclear Information System (INIS)

    Stein, J.C.

    1985-01-01

    Tryptic peptides derived from the regulatory subunits of brain and heart cAMP-dependent protein kinase II were mapped by reverse phase HPLC. At 280 nm, 15 unique peptides were found only in the heart RII digest, while 5 other peptides were obtained only from brain RII. At 210 nm, 13 brain-RII specific and 15 heart-RII specific tryptic peptides were identified and resolved. Two-dimensional mapping analyses revealed that several 37 P-labeled tryptic fragments derived from the autophosphorylation and the photoaffinity labeled cAMP-binding sites of brain RII were separate and distinct from the 32 P-peptides isolated from similarly treated heart RII. The tryptic phosphopeptide containing the autophosphorylation site in brain RII was purified. The sequence and phosphorylation site is: Arg-Ala-Ser(P)-Val-Cys-Ala-Glu-Ala-Tyr-Asn-Pro-Asp-Glu-Glu-Glu-Asp-Asp-Ala-Glu. Astrocytes and neurons exhibit high levels of the brain RII enzyme, while oligodendrocytes contain the heart RII enzyme. Monoclonal antibodies to bovine cerebral cortex RII were made and characterized. The antibodies elucidated a subtle difference between membrane-associated and cytosolic RII from cerebral cortex

  3. Prognostic value of cell cycle regulatory proteins in muscle-infiltrating bladder cancer.

    Science.gov (United States)

    Galmozzi, Fabia; Rubagotti, Alessandra; Romagnoli, Andrea; Carmignani, Giorgio; Perdelli, Luisa; Gatteschi, Beatrice; Boccardo, Francesco

    2006-12-01

    The aims of this study were to investigate the expression levels of proteins involved in cell cycle regulation in specimens of bladder cancer and to correlate them with the clinicopathological characteristics, proliferative activity and survival. Eighty-two specimens obtained from patients affected by muscle-invasive bladder cancer were evaluated immunohistochemically for p53, p21 and cyclin D1 expression, as well as for the tumour proliferation index, Ki-67. The statistical analysis included Kaplan-Meier curves with log-rank test and Cox proportional hazards models. In univariate analyses, low Ki-67 proliferation index (P = 0.045) and negative p21 immunoreactivity (P = 0.04) were associated to patient's overall survival (OS), but in multivariate models p21 did not reach statistical significance. When the combinations of the variables were assessed in two separate multivariate models that included tumour stage, grading, lymph node status, vascular invasion and perineural invasion, the combined variables p21/Ki-67 or p21/cyclin D1 expression were independent predictors for OS; in particular, patients with positive p21/high Ki-67 (P = 0.015) or positive p21/negative cyclin D1 (P = 0.04) showed the worst survival outcome. Important alterations in the cell cycle regulatory pathways occur in muscle-invasive bladder cancer and the combined use of cell cycle regulators appears to provide significant prognostic information that could be used to select the patients most suitable for multimodal therapeutic approaches.

  4. Cross-phosphorylation of bacterial serine/threonine and tyrosine protein kinases on key regulatory residues

    Directory of Open Access Journals (Sweden)

    Lei eShi

    2014-09-01

    Full Text Available Bacteria possess protein serine/threonine and tyrosine kinases which resemble eukaryal kinases in their capacity to phosphorylate multiple substrates. We hypothesized that the analogy might extend further, and bacterial kinases may also undergo mutual phosphorylation and activation, which is currently considered as a hallmark of eukaryal kinase networks. In order to test this hypothesis, we explored the capacity of all members of four different classes of serine/threonine and tyrosine kinases present in the firmicute model organism Bacillus subtilis to phosphorylate each other in vitro and interact with each other in vivo. The interactomics data suggested a high degree of connectivity among all types of kinases, while phosphorylation assays revealed equally wide-spread cross-phosphorylation events. Our findings suggest that the Hanks-type kinases PrkC, PrkD and YabT exhibit the highest capacity to phosphorylate other B. subtilis kinases, while the BY-kinase PtkA and the two-component-like kinases RsbW and SpoIIAB show the highest propensity to be phosphorylated by other kinases. Analysis of phosphorylated residues on several selected recipient kinases suggests that most cross-phosphorylation events concern key regulatory residues. Therefore, cross-phosphorylation events are very likely to influence the capacity of recipient kinases to phosphorylate substrates downstream in the signal transduction cascade. We therefore conclude that bacterial serine/threonine and tyrosine kinases probably engage in a network-type behavior previously described only in eukaryal cells.

  5. Coordinate Regulation of Yeast Sterol Regulatory Element-binding Protein (SREBP) and Mga2 Transcription Factors.

    Science.gov (United States)

    Burr, Risa; Stewart, Emerson V; Espenshade, Peter J

    2017-03-31

    The Mga2 and Sre1 transcription factors regulate oxygen-responsive lipid homeostasis in the fission yeast Schizosaccharomyces pombe in a manner analogous to the mammalian sterol regulatory element-binding protein (SREBP)-1 and SREBP-2 transcription factors. Mga2 and SREBP-1 regulate triacylglycerol and glycerophospholipid synthesis, whereas Sre1 and SREBP-2 regulate sterol synthesis. In mammals, a shared activation mechanism allows for coordinate regulation of SREBP-1 and SREBP-2. In contrast, distinct pathways activate fission yeast Mga2 and Sre1. Therefore, it is unclear whether and how these two related pathways are coordinated to maintain lipid balance in fission yeast. Previously, we showed that Sre1 cleavage is defective in the absence of mga2 Here, we report that this defect is due to deficient unsaturated fatty acid synthesis, resulting in aberrant membrane transport. This defect is recapitulated by treatment with the fatty acid synthase inhibitor cerulenin and is rescued by addition of exogenous unsaturated fatty acids. Furthermore, sterol synthesis inhibition blocks Mga2 pathway activation. Together, these data demonstrate that Sre1 and Mga2 are each regulated by the lipid product of the other transcription factor pathway, providing a source of coordination for these two branches of lipid synthesis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Fas/CD95 regulatory protein Faim2 is neuroprotective after transient brain ischemia.

    Science.gov (United States)

    Reich, Arno; Spering, Christopher; Gertz, Karen; Harms, Christoph; Gerhardt, Ellen; Kronenberg, Golo; Nave, Klaus A; Schwab, Markus; Tauber, Simone C; Drinkut, Anja; Harms, Kristian; Beier, Chrstioph P; Voigt, Aaron; Göbbels, Sandra; Endres, Matthias; Schulz, Jörg B

    2011-01-05

    Death receptor (DR) signaling has a major impact on the outcome of numerous neurological diseases, including ischemic stroke. DRs mediate not only cell death signals, but also proinflammatory responses and cell proliferation. Identification of regulatory proteins that control the switch between apoptotic and alternative DR signaling opens new therapeutic opportunities. Fas apoptotic inhibitory molecule 2 (Faim2) is an evolutionary conserved, neuron-specific inhibitor of Fas/CD95-mediated apoptosis. To investigate its role during development and in disease models, we generated Faim2-deficient mice. The ubiquitous null mutation displayed a viable and fertile phenotype without overt deficiencies. However, lack of Faim2 caused an increase in susceptibility to combined oxygen-glucose deprivation in primary neurons in vitro as well as in caspase-associated cell death, stroke volume, and neurological impairment after cerebral ischemia in vivo. These processes were rescued by lentiviral Faim2 gene transfer. In summary, we provide evidence that Faim2 is a novel neuroprotective molecule in the context of cerebral ischemia.

  7. [Glucokinase and glucokinase regulatory proteins as molecular targets for novel antidiabetic drugs].

    Science.gov (United States)

    Rubtsov, P M; Igudin, E L; Tiulpakov, A N

    2015-01-01

    The impairment of glucose homeostasis leads to hyperglycemia and type-2 diabetes mellitus. Glucokinase (GK), an enzyme that catalyzes the conversion of glucose to glucose-6-phosphate in pancreatic ß-cells, liver hepatocytes, specific hypothalamic neurons, and intestine enterocytes, is a key regulator of glucose homeostasis. In hepatocytes, GK controls the glucose uptake and glycogen synthesis and inhibits the glucose synthesis via the gluconeogenesis pathway. Glucokinase regulatory protein (GKRP) synthesized in hepatocytes acts as an endogenous GK inhibitor. During fasting, GKRP binds GK, inactivates it, and transports it into the cell nucleus, thus isolating it from the hepatocyte carbohydrate metabolism. In the beginning of the 2000s, the research was mainly focused on the development and trials of the small molecule GK activators as potential antidiabetic glucose-lowering drugs. However, the use of such substances increased the risk of hypoglycemia, and clinical studies of most synthetic GK activators are currently discontinued. Allosteric inhibitors of the GK-GKRP interaction are coming as alternative agents increasing the GK activity that can substitute GKA. In this review, we discuss the recent advances and the current state of art in the development of potential antidiabetic drugs targeted to GK as a key regulator of glucose homeostasis.

  8. Delay-independent stability of genetic regulatory networks.

    Science.gov (United States)

    Wu, Fang-Xiang

    2011-11-01

    Genetic regulatory networks can be described by nonlinear differential equations with time delays. In this paper, we study both locally and globally delay-independent stability of genetic regulatory networks, taking messenger ribonucleic acid alternative splicing into consideration. Based on nonnegative matrix theory, we first develop necessary and sufficient conditions for locally delay-independent stability of genetic regulatory networks with multiple time delays. Compared to the previous results, these conditions are easy to verify. Then we develop sufficient conditions for global delay-independent stability for genetic regulatory networks. Compared to the previous results, this sufficient condition is less conservative. To illustrate theorems developed in this paper, we analyze delay-independent stability of two genetic regulatory networks: a real-life repressilatory network with three genes and three proteins, and a synthetic gene regulatory network with five genes and seven proteins. The simulation results show that the theorems developed in this paper can effectively determine the delay-independent stability of genetic regulatory networks.

  9. Identification of a novel function of CX-4945 as a splicing regulator.

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    Hyeongki Kim

    Full Text Available Alternative splicing is a nearly ubiquitous versatile process that controls gene expression and creates numerous protein isoforms with different functions from a single gene. The significance of alternative splicing has been confirmed by the increasing number of human diseases that are caused by misregulation of splicing events. Very few compounds, however, have been reported to act as inhibitors of alternative splicing, and their potential clinical use needs to be evaluated. Here, we report that CX-4945, a previously well-characterized inhibitor of casein kinase 2 (CK2 and a molecule currently in clinical trials (Phase II for cancer treatment, regulates splicing in mammalian cells in a CK2-independent manner. Transcriptome-wide analysis using exon array also showed a widespread alteration in alternative splicing of numerous genes. We found that CX-4945 potently inhibits the Cdc2-like kinases (Clks in vitro and in turn, leads to suppression of the phosphorylation of serine/arginine-rich (SR proteins in mammalian cells. Surprisingly, the overall efficacy of CX-4945 on Clks (IC50 = 3-90 nM was stronger than that of TG-003, the strongest inhibitor reported to date. Of the Clks, Clk2 was most strongly inhibited by CX-4945 in an ATP-competitive manner. Our research revealed an unexpected activity of the drug candidate CX-4945 as a potent splicing modulator and also suggested a potential application for therapy of diseases caused by abnormal splicing.

  10. IBTK Differently Modulates Gene Expression and RNA Splicing in HeLa and K562 Cells

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    Giuseppe Fiume

    2016-11-01

    Full Text Available The IBTK gene encodes the major protein isoform IBTKα that was recently characterized as substrate receptor of Cul3-dependent E3 ligase, regulating ubiquitination coupled to proteasomal degradation of Pdcd4, an inhibitor of translation. Due to the presence of Ankyrin-BTB-RCC1 domains that mediate several protein-protein interactions, IBTKα could exert expanded regulatory roles, including interaction with transcription regulators. To verify the effects of IBTKα on gene expression, we analyzed HeLa and K562 cell transcriptomes by RNA-Sequencing before and after IBTK knock-down by shRNA transduction. In HeLa cells, 1285 (2.03% of 63,128 mapped transcripts were differentially expressed in IBTK-shRNA-transduced cells, as compared to cells treated with control-shRNA, with 587 upregulated (45.7% and 698 downregulated (54.3% RNAs. In K562 cells, 1959 (3.1% of 63128 mapped RNAs were differentially expressed in IBTK-shRNA-transduced cells, including 1053 upregulated (53.7% and 906 downregulated (46.3%. Only 137 transcripts (0.22% were commonly deregulated by IBTK silencing in both HeLa and K562 cells, indicating that most IBTKα effects on gene expression are cell type-specific. Based on gene ontology classification, the genes responsive to IBTK are involved in different biological processes, including in particular chromatin and nucleosomal organization, gene expression regulation, and cellular traffic and migration. In addition, IBTK RNA interference affected RNA maturation in both cell lines, as shown by the evidence of alternative 3′- and 5′-splicing, mutually exclusive exons, retained introns, and skipped exons. Altogether, these results indicate that IBTK differently modulates gene expression and RNA splicing in HeLa and K562 cells, demonstrating a novel biological role of this protein.

  11. IBTK Differently Modulates Gene Expression and RNA Splicing in HeLa and K562 Cells.

    Science.gov (United States)

    Fiume, Giuseppe; Scialdone, Annarita; Rizzo, Francesca; De Filippo, Maria Rosaria; Laudanna, Carmelo; Albano, Francesco; Golino, Gaetanina; Vecchio, Eleonora; Pontoriero, Marilena; Mimmi, Selena; Ceglia, Simona; Pisano, Antonio; Iaccino, Enrico; Palmieri, Camillo; Paduano, Sergio; Viglietto, Giuseppe; Weisz, Alessandro; Scala, Giuseppe; Quinto, Ileana

    2016-11-07

    The IBTK gene encodes the major protein isoform IBTKα that was recently characterized as substrate receptor of Cul3-dependent E3 ligase, regulating ubiquitination coupled to proteasomal degradation of Pdcd4, an inhibitor of translation. Due to the presence of Ankyrin-BTB-RCC1 domains that mediate several protein-protein interactions, IBTKα could exert expanded regulatory roles, including interaction with transcription regulators. To verify the effects of IBTKα on gene expression, we analyzed HeLa and K562 cell transcriptomes by RNA-Sequencing before and after IBTK knock-down by shRNA transduction. In HeLa cells, 1285 (2.03%) of 63,128 mapped transcripts were differentially expressed in IBTK -shRNA-transduced cells, as compared to cells treated with control-shRNA, with 587 upregulated (45.7%) and 698 downregulated (54.3%) RNAs. In K562 cells, 1959 (3.1%) of 63128 mapped RNAs were differentially expressed in IBTK -shRNA-transduced cells, including 1053 upregulated (53.7%) and 906 downregulated (46.3%). Only 137 transcripts (0.22%) were commonly deregulated by IBTK silencing in both HeLa and K562 cells, indicating that most IBTKα effects on gene expression are cell type-specific. Based on gene ontology classification, the genes responsive to IBTK are involved in different biological processes, including in particular chromatin and nucleosomal organization, gene expression regulation, and cellular traffic and migration. In addition, IBTK RNA interference affected RNA maturation in both cell lines, as shown by the evidence of alternative 3'- and 5'-splicing, mutually exclusive exons, retained introns, and skipped exons. Altogether, these results indicate that IBTK differently modulates gene expression and RNA splicing in HeLa and K562 cells, demonstrating a novel biological role of this protein.

  12. Nickel decreases cellular iron level and converts cytosolic aconitase to iron-regulatory protein 1 in A549 cells

    International Nuclear Information System (INIS)

    Chen Haobin; Davidson, Todd; Singleton, Steven; Garrick, Michael D.; Costa, Max

    2005-01-01

    Nickel (Ni) compounds are well-established carcinogens and are known to initiate a hypoxic response in cells via the stabilization and transactivation of hypoxia-inducible factor-1 alpha (HIF-1α). This change may be the consequence of nickel's interference with the function of several Fe(II)-dependent enzymes. In this study, the effects of soluble nickel exposure on cellular iron homeostasis were investigated. Nickel treatment decreased both mitochondrial and cytosolic aconitase (c-aconitase) activity in A549 cells. Cytosolic aconitase was converted to iron-regulatory protein 1, a form critical for the regulation of cellular iron homeostasis. The increased activity of iron-regulatory protein 1 after nickel exposure stabilized and increased transferrin receptor (Tfr) mRNA and antagonized the iron-induced ferritin light chain protein synthesis. The decrease of aconitase activity after nickel treatment reflected neither direct interference with aconitase function nor obstruction of [4Fe-4S] cluster reconstitution by nickel. Exposure of A549 cells to soluble nickel decreased total cellular iron by about 40%, a decrease that likely caused the observed decrease in aconitase activity and the increase of iron-regulatory protein 1 activity. Iron treatment reversed the effect of nickel on cytosolic aconitase and iron-regulatory protein 1. To assess the mechanism for the observed effects, human embryonic kidney (HEK) cells over expressing divalent metal transporter-1 (DMT1) were compared to A549 cells expressing only endogenous transporters for inhibition of iron uptake by nickel. The inhibition data suggest that nickel can enter via DMT1 and compete with iron for entry into the cell. This disturbance of cellular iron homeostasis by nickel may have a great impact on the ability of the cell to regulate a variety of cell functions, as well as create a state of hypoxia in cells under normal oxygen tension. These effects may be very important in how nickel exerts phenotypic

  13. Phosphoproteomics reveals that glycogen synthase kinase-3 phosphorylates multiple splicing factors and is associated with alternative splicing

    Science.gov (United States)

    Shinde, Mansi Y.; Sidoli, Simone; Kulej, Katarzyna; Mallory, Michael J.; Radens, Caleb M.; Reicherter, Amanda L.; Myers, Rebecca L.; Barash, Yoseph; Lynch, Kristen W.; Garcia, Benjamin A.; Klein, Peter S.

    2017-01-01

    Glycogen synthase kinase-3 (GSK-3) is a constitutively active, ubiquitously expressed protein kinase that regulates multiple signaling pathways. In vitro kinase assays and genetic and pharmacological manipulations of GSK-3 have identified more than 100 putative GSK-3 substrates in diverse cell types. Many more have been predicted on the basis of a recurrent GSK-3 consensus motif ((pS/pT)XXX(S/T)), but this prediction has not been tested by analyzing the GSK-3 phosphoproteome. Using stable isotope labeling of amino acids in culture (SILAC) and MS techniques to analyze the repertoire of GSK-3–dependent phosphorylation in mouse embryonic stem cells (ESCs), we found that ∼2.4% of (pS/pT)XXX(S/T) sites are phosphorylated in a GSK-3–dependent manner. A comparison of WT and Gsk3a;Gsk3b knock-out (Gsk3 DKO) ESCs revealed prominent GSK-3–dependent phosphorylation of multiple splicing factors and regulators of RNA biosynthesis as well as proteins that regulate transcription, translation, and cell division. Gsk3 DKO reduced phosphorylation of the splicing factors RBM8A, SRSF9, and PSF as well as the nucleolar proteins NPM1 and PHF6, and recombinant GSK-3β phosphorylated these proteins in vitro. RNA-Seq of WT and Gsk3 DKO ESCs identified ∼190 genes that are alternatively spliced in a GSK-3–dependent manner, supporting a broad role for GSK-3 in regulating alternative splicing. The MS data also identified posttranscriptional regulation of protein abundance by GSK-3, with ∼47 proteins (1.4%) whose levels increased and ∼78 (2.4%) whose levels decreased in the absence of GSK-3. This study provides the first unbiased analysis of the GSK-3 phosphoproteome and strong evidence that GSK-3 broadly regulates alternative splicing. PMID:28916722

  14. RNA splicing in a new rhabdovirus from Culex mosquitoes.

    Science.gov (United States)

    Kuwata, Ryusei; Isawa, Haruhiko; Hoshino, Keita; Tsuda, Yoshio; Yanase, Tohru; Sasaki, Toshinori; Kobayashi, Mutsuo; Sawabe, Kyoko

    2011-07-01

    Among members of the order Mononegavirales, RNA splicing events have been found only in the family Bornaviridae. Here, we report that a new rhabdovirus isolated from the mosquito Culex tritaeniorhynchus replicates in the nuclei of infected cells and requires RNA splicing for viral mRNA maturation. The virus, designated Culex tritaeniorhynchus rhabdovirus (CTRV), shares a similar genome organization with other rhabdoviruses, except for the presence of a putative intron in the coding region for the L protein. Molecular phylogenetic studies indicated that CTRV belongs to the family Rhabdoviridae, but it is yet to be assigned a genus. Electron microscopic analysis revealed that the CTRV virion is extremely elongated, unlike virions of rhabdoviruses, which are generally bullet shaped. Northern hybridization confirmed that a large transcript (approximately 6,500 nucleotides [nt]) from the CTRV L gene was present in the infected cells. Strand-specific reverse transcription-PCR (RT-PCR) analyses identified the intron-exon boundaries and the 76-nt intron sequence, which contains the typical motif for eukaryotic spliceosomal intron-splice donor/acceptor sites (GU-AG), a predicted branch point, and a polypyrimidine tract. In situ hybridization exhibited that viral RNAs are primarily localized in the nucleus of infected cells, indicating that CTRV replicates in the nucleus and is allowed to utilize the host's nuclear splicing machinery. This is the first report of RNA splicing among the members of the family Rhabdoviridae.

  15. The determinants of alternative RNA splicing in human cells.

    Science.gov (United States)

    Ramanouskaya, Tatsiana V; Grinev, Vasily V

    2017-12-01

    Alternative splicing represents an important level of the regulation of gene function in eukaryotic organisms. It plays a critical role in virtually every biological process within an organism, including regulation of cell division and cell death, differentiation of tissues in the embryo and the adult organism, as well as in cellular response to diverse environmental factors. In turn, studies of the last decade have shown that alternative splicing itself is controlled by different mechanisms. Unfortunately, there is no clear understanding of how these diverse mechanisms, or determinants, regulate and constrain the set of alternative RNA species produced from any particular gene in every cell of the human body. Here, we provide a consolidated overview of alternative splicing determinants including RNA-protein interactions, epigenetic regulation via chromatin remodeling, coupling of transcription-to-alternative splicing, effect of secondary structures in pre-RNA, and function of the RNA quality control systems. We also extensively and critically discuss some mechanistic insights on coordinated inclusion/exclusion of exons during the formation of mature RNA molecules. We conclude that the final structure of RNA is pre-determined by a complex interplay between cis- and trans-acting factors. Altogether, currently available empirical data significantly expand our understanding of the functioning of the alternative splicing machinery of cells in normal and pathological conditions. On the other hand, there are still many blind spots that require further deep investigations.

  16. The Prognostic Impact of Some Cell Cycle Regulatory Proteins in Egyptian Breast Cancer Patients

    International Nuclear Information System (INIS)

    KAMEL, A.; Mokhtar, N.; Elshaknkiry, N.; Yassin, D.; Elnahass, Y.; Zakarya, O.; Elbasmy, A.; Elmetenawy, W.

    2006-01-01

    Purpose: The particular goal of this work is to study some cell cycle regulatory proteins and their potential impact on prognosis of breast cancer; p53, cyclin D 1 and p27 are potential effectors being the major contributors to the control of the restriction (R) check point of the cell cycle. We also aimed to evaluate different techniques used to detect these cell cycle proteins. Material and Methods: Forty five breast cancer patients as well as 10 controls with non malignant pathology were assessed for cell cycle regulators each by 2 different techniques; p53 was assessed by enzyme immunoassay (EJA) and immunohistochemistry (lHC), cyclin D1 by Western Blotting (WB) and IHC and p27 by WB and me. The cut-off was calculated as the mean of the normal controls +2 SD. Patients were followed up for 4 years and their laboratory data were correlated with different clinical parameters and with other studied regulators. Results: Eighty seven percent of cases (39/45) were positive for p53 by EIA with a range from 20 to 4300, and a mean of 464±97 I pg/mg protein. By mc, 80% (24/30) of the cases showed varying degrees of positivity. Using WB, cyclin D 1 showed high expression levels above cut off values in 69% of patients (31/45) and in 67% (20/30) by me. The corresponding positive figures for p27 were 82% (37/45) and 73% (22/30) using the two techniques, respectively. No significant association was found between p53, cyclin 01 and p27 on one side and different clinical parameters as lymph node status, tumor size or presence of distant metastases on the other side. Survival was poor in patients with high p53 expression. Cyclin D1 positive cases showed comparable survival with negative cases, whereas high p27 levels favored a longer disease free survival. Conclusions: Techniques more suitable for assessment of each of these markers in our consideration were EIA for p53, WB for cyclin D1 and IHC for p27. Moreover, this study demonstrated that these markers were relevant to the

  17. RNA regulatory elements and polyadenylation in plants

    Directory of Open Access Journals (Sweden)

    Arthur G. Hunt

    2012-01-01

    Full Text Available Alternative poly(A site choice (also known as alternative polyadenylation, or APA has the potential to affect gene expression in qualitative and quantitative ways. Alternative polyadenylation may affect as many as 82% of all expressed genes in a plant. The consequences of APA include the generation of transcripts with differing 3’-UTRs (and thus differing potential regulatory potential and of transcripts with differing protein-coding potential. Genome-wide studies of possible APA suggest a linkage with pre-mRNA splicing, and indicate a coincidence of and perhaps cooperation between RNA regulatory elements that affect splicing efficiency and the recognition of novel intronic poly(A sites. These studies also raise the possibility of the existence of a novel class of polyadenylation-related cis elements that are distinct from the well-characterized plant polyadenylation signal. Many potential APA events, however, have not been associated with identifiable cis elements. The present state of the field reveals a broad scope of APA, and also numerous opportunities for research into mechanisms that govern both choice and regulation of poly(A sites in plants.

  18. Splicing Analysis of Exonic OCRL Mutations Causing Lowe Syndrome or Dent-2 Disease

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    Lorena Suarez-Artiles

    2018-01-01

    Full Text Available Mutations in the OCRL gene are associated with both Lowe syndrome and Dent-2 disease. Patients with Lowe syndrome present congenital cataracts, mental disabilities and a renal proximal tubulopathy, whereas patients with Dent-2 disease exhibit similar proximal tubule dysfunction but only mild, or no additional clinical defects. It is not yet understood why some OCRL mutations cause the phenotype of Lowe syndrome, while others develop the milder phenotype of Dent-2 disease. Our goal was to gain new insights into the consequences of OCRL exonic mutations on pre-mRNA splicing. Using predictive bioinformatics tools, we selected thirteen missense mutations and one synonymous mutation based on their potential effects on splicing regulatory elements or splice sites. These mutations were analyzed in a minigene splicing assay. Results of the RNA analysis showed that three presumed missense mutations caused alterations in pre-mRNA splicing. Mutation c.741G>T; p.(Trp247Cys generated splicing silencer sequences and disrupted splicing enhancer motifs that resulted in skipping of exon 9, while mutations c.2581G>A; p.(Ala861Thr and c.2581G>C; p.(Ala861Pro abolished a 5′ splice site leading to skipping of exon 23. Mutation c.741G>T represents the first OCRL exonic variant outside the conserved splice site dinucleotides that results in alteration of pre-mRNA splicing. Our results highlight the importance of evaluating the effects of OCRL exonic mutations at the mRNA level.

  19. The chaperone-like activity of α-synuclein attenuates aggregation of its alternatively spliced isoform, 112-synuclein in vitro: plausible cross-talk between isoforms in protein aggregation.

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    Krishna Madhuri Manda

    Full Text Available Abnormal oligomerization and aggregation of α-synuclein (α-syn/WT-syn has been shown to be a precipitating factor in the pathophysiology of Parkinson's disease (PD. Earlier observations on the induced-alternative splicing of α-syn by Parkinsonism mimetics as well as identification of region specific abnormalities in the transcript levels of 112-synuclein (112-syn in diseased subjects underscores the role of 112-syn in the pathophysiology of PD. In the present study, we sought to identify the aggregation potential of 112-syn in the presence or absence of WT-syn to predict its plausible role in protein aggregation events. Results demonstrate that unlike WT-syn, lack of 28 aa in the C-terminus results in the loss of chaperone-like activity with a concomitant gain in vulnerability to heat-induced aggregation and time-dependent fibrillation. The effects were dose and time-dependent and a significant aggregation of 112-syn was evident at as low as 45 °C following 10 min of incubation. The heat-induced aggregates were found to be ill-defined structures and weakly positive towards Thioflavin-T (ThT staining as compared to clearly distinguishable ThT positive extended fibrils resulting upon 24 h of incubation at 37 °C. Further, the chaperone-like activity of WT-syn significantly attenuated heat-induced aggregation of 112-syn in a dose and time-dependent manner. On contrary, WT-syn synergistically enhanced fibrillation of 112-syn. Overall, the present findings highlight a plausible cross-talk between isoforms of α-syn and the relative abundance of these isoforms may dictate the nature and fate of protein aggregates.

  20. Alternative Splicing of the Pituitary Adenylate Cyclase-Activating Polypeptide Receptor PAC1: Mechanisms of Fine Tuning of Brain Activity

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    Janna eBlechman

    2013-05-01

    Full Text Available Alternative splicing of the precursor mRNA encoding for the neuropeptide receptor PAC1/ADCYAP1R1 generates multiple protein products that exhibit pleiotropic activities. Recent studies in mammals and zebrafish have implicated some of these splice isoforms in control of both cellular and body homeostasis. Here, we review the regulation of PAC1 splice variants and their underlying signal transduction and physiological processes in the nervous system.

  1. Acute Endoplasmic Reticulum Stress-Independent Unconventional Splicing of XBP1 mRNA in the Nucleus of Mammalian Cells

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    Yuanyuan Wang

    2015-06-01

    Full Text Available The regulation of expression of X-box-binding protein-1 (XBP1, a transcriptional factor, involves an unconventional mRNA splicing that removes the 26 nucleotides intron. In contrast to the conventional splicing that exclusively takes place in the nucleus, determining the location of unconventional splicing still remains controversial. This study was designed to examine whether the unconventional spicing of XBP1 mRNA could occur in the nucleus and its possible biological relevance. We use RT-PCR reverse transcription system and the expand high fidelity PCR system to detect spliced XBP1 mRNA, and fraction cells to determine the location of the unconventional splicing of XBP1 mRNA. We employ reporter constructs to show the presence of unconventional splicing machinery in mammal cells independently of acute endoplasmic reticulum (ER stress. Our results reveal the presence of basal unconventional splicing of XBP1 mRNA in the nucleus that also requires inositol-requiring transmembrane kinase and endonuclease 1α (IRE1α and can occur independently of acute ER stress. Furthermore, we confirm that acute ER stress induces the splicing of XBP1 mRNA predominantly occurring in the cytoplasm, but it also promotes the splicing in the nucleus. The deletion of 5′-nucleotides in XBP1 mRNA significantly increases its basal unconventional splicing, suggesting that the secondary structure of XBP1 mRNA may determine the location of unconventional splicing. These results suggest that the unconventional splicing of XBP1 mRNA can take place in the nucleus and/or cytoplasm, which possibly depends on the elaborate regulation. The acute ER stress-independent unconventional splicing in the nucleus is most likely required for the maintaining of day-to-day folding protein homeostasis.

  2. Identification of high-confidence RNA regulatory elements by combinatorial classification of RNA-protein binding sites.

    Science.gov (United States)

    Li, Yang Eric; Xiao, Mu; Shi, Binbin; Yang, Yu-Cheng T; Wang, Dong; Wang, Fei; Marcia, Marco; Lu, Zhi John

    2017-09-08

    Crosslinking immunoprecipitation sequencing (CLIP-seq) technologies have enabled researchers to characterize transcriptome-wide binding sites of RNA-binding protein (RBP) with high resolution. We apply a soft-clustering method, RBPgroup, to various CLIP-seq datasets to group together RBPs that specifically bind the same RNA sites. Such combinatorial clustering of RBPs helps interpret CLIP-seq data and suggests functional RNA regulatory elements. Furthermore, we validate two RBP-RBP interactions in cell lines. Our approach links proteins and RNA motifs known to possess similar biochemical and cellular properties and can, when used in conjunction with additional experimental data, identify high-confidence RBP groups and their associated RNA regulatory elements.

  3. Role of regulatory subunits and protein kinase inhibitor (PKI) in determining nuclear localization and activity of the catalytic subunit of protein kinase A.

    Science.gov (United States)

    Wiley, J C; Wailes, L A; Idzerda, R L; McKnight, G S

    1999-03-05

    Regulation of protein kinase A by subcellular localization may be critical to target catalytic subunits to specific substrates. We employed epitope-tagged catalytic subunit to correlate subcellular localization and gene-inducing activity in the presence of regulatory subunit or protein kinase inhibitor (PKI). Transiently expressed catalytic subunit distributed throughout the cell and induced gene expression. Co-expression of regulatory subunit or PKI blocked gene induction and prevented nuclear accumulation. A mutant PKI lacking the nuclear export signal blocked gene induction but not nuclear accumulation, demonstrating that nuclear export is not essential to inhibit gene induction. When the catalytic subunit was targeted to the nucleus with a nuclear localization signal, it was not sequestered in the cytoplasm by regulatory subunit, although its activity was completely inhibited. PKI redistributed the nuclear catalytic subunit to the cytoplasm and blocked gene induction, demonstrating that the nuclear export signal of PKI can override a strong nuclear localization signal. With increasing PKI, the export process appeared to saturate, resulting in the return of catalytic subunit to the nucleus. These results demonstrate that both the regulatory subunit and PKI are able to completely inhibit the gene-inducing activity of the catalytic subunit even when the catalytic subunit is forced to concentrate in the nuclear compartment.

  4. On splice site prediction using weight array models: a comparison of smoothing techniques

    International Nuclear Information System (INIS)

    Taher, Leila; Meinicke, Peter; Morgenstern, Burkhard

    2007-01-01

    In most eukaryotic genes, protein-coding exons are separated by non-coding introns which are removed from the primary transcript by a process called 'splicing'. The positions where introns are cut and exons are spliced together are called 'splice sites'. Thus, computational prediction of splice sites is crucial for gene finding in eukaryotes. Weight array models are a powerful probabilistic approach to splice site detection. Parameters for these models are usually derived from m-tuple frequencies in trusted training data and subsequently smoothed to avoid zero probabilities. In this study we compare three different ways of parameter estimation for m-tuple frequencies, namely (a) non-smoothed probability estimation, (b) standard pseudo counts and (c) a Gaussian smoothing procedure that we recently developed

  5. Involvement of the iron regulatory protein from Eisenia andrei earthworms in the regulation of cellular iron homeostasis.

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    Petra Procházková

    Full Text Available Iron homeostasis in cells is regulated by iron regulatory proteins (IRPs that exist in different organisms. IRPs are cytosolic proteins that bind to iron-responsive elements (IREs of the 5'- or 3'-untranslated regions (UTR of mRNAs that encode many proteins involved in iron metabolism. In this study, we have cloned and described a new regulatory protein belonging to the family of IRPs from the earthworm Eisenia andrei (EaIRP. The earthworm IRE site in 5'-UTR of ferritin mRNA most likely folds into a secondary structure that differs from the conventional IRE structures of ferritin due to the absence of a typically unpaired cytosine that participates in protein binding. Prepared recombinant EaIRP and proteins from mammalian liver extracts are able to bind both mammalian and Eisenia IRE structures of ferritin mRNA, although the affinity of the rEaIRP/Eisenia IRE structure is rather low. This result suggests the possible contribution of a conventional IRE structure. When IRP is supplemented with a Fe-S cluster, it can function as a cytosolic aconitase. Cellular cytosolic and mitochondrial fractions, as well as recombinant EaIRP, exhibit aconitase activity that can be abolished by the action of oxygen radicals. The highest expression of EaIRP was detected in parts of the digestive tract. We can assume that earthworms may possess an IRE/IRP regulatory network as a potential mechanism for maintaining cellular iron homeostasis, although the aconitase function of EaIRP is most likely more relevant.

  6. Involvement of the Iron Regulatory Protein from Eisenia andrei Earthworms in the Regulation of Cellular Iron Homeostasis

    Science.gov (United States)

    Procházková, Petra; Škanta, František; Roubalová, Radka; Šilerová, Marcela; Dvořák, Jiří; Bilej, Martin

    2014-01-01

    Iron homeostasis in cells is regulated by iron regulatory proteins (IRPs) that exist in different organisms. IRPs are cytosolic proteins that bind to iron-responsive elements (IREs) of the 5′- or 3′-untranslated regions (UTR) of mRNAs that encode many proteins involved in iron metabolism. In this study, we have cloned and described a new regulatory protein belonging to the family of IRPs from the earthworm Eisenia andrei (EaIRP). The earthworm IRE site in 5′-UTR of ferritin mRNA most likely folds into a secondary structure that differs from the conventional IRE structures of ferritin due to the absence of a typically unpaired cytosine that participates in protein binding. Prepared recombinant EaIRP and proteins from mammalian liver extracts are able to bind both mammalian and Eisenia IRE structures of ferritin mRNA, although the affinity of the rEaIRP/Eisenia IRE structure is rather low. This result suggests the possible contribution of a conventional IRE structure. When IRP is supplemented with a Fe-S cluster, it can function as a cytosolic aconitase. Cellular cytosolic and mitochondrial fractions, as well as recombinant EaIRP, exhibit aconitase activity that can be abolished by the action of oxygen radicals. The highest expression of EaIRP was detected in parts of the digestive tract. We can assume that earthworms may possess an IRE/IRP regulatory network as a potential mechanism for maintaining cellular iron homeostasis, although the aconitase function of EaIRP is most likely more relevant. PMID:25279857

  7. Inhibitors Alter the Stochasticity of Regulatory Proteins to Force Cells to Switch to the Other State in the Bistable System.

    Science.gov (United States)

    Jhang, Wun-Sin; Lo, Shih-Chiang; Yeh, Chen-Chao; Shu, Che-Chi

    2017-06-30

    The cellular behaviors under the control of genetic circuits are subject to stochastic fluctuations, or noise. The stochasticity in gene regulation, far from a nuisance, has been gradually appreciated for its unusual function in cellular activities. In this work, with Chemical Master Equation (CME), we discovered that the addition of inhibitors altered the stochasticity of regulatory proteins. For a bistable system of a mutually inhibitory network, such a change of noise led to the migration of cells in the bimodal distribution. We proposed that the consumption of regulatory protein caused by the addition of inhibitor is not the only reason for pushing cells to the specific state; the change of the intracellular stochasticity is also the main cause for the redistribution. For the level of the inhibitor capable of driving 99% of cells, if there is no consumption of regulatory protein, 88% of cells were guided to the specific state. It implied that cells were pushed, by the inhibitor, to the specific state due to the change of stochasticity.

  8. Regulatory CD4 T cells inhibit HIV-1 expression of other CD4 T cell subsets via interactions with cell surface regulatory proteins.

    Science.gov (United States)

    Zhang, Mingce; Robinson, Tanya O; Duverger, Alexandra; Kutsch, Olaf; Heath, Sonya L; Cron, Randy Q

    2018-03-01

    During chronic HIV-1 infection, regulatory CD4 T cells (Tregs) frequently represent the largest subpopulation of CD4 T cell subsets, implying relative resistant to HIV-1. When HIV-1 infection of CD4 T cells was explored in vitro and ex vivo from patient samples, Tregs possessed lower levels of HIV-1 DNA and RNA in comparison with conventional effector and memory CD4 T cells. Moreover, Tregs suppressed HIV-1 expression in other CD4 T cells in an in vitro co-culture system. This suppression was mediated in part via multiple inhibitory surface proteins expressed on Tregs. Antibody blockade of CTLA-4, PD-1, and GARP on Tregs resulted in increased HIV-1 DNA integration and mRNA expression in neighboring CD4 T cells. Moreover, antibody blockade of Tregs inhibitory proteins resulted in increased HIV-1 LTR transcription in co-cultured CD4 T cells. Thus, Tregs inhibit HIV-1 infection of other CD4 T cell subsets via interactions with inhibitory cell surface proteins. Copyright © 2018 Elsevier Inc. All rights reserved.

  9. Reprogramming the Dynamin 2 mRNA by Spliceosome-mediated RNA Trans-splicing

    Directory of Open Access Journals (Sweden)

    Delphine Trochet

    2016-01-01

    Full Text Available Dynamin 2 (DNM2 is a large GTPase, ubiquitously expressed, involved in membrane trafficking and regulation of actin and microtubule cytoskeletons. DNM2 mutations cause autosomal dominant centronuclear myopathy which is a rare congenital myopathy characterized by skeletal muscle weakness and histopathological features including nuclear centralization in absence of regeneration. No curative treatment is currently available for the DNM2-related autosomal dominant centronuclear myopathy. In order to develop therapeutic strategy, we evaluated here the potential of Spliceosome-Mediated RNA Trans-splicing technology to reprogram the Dnm2-mRNA in vitro and in vivo in mice. We show that classical 3′-trans-splicing strategy cannot be considered as accurate therapeutic strategy regarding toxicity of the pre-trans-splicing molecules leading to low rate of trans-splicing in vivo. Thus, we tested alternative strategies devoted to prevent this toxicity and enhance frequency of trans-splicing events. We succeeded to overcome the toxicity through a 5′-trans-splicing strategy which also allows detection of trans-splicing events at mRNA and protein levels in vitro and in vivo. These results suggest that the Spliceosome-Mediated RNA Trans-splicing strategy may be used to reprogram mutated Dnm2-mRNA but highlight the potential toxicity linked to the molecular tools which have to be carefully investigated during preclinical development.

  10. Zinc deficiency-induced iron accumulation, a consequence of alterations in iron regulatory protein-binding activity, iron transporters, and iron storage proteins.

    Science.gov (United States)

    Niles, Brad J; Clegg, Michael S; Hanna, Lynn A; Chou, Susan S; Momma, Tony Y; Hong, Heeok; Keen, Carl L

    2008-02-22

    One consequence of zinc deficiency is an elevation in cell and tissue iron concentrations. To examine the mechanism(s) underlying this phenomenon, Swiss 3T3 cells were cultured in zinc-deficient (D, 0.5 microM zinc), zinc-supplemented (S, 50 microM zinc), or control (C, 4 microM zinc) media. After 24 h of culture, cells in the D group were characterized by a 50% decrease in intracellular zinc and a 35% increase in intracellular iron relative to cells in the S and C groups. The increase in cellular iron was associated with increased transferrin receptor 1 protein and mRNA levels and increased ferritin light chain expression. The divalent metal transporter 1(+)iron-responsive element isoform mRNA was decreased during zinc deficiency-induced iron accumulation. Examination of zinc-deficient cells revealed increased binding of iron regulatory protein 2 (IRP2) and decreased binding of IRP1 to a consensus iron-responsive element. The increased IRP2-binding activity in zinc-deficient cells coincided with an increased level of IRP2 protein. The accumulation of IRP2 protein was independent of zinc deficiency-induced intracellular nitric oxide production but was attenuated by the addition of the antioxidant N-acetylcysteine or ascorbate to the D medium. These data support the concept that zinc deficiency can result in alterations in iron transporter, storage, and regulatory proteins, which facilitate iron accumulation.

  11. Diverse alternative back-splicing and alternative splicing landscape of circular RNAs

    Science.gov (United States)

    Zhang, Xiao-Ou; Dong, Rui; Zhang, Yang; Zhang, Jia-Lin; Luo, Zheng; Zhang, Jun; Chen, Ling-Ling; Yang, Li

    2016-01-01

    Circular RNAs (circRNAs) derived from back-spliced exons have been widely identified as being co-expressed with their linear counterparts. A single gene locus can produce multiple circRNAs through alternative back-splice site selection and/or alternative splice site selection; however, a detailed map of alternative back-splicing/splicing in circRNAs is lacking. Here, with the upgraded CIRCexplorer2 pipeline, we systematically annotated different types of alternative back-splicing and alternative splicing events in circRNAs from various cell lines. Compared with their linear cognate RNAs, circRNAs exhibited distinct patterns of alternative back-splicing and alternative splicing. Alternative back-splice site selection was correlated with the competition of putative RNA pairs across introns that bracket alternative back-splice sites. In addition, all four basic types of alternative splicing that have been identified in the (linear) mRNA process were found within circRNAs, and many exons were predominantly spliced in circRNAs. Unexpectedly, thousands of previously unannotated exons were detected in circRNAs from the examined cell lines. Although these novel exons had similar splice site strength, they were much less conserved than known exons in sequences. Finally, both alternative back-splicing and circRNA-predominant alternative splicing were highly diverse among the examined cell lines. All of the identified alternative back-splicing and alternative splicing in circRNAs are available in the CIRCpedia database (http://www.picb.ac.cn/rnomics/circpedia). Collectively, the annotation of alternative back-splicing and alternative splicing in circRNAs provides a valuable resource for depicting the complexity of circRNA biogenesis and for studying the potential functions of circRNAs in different cells. PMID:27365365

  12. The splicing regulator PTBP1 controls the activity of the transcription factor Pbx1 during neuronal differentiation.

    Science.gov (United States)

    Linares, Anthony J; Lin, Chia-Ho; Damianov, Andrey; Adams, Katrina L; Novitch, Bennett G; Black, Douglas L

    2015-12-24

    The RNA-binding proteins PTBP1 and PTBP2 control programs of alternative splicing during neuronal development. PTBP2 was found to maintain embryonic splicing patterns of many synaptic and cytoskeletal proteins during differentiation of neuronal progenitor cells (NPCs) into early neurons. However, the role of the earlier PTBP1 program in embryonic stem cells (ESCs) and NPCs was not clear. We show that PTBP1 controls a program of neuronal gene expression that includes the transcription factor Pbx1. We identify exons specifically regulated by PTBP1 and not PTBP2 as mouse ESCs differentiate into NPCs. We find that PTBP1 represses Pbx1 exon 7 and the expression of the neuronal Pbx1a isoform in ESCs. Using CRISPR-Cas9 to delete regulatory elements for exon 7, we induce Pbx1a expression in ESCs, finding that this activates transcription of neuronal genes. Thus, PTBP1 controls the activity of Pbx1 to suppress its neuronal transcriptional program prior to induction of NPC development.

  13. Changes in exon–intron structure during vertebrate evolution affect the splicing pattern of exons

    Science.gov (United States)

    Gelfman, Sahar; Burstein, David; Penn, Osnat; Savchenko, Anna; Amit, Maayan; Schwartz, Schraga; Pupko, Tal; Ast, Gil

    2012-01-01

    Exon–intron architecture is one of the major features directing the splicing machinery to the short exons that are located within long flanking introns. However, the evolutionary dynamics of exon–intron architecture and its impact on splicing is largely unknown. Using a comparative genomic approach, we analyzed 17 vertebrate genomes and reconstructed the ancestral motifs of both 3′ and 5′ splice sites, as also the ancestral length of exons and introns. Our analyses suggest that vertebrate introns increased in length from the shortest ancestral introns to the longest primate introns. An evolutionary analysis of splice sites revealed that weak splice sites act as a restrictive force keeping introns short. In contrast, strong splice sites allow recognition of exons flanked by long introns. Reconstruction of the ancestral state suggests these phenomena were not prevalent in the vertebrate ancestor, but appeared during vertebrate evolution. By calculating evolutionary rate shifts in exons, we identified cis-acting regulatory sequences that became fixed during the transition from early vertebrates to mammals. Experimental validations performed on a selection of these hexamers confirmed their regulatory function. We additionally revealed many features of exons that can discriminate alternative from constitutive exons. These features were integrated into a machine-learning approach to predict whether an exon is alternative. Our algorithm obtains very high predictive power (AUC of 0.91), and using these predictions we have identified and successfully validated novel alternatively spliced exons. Overall, we provide novel insights regarding the evolutionary constraints acting upon exons and their recognition by the splicing machinery. PMID:21974994

  14. The Spliced Leader Trans-Splicing Mechanism in Different Organisms: Molecular Details and Possible Biological Roles

    Directory of Open Access Journals (Sweden)

    Mainá eBitar

    2013-10-01

    Full Text Available The spliced leader (SL is a gene that generates a functional ncRNA that is composed of two regions: an intronic region of unknown function (SLi and an exonic region (SLe, which is transferred to the 5’ end of independent transcripts yielding mature mRNAs, in a process known as spliced leader trans-splicing (SLTS. The best described function for SLTS is to solve polycistronic transcripts into monocistronic units, specifically in Trypanosomatids. In other metazoans, it is speculated that the SLe addition could lead to increased mRNA stability, differential recruitment of the translational machinery, modification of the 5' region or a combination of these effects. Although important aspects of this mechanism have been revealed, several features remain to be elucidated. We have analyzed 157 SLe sequences from 148 species from 7 phyla and found a high degree of conservation among the sequences of species from the same phylum, although no considerable similarity seems to exist between sequences of species from different phyla. When analyzing case studies, we found evidence that a given SLe will always be related to a given set of transcripts in different species from the same phylum, and therefore, different SLe sequences from the same species would regulate different sets of transcripts. In addition, we have observed distinct transcript categories to be preferential targets for the SLe addition in different phyla. This work sheds light into crucial and controversial aspects of the SLTS mechanism. It represents a comprehensive study concerning various species and different characteristics of this important post-transcriptional regulatory mechanism.

  15. SACE_5599, a putative regulatory protein, is involved in morphological differentiation and erythromycin production in Saccharopolyspora erythraea.

    Science.gov (United States)

    Kirm, Benjamin; Magdevska, Vasilka; Tome, Miha; Horvat, Marinka; Karničar, Katarina; Petek, Marko; Vidmar, Robert; Baebler, Spela; Jamnik, Polona; Fujs, Štefan; Horvat, Jaka; Fonovič, Marko; Turk, Boris; Gruden, Kristina; Petković, Hrvoje; Kosec, Gregor

    2013-12-17

    Erythromycin is a medically important antibiotic, biosynthesized by the actinomycete Saccharopolyspora erythraea. Genes encoding erythromycin biosynthesis are organized in a gene cluster, spanning over 60 kbp of DNA. Most often, gene clusters encoding biosynthesis of secondary metabolites contain regulatory genes. In contrast, the erythromycin gene cluster does not contain regulatory genes and regulation of its biosynthesis has therefore remained poorly understood, which has for a long time limited genetic engineering approaches for erythromycin yield improvement. We used a comparative proteomic approach to screen for potential regulatory proteins involved in erythromycin biosynthesis. We have identified a putative regulatory protein SACE_5599 which shows significantly higher levels of expression in an erythromycin high-producing strain, compared to the wild type S. erythraea strain. SACE_5599 is a member of an uncharacterized family of putative regulatory genes, located in several actinomycete biosynthetic gene clusters. Importantly, increased expression of SACE_5599 was observed in the complex fermentation medium and at controlled bioprocess conditions, simulating a high-yield industrial fermentation process in the bioreactor. Inactivation of SACE_5599 in the high-producing strain significantly reduced erythromycin yield, in addition to drastically decreasing sporulation intensity of the SACE_5599-inactivated strains when cultivated on ABSM4 agar medium. In contrast, constitutive overexpression of SACE_5599 in the wild type NRRL23338 strain resulted in an increase of erythromycin yield by 32%. Similar yield increase was also observed when we overexpressed the bldD gene, a previously identified regulator of erythromycin biosynthesis, thereby for the first time revealing its potential for improving erythromycin biosynthesis. SACE_5599 is the second putative regulatory gene to be identified in S. erythraea which has positive influence on erythromycin yield. Like bld

  16. BAP1 missense mutation c.2054 A>T (p.E685V completely disrupts normal splicing through creation of a novel 5' splice site in a human mesothelioma cell line.

    Directory of Open Access Journals (Sweden)

    Arianne Morrison

    Full Text Available BAP1 is a tumor suppressor gene that is lost or deleted in diverse cancers, including uveal mela¬noma, malignant pleural mesothelioma (MPM, clear cell renal carcinoma, and cholangiocarcinoma. Recently, BAP1 germline mutations have been reported in families with combinations of these same cancers. A particular challenge for mutation screening is the classification of non-truncating BAP1 sequence variants because it is not known whether these subtle changes can affect the protein function sufficiently to predispose to cancer development. Here we report mRNA splicing analysis on a homozygous substitution mutation, BAP1 c. 2054 A&T (p.Glu685Val, identified in an MPM cell line derived from a mesothelioma patient. The mutation occurred at the 3rd nucleotide from the 3' end of exon 16. RT-PCR, cloning and subsequent sequencing revealed several aberrant splicing products not observed in the controls: 1 a 4 bp deletion at the end of exon 16 in all clones derived from the major splicing product. The BAP1 c. 2054 A&T mutation introduced a new 5' splice site (GU, which resulted in the deletion of 4 base pairs and presumably protein truncation; 2 a variety of alternative splicing products that led to retention of different introns: introns 14-16; introns 15-16; intron 14 and intron 16; 3 partial intron 14 and 15 retentions caused by activation of alternative 3' splice acceptor sites (AG in the introns. Taken together, we were unable to detect any correctly spliced mRNA transcripts in this cell line. These results suggest that aberrant splicing caused by this mutation is quite efficient as it completely abolishes normal splicing through creation of a novel 5' splice site and activation of cryptic splice sites. These data support the conclusion that BAP1 c.2054 A&T (p.E685V variant is a pathogenic mutation and contributes to MPM through disruption of normal splicing.

  17. Finding trans-regulatory genes and protein complexes modulating meiotic recombination hotspots of human, mouse and yeast.

    Science.gov (United States)

    Wu, Min; Kwoh, Chee-Keong; Li, Xiaoli; Zheng, Jie

    2014-09-11

    The regulatory mechanism of recombination is one of the most fundamental problems in genomics, with wide applications in genome wide association studies (GWAS), birth-defect diseases, molecular evolution, cancer research, etc. Recombination events cluster into short genomic regions called "recombination hotspots". Recently, a zinc finger protein PRDM9 was reported to regulate recombination hotspots in human and mouse genomes. In addition, a 13-mer motif contained in the binding sites of PRDM9 is found to be enriched in human hotspots. However, this 13-mer motif only covers a fraction of hotspots, indicating that PRDM9 is not the only regulator of recombination hotspots. Therefore, the challenge of discovering other regulators of recombination hotspots becomes significant. Furthermore, recombination is a complex process. Hence, multiple proteins acting as machinery, rather than individual proteins, are more likely to carry out this process in a precise and stable manner. Therefore, the extension of the prediction of individual trans-regulators to protein complexes is also highly desired. In this paper, we introduce a pipeline to identify genes and protein complexes associated with recombination hotspots. First, we prioritize proteins associated with hotspots based on their preference of binding to hotspots and coldspots. Second, using the above identified genes as seeds, we apply the Random Walk with Restart algorithm (RWR) to propagate their influences to other proteins in protein-protein interaction (PPI) networks. Hence, many proteins without DNA-binding information will also be assigned a score to implicate their roles in recombination hotspots. Third, we construct sub-PPI networks induced by top genes ranked by RWR for various species (e.g., yeast, human and mouse) and detect protein complexes in those sub-PPI networks. The GO term analysis show that our prioritizing methods and the RWR algorithm are capable of identifying novel genes associated with

  18. Partitioning of genetic variation between regulatory and coding gene segments: the predominance of software variation in genes encoding introvert proteins.

    Science.gov (United States)

    Mitchison, A

    1997-01-01

    In considering genetic variation in eukaryotes, a fundamental distinction can be made between variation in regulatory (software) and coding (hardware) gene segments. For quantitative traits the bulk of variation, particularly that near the population mean, appears to reside in regulatory segments. The main exceptions to this rule concern proteins which handle extrinsic substances, here termed extrovert proteins. The immune system includes an unusually large proportion of this exceptional category, but even so its chief source of variation may well be polymorphism in regulatory gene segments. The main evidence for this view emerges from genome scanning for quantitative trait loci (QTL), which in the case of the immune system points to a major contribution of pro-inflammatory cytokine genes. Further support comes from sequencing of major histocompatibility complex (Mhc) class II promoters, where a high level of polymorphism has been detected. These Mhc promoters appear to act, in part at least, by gating the back-signal from T cells into antigen-presenting cells. Both these forms of polymorphism are likely to be sustained by the need for flexibility in the immune response. Future work on promoter polymorphism is likely to benefit from the input from genome informatics.

  19. [Knocking-out extra domain A alternative splice fragment of fibronectin using a clustered regularly interspaced short palindromic repeats/associated proteins 9 system].

    Science.gov (United States)

    Yang, Yue; Wang, Haicheng; Xu, Shuyu; Peng, Jing; Jiang, Jiuhui; Li, Cuiying

    2015-08-01

    To investigate the effect of the fibronectin extra domain A on the aggressiveness of salivary adenoid cystic carcinoma (SACC) cells, via the clustered regularly interspaced short palindromic repeats (CRISPR)/ associated proteins (Cas) system. One sgRNA was designed to target the upstream of the genome sequences of extra domain A(EDA) exon and the downstream. Then the sgRNA was linked into plasmid PX-330 and transfected into SACC-83 cells. PCR and DNA sequence were used to testify the knockout cells, and the monoclones of EDA absent SACC cells were selected (A+C-2, A+C-6, B+C-10). CCK-8 cell proliferation and invasion was then tested in control group and the experimental group. The sgRNA was successfully linked into PX-330 plasmid. Part of adenoid cystic carcinoma cells' SACC-83 genomic EDA exon was knocked out, and the knockdown efficiency was above 70%, but the total amount of fibronectin did not change significantly. Three monoclones of EDA absent SACC- 83 cells were successfully selected with diminished migration and proliferation. The CRISPR/Cas9 system was a simplified system with relatively high knockout efficiency and EDA knockout could inhibiting SACC cell's mobility and invasiveness.

  20. Changes in RNA Splicing in Developing Soybean (Glycine max Embryos

    Directory of Open Access Journals (Sweden)

    Delasa Aghamirzaie

    2013-11-01

    Full Text Available Developing soybean seeds accumulate oils, proteins, and carbohydrates that are used as oxidizable substrates providing metabolic precursors and energy during seed germination. The accumulation of these storage compounds in developing seeds is highly regulated at multiple levels, including at transcriptional and post-transcriptional regulation. RNA sequencing was used to provide comprehensive information about transcriptional and post-transcriptional events that take place in developing soybean embryos. Bioinformatics analyses lead to the identification of different classes of alternatively spliced isoforms and corresponding changes in their levels on a global scale during soybean embryo development. Alternative splicing was associated with transcripts involved in various metabolic and developmental processes, including central carbon and nitrogen metabolism, induction of maturation and dormancy, and splicing itself. Detailed examination of selected RNA isoforms revealed alterations in individual domains that could result in changes in subcellular localization of the resulting proteins, protein-protein and enzyme-substrate interactions, and regulation of protein activities. Different isoforms may play an important role in regulating developmental and metabolic processes occurring at different stages in developing oilseed embryos.

  1. HOXA1 and TALE proteins display cross-regulatory interactions and form a combinatorial binding code on HOXA1 targets.

    Science.gov (United States)

    De Kumar, Bony; Parker, Hugo J; Paulson, Ariel; Parrish, Mark E; Pushel, Irina; Singh, Narendra Pratap; Zhang, Ying; Slaughter, Brian D; Unruh, Jay R; Florens, Laurence; Zeitlinger, Julia; Krumlauf, Robb

    2017-09-01

    Hoxa1 has diverse functional roles in differentiation and development. We identify and characterize properties of regions bound by HOXA1 on a genome-wide basis in differentiating mouse ES cells. HOXA1-bound regions are enriched for clusters of consensus binding motifs for HOX, PBX, and MEIS, and many display co-occupancy of PBX and MEIS. PBX and MEIS are members of the TALE family and genome-wide analysis of multiple TALE members (PBX, MEIS, TGIF, PREP1, and PREP2) shows that nearly all HOXA1 targets display occupancy of one or more TALE members. The combinatorial binding patterns of TALE proteins define distinct classes of HOXA1 targets, which may create functional diversity. Transgenic reporter assays in zebrafish confirm enhancer activities for many HOXA1-bound regions and the importance of HOX-PBX and TGIF motifs for their regulation. Proteomic analyses show that HOXA1 physically interacts on chromatin with PBX, MEIS, and PREP family members, but not with TGIF, suggesting that TGIF may have an independent input into HOXA1-bound regions. Therefore, TALE proteins appear to represent a wide repertoire of HOX cofactors, which may coregulate enhancers through distinct mechanisms. We also discover extensive auto- and cross-regulatory interactions among the Hoxa1 and TALE genes, indicating that the specificity of HOXA1 during development may be regulated though a complex cross-regulatory network of HOXA1 and TALE proteins. This study provides new insight into a regulatory network involving combinatorial interactions between HOXA1 and TALE proteins. © 2017 De Kumar et al.; Published by Cold Spring Harbor Laboratory Press.

  2. Spliced RNA of woodchuck hepatitis virus.

    Science.gov (United States)

    Ogston, C W; Razman, D G

    1992-07-01

    Polymerase chain reaction was used to investigate RNA splicing in liver of woodchucks infected with woodchuck hepatitis virus (WHV). Two spliced species were detected, and the splice junctions were sequenced. The larger spliced RNA has an intron of 1300 nucleotides, and the smaller spliced sequence shows an additional downstream intron of 1104 nucleotides. We did not detect singly spliced sequences from which the smaller intron alone was removed. Control experiments showed that spliced sequences are present in both RNA and DNA in infected liver, showing that the viral reverse transcriptase can use spliced RNA as template. Spliced sequences were detected also in virion DNA prepared from serum. The upstream intron produces a reading frame that fuses the core to the polymerase polypeptide, while the downstream intron causes an inframe deletion in the polymerase open reading frame. Whereas the splicing patterns in WHV are superficially similar to those reported recently in hepatitis B virus, we detected no obvious homology in the coding capacity of spliced RNAs from these two viruses.

  3. Structure of the Na,K-ATPase regulatory protein FXYD2b in micelles: implications for membrane-water interfacial arginines.

    Science.gov (United States)

    Gong, Xiao-Min; Ding, Yi; Yu, Jinghua; Yao, Yong; Marassi, Francesca M

    2015-01-01

    FXYD2 is a membrane protein responsible for regulating the function of the Na,K-ATPase in mammalian kidney epithelial cells. Here we report the structure of FXYD2b, one of two splice variants of the protein, determined by NMR spectroscopy in detergent micelles. Solid-state NMR characterization of the protein embedded in phospholipid bilayers indicates that several arginine side chains may be involved in hydrogen bond interactions with the phospholipid polar head groups. The structure and the NMR data suggest that FXYD2b could regulate the Na,K-ATPase by modulating the effective membrane surface electrostatics near the ion binding sites of the pump. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. Detection of alternative splice variants at the proteome level in Aspergillus flavus.

    Science.gov (United States)

    Chang, Kung-Yen; Georgianna, D Ryan; Heber, Steffen; Payne, Gary A; Muddiman, David C

    2010-03-05

    Identification of proteins from proteolytic peptides or intact proteins plays an essential role in proteomics. Researchers use search engines to match the acquired peptide sequences to the target proteins. However, search engines depend on protein databases to provide candidates for consideration. Alternative splicing (AS), the mechanism where the exon of pre-mRNAs can be spliced and rearranged to generate distinct mRNA and therefore protein variants, enable higher eukaryotic organisms, with only a limited number of genes, to have the requisite complexity and diversity at the proteome level. Multiple alternative isoforms from one gene often share common segments of sequences. However, many protein databases only include a limited number of isoforms to keep minimal redundancy. As a result, the database search might not identify a target protein even with high quality tandem MS data and accurate intact precursor ion mass. We computationally predicted an exhaustive list of putative isoforms of Aspergillus flavus proteins from 20 371 expressed sequence tags to investigate whether an alternative splicing protein database can assign a greater proportion of mass spectrometry data. The newly constructed AS database provided 9807 new alternatively spliced variants in addition to 12 832 previously annotated proteins. The searches of the existing tandem MS spectra data set using the AS database identified 29 new proteins encoded by 26 genes. Nine fungal genes appeared to have multiple protein isoforms. In addition to the discovery of splice variants, AS database also showed potential to improve genome annotation. In summary, the introduction of an alternative splicing database helps identify more proteins and unveils more information about a proteome.

  5. Both sides of the same coin: Rac1 splicing regulating by EGF signaling.

    Science.gov (United States)

    Fu, Xiang-Dong

    2017-04-01

    EGF, a well-studied mitogen for cancer cells, is revealed to induce an E3 ubiquitin ligase adaptor SPSB1, which recruits the Elongin B/C-Collin complex to trigger ubiquitylation of the negative splicing regulator hnRNP A1. This event is synergized with EGF-activated SR proteins to alter alternative splicing of a key small GTPase Rac1 to enhance cell migration, highlighting converging EGF signals on both negative and positive splicing regulators to jointly promote a key cancer pathway.

  6. Splicing factor 1 modulates dietary restriction and TORC1 pathway longevity in C. elegans

    DEFF Research Database (Denmark)

    Heintz, Caroline; Doktor, Thomas K; Lanjuin, Anne

    2017-01-01

    via splicing factor 1 (SFA-1; the C. elegans homologue of SF1, also known as branchpoint binding protein, BBP). We show that SFA-1 is specifically required for lifespan extension by dietary restriction and by modulation of the TORC1 pathway components AMPK, RAGA-1 and RSKS-1/S6 kinase. We also...... homeostasis is a biomarker and predictor of life expectancy in Caenorhabditis elegans. Using transcriptomics and in-depth splicing analysis in young and old animals fed ad libitum or subjected to dietary restriction, we find defects in global pre-mRNA splicing with age that are reduced by dietary restriction...

  7. The RNA-binding protein HOS5 and serine/arginine-rich proteins RS40 and RS41 participate in miRNA biogenesis in Arabidopsis

    KAUST Repository

    Chen, Tao

    2015-07-30

    MicroRNAs are a class of small regulatory RNAs that are generated from primary miRNA (pri-miRNA) transcripts with a stem-loop structure. Accuracy of the processing of pri-miRNA into mature miRNA in plants can be enhanced by SERRATE (SE) and HYPONASTIC LEAVES 1 (HYL1). HYL1 activity is regulated by the FIERY2 (FRY2)/RNA polymerase II C-terminal domain phosphatase-like 1 (CPL1). Here, we discover that HIGH OSMOTIC STRESS GENE EXPRESSION 5 (HOS5) and two serine/arginine-rich splicing factors RS40 and RS41, previously shown to be involved in pre-mRNA splicing, affect the biogenesis of a subset of miRNA. These proteins are required for correct miRNA strand selection and the maintenance of miRNA levels. FRY2 dephosphorylates HOS5 whose phosphorylation status affects its subnuclear localization. HOS5 and the RS proteins bind both intronless and intron-containing pri-miRNAs. Importantly, all of these splicing-related factors directly interact with both HYL1 and SE in nuclear splicing speckles. Our results indicate that these splicing factors are directly involved in the biogenesis of a group of miRNA.

  8. Heat shock protein 70 inhibits shrinkage-induced programmed cell death via mechanisms independent of effects on cell volume-regulatory membrane transport proteins

    DEFF Research Database (Denmark)

    Nylandsted, J; Jäättelä, M; Hoffmann, E K

    2004-01-01

    Cell shrinkage is a ubiquitous feature of programmed cell death (PCD), but whether it is an obligatory signalling event in PCD is unclear. Heat shock protein 70 (Hsp70) potently counteracts PCD in many cells, by mechanisms that are incompletely understood. In the present investigation, we found...... that severe hypertonic stress greatly diminished the viability of murine fibrosarcoma cells (WEHI-902) and immortalized murine embryonic fibroblasts (iMEFs). This effect was attenuated markedly by Hsp70 over-expression. To determine whether the protective effect of Hsp70 was mediated via an effect on volume...... regulatory ion transport, we compared regulatory volume decrease (RVD) and increase (RVI) in control WEHI-902 cells and after increasing Hsp70 levels by heat shock or over-expression (WEHI-912). Hsp70 levels affected neither RVD, RVI nor the relative contributions of the Na(+)/H(+)-exchanger (NHE1) and Na...

  9. A novel CDX2 isoform regulates alternative splicing.

    Directory of Open Access Journals (Sweden)

    Matthew E Witek

    Full Text Available Gene expression is a dynamic and coordinated process coupling transcription with pre-mRNA processing. This regulation enables tissue-specific transcription factors to induce expression of specific transcripts that are subsequently amplified by alternative splicing allowing for increased proteome complexity and functional diversity. The intestine-specific transcription factor CDX2 regulates development and maintenance of the intestinal epithelium by inducing expression of genes characteristic of the mature enterocyte phenotype. Here, sequence analysis of CDX2 mRNA from colonic mucosa-derived tissues revealed an alternatively spliced transcript (CDX2/AS that encodes a protein with a truncated homeodomain and a novel carboxy-terminal domain enriched in serine and arginine residues (RS domain. CDX2 and CDX2/AS exhibited distinct nuclear expression patterns with minimal areas of co-localization. CDX2/AS did not activate the CDX2-dependent promoter of guanylyl cyclase C nor inhibit transcriptional activity of CDX2. Unlike CDX2, CDX2/AS co-localized with the putative splicing factors ASF/SF2 and SC35. CDX2/AS altered splicing patterns of CD44v5 and Tra2-β1 minigenes in Lovo colon cancer cells independent of CDX2 expression. These data demonstrate unique dual functions of the CDX2 gene enabling it to regulate gene expression through both transcription (CDX2 and pre-mRNA processing (CDX2/AS.

  10. Automated Eukaryotic Gene Structure Annotation Using EVidenceModeler and the Program to Assemble Spliced Alignments

    Energy Technology Data Exchange (ETDEWEB)

    Haas, B J; Salzberg, S L; Zhu, W; Pertea, M; Allen, J E; Orvis, J; White, O; Buell, C R; Wortman, J R

    2007-12-10

    EVidenceModeler (EVM) is presented as an automated eukaryotic gene structure annotation tool that reports eukaryotic gene structures as a weighted consensus of all available evidence. EVM, when combined with the Program to Assemble Spliced Alignments (PASA), yields a comprehensive, configurable annotation system that predicts protein-coding genes and alternatively spliced isoforms. Our experiments on both rice and human genome sequences demonstrate that EVM produces automated gene structure annotation approaching the quality of manual curation.

  11. TCR Signal Strength Regulates Akt Substrate Specificity To Induce Alternate Murine Th and T Regulatory Cell Differentiation Programs.

    Science.gov (United States)

    Hawse, William F; Boggess, William C; Morel, Penelope A

    2017-07-15

    The Akt/mTOR pathway is a key driver of murine CD4 + T cell differentiation, and induction of regulatory T (Treg) cells results from low TCR signal strength and low Akt/mTOR signaling. However, strong TCR signals induce high Akt activity that promotes Th cell induction. Yet, it is unclear how Akt controls alternate T cell fate decisions. We find that the strength of the TCR signal results in differential Akt enzymatic activity. Surprisingly, the Akt substrate networks associated with T cell fate decisions are qualitatively different. Proteomic profiling of Akt signaling networks during Treg versus Th induction demonstrates that Akt differentially regulates RNA processing and splicing factors to drive T cell differentiation. Interestingly, heterogeneous nuclear ribonucleoprotein (hnRNP) L or hnRNP A1 are Akt substrates during Treg induction and have known roles in regulating the stability and splicing of key mRNAs that code for proteins in the canonical TCR signaling pathway, including CD3ζ and CD45. Functionally, inhibition of Akt enzymatic activity results in the dysregulation of splicing during T cell differentiation, and knockdown of hnRNP L or hnRNP A1 results in the lower induction of Treg cells. Together, this work suggests that a switch in substrate specificity coupled to the phosphorylation status of Akt may lead to alternative cell fates and demonstrates that proteins involved with alternative splicing are important factors in T cell fate decisions. Copyright © 2017 by The American Association of Immunologists, Inc.

  12. Crystallization and preliminary X-ray diffraction analysis of iron regulatory protein 1 in complex with ferritin IRE RNA

    International Nuclear Information System (INIS)

    Selezneva, Anna I.; Cavigiolio, Giorgio; Theil, Elizabeth C.; Walden, William E.; Volz, Karl

    2006-01-01

    The iron regulatory protein IRP1 has been crystallized in a complex with ferritin IRE RNA and a complete data set has been collected to 2.8 Å resolution. Iron regulatory protein 1 (IRP1) is a bifunctional protein with activity as an RNA-binding protein or as a cytoplasmic aconitase. Interconversion of IRP1 between these mutually exclusive states is central to cellular iron regulation and is accomplished through iron-responsive assembly and disassembly of a [4Fe–4S] cluster. When in its apo form, IRP1 binds to iron responsive elements (IREs) found in mRNAs encoding proteins of iron storage and transport and either prevents translation or degradation of the bound mRNA. Excess cellular iron stimulates the assembly of a [4Fe–4S] cluster in IRP1, inhibiting its IRE-binding ability and converting it to an aconitase. The three-dimensional structure of IRP1 in its different active forms will provide details of the interconversion process and clarify the selective recognition of mRNA, Fe–S sites and catalytic activity. To this end, the apo form of IRP1 bound to a ferritin IRE was crystallized. Crystals belong to the monoclinic space group P2 1 , with unit-cell parameters a = 109.6, b = 80.9, c = 142.9 Å, β = 92.0°. Native data sets have been collected from several crystals with resolution extending to 2.8 Å and the structure has been solved by molecular replacement

  13. Structural and regulatory diversity shape HLA-C protein expression levels

    DEFF Research Database (Denmark)

    Kaur, Gurman; Gras, Stephanie; Mobbs, Jesse I

    2017-01-01

    expression of HLA-C allomorphs at the cell surface by influencing the structure of the peptide-binding cleft and the diversity of peptides bound by the HLA-C molecules. Together with a phylogenetic analysis, these results highlight the diversity and long-term balancing selection of regulatory factors...

  14. Tumorigenic Properties of Iron Regulatory Protein 2 (IRP2) Mediated by Its Specific 73-Amino Acids Insert

    OpenAIRE

    Maffettone, Carmen; Chen, Guohua; Drozdov, Ignat; Ouzounis, Christos; Pantopoulos, Kostas

    2010-01-01

    Iron regulatory proteins, IRP1 and IRP2, bind to mRNAs harboring iron responsive elements and control their expression. IRPs may also perform additional functions. Thus, IRP1 exhibited apparent tumor suppressor properties in a tumor xenograft model. Here we examined the effects of IRP2 in a similar setting. Human H1299 lung cancer cells or clones engineered for tetracycline-inducible expression of wild type IRP2, or the deletion mutant IRP2(Delta73) (lacking a specific insert of 73 amino acid...

  15. Novel RNA-binding properties of the MTG chromatin regulatory proteins

    NARCIS (Netherlands)

    S. Rossetti (Stefano); L. van Unen (Leontine); N. Sacchi; A.T. Hoogeveen (Andre)

    2008-01-01

    textabstractBackground: The myeloid translocation gene (MTG) proteins are non-DNA-binding transcriptional regulators capable of interacting with chromatin modifying proteins. As a consequence of leukemia-associated chromosomal translocations, two of the MTG proteins, MTG8 and MTG16, are fused to the

  16. Cell Cycle and Apoptosis Regulatory Protein (CARP)-1 is Expressed inOsteoblasts and Regulated by PTH

    International Nuclear Information System (INIS)

    Sharma, Sonali; Mahalingam, Chandrika D.; Das, Varsha; Jamal, Shazia; Levi, Edi; Rishi, Arun K.; Datta, Nabanita S.

    2013-01-01

    Highlights: •CARP-1 is identified for the first time in bone cells. •PTH downregulates CARP-1 expression in differentiated osteoblasts. •PTH displaces CARP-1 from nucleus to the cytoplasm in differentiated osteoblasts. •Downregulation of CARP-1 by PTH involves PKA, PKC and P-p38 MAPK pathways. -- Abstract: Bone mass is dependent on osteoblast proliferation, differentiation and life-span of osteoblasts. Parathyroid hormone (PTH) controls osteoblast cell cycle regulatory proteins and suppresses mature osteoblasts apoptosis. Intermittent administration of PTH increases bone mass but the mechanism of action are complex and incompletely understood. Cell Cycle and Apoptosis Regulatory Protein (CARP)-1 (aka CCAR1) is a novel transducer of signaling by diverse agents including cell growth and differentiation factors. To gain further insight into the molecular mechanism, we investigated involvement of CARP-1 in PTH signaling in osteoblasts. Immunostaining studies revealed presence of CARP-1 in osteoblasts and osteocytes, while a minimal to absent levels were noted in the chondrocytes of femora from 10 to 12-week old mice. Treatment of 7-day differentiated MC3T3-E1 clone-4 (MC-4) mouse osteoblastic cells and primary calvarial osteoblasts with PTH for 30 min to 5 h followed by Western blot analysis showed 2- to 3-fold down-regulation of CARP-1 protein expression in a dose- and time-dependent manner compared to the respective vehicle treated control cells. H-89, a Protein Kinase A (PKA) inhibitor, suppressed PTH action on CARP-1 protein expression indicating PKA-dependent mechanism. PMA, a Protein Kinase C (PKC) agonist, mimicked PTH action, and the PKC inhibitor, GF109203X, partially blocked PTH-dependent downregulation of CARP-1, implying involvement of PKC. U0126, a Mitogen-Activated Protein Kinase (MAPK) Kinase (MEK) inhibitor, failed to interfere with CARP-1 suppression by PTH. In contrast, SB203580, p38 inhibitor, attenuated PTH down-regulation of CARP-1

  17. Cell Cycle and Apoptosis Regulatory Protein (CARP)-1 is Expressed inOsteoblasts and Regulated by PTH

    Energy Technology Data Exchange (ETDEWEB)

    Sharma, Sonali; Mahalingam, Chandrika D.; Das, Varsha [Department of Internal Medicine/Endocrinology, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Jamal, Shazia [Department of Oncology, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Levi, Edi [Department of Oncology, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Department of Pathology, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Rishi, Arun K. [Department of Oncology, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, MI 48201 (United States); VA Medical Center, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Datta, Nabanita S., E-mail: ndatta@med.wayne.edu [Department of Internal Medicine/Endocrinology, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Cardiovascular Research Institute, Wayne State University School of Medicine, Detroit, MI 48201 (United States)

    2013-07-12

    Highlights: •CARP-1 is identified for the first time in bone cells. •PTH downregulates CARP-1 expression in differentiated osteoblasts. •PTH displaces CARP-1 from nucleus to the cytoplasm in differentiated osteoblasts. •Downregulation of CARP-1 by PTH involves PKA, PKC and P-p38 MAPK pathways. -- Abstract: Bone mass is dependent on osteoblast proliferation, differentiation and life-span of osteoblasts. Parathyroid hormone (PTH) controls osteoblast cell cycle regulatory proteins and suppresses mature osteoblasts apoptosis. Intermittent administration of PTH increases bone mass but the mechanism of action are complex and incompletely understood. Cell Cycle and Apoptosis Regulatory Protein (CARP)-1 (aka CCAR1) is a novel transducer of signaling by diverse agents including cell growth and differentiation factors. To gain further insight into the molecular mechanism, we investigated involvement of CARP-1 in PTH signaling in osteoblasts. Immunostaining studies revealed presence of CARP-1 in osteoblasts and osteocytes, while a minimal to absent levels were noted in the chondrocytes of femora from 10 to 12-week old mice. Treatment of 7-day differentiated MC3T3-E1 clone-4 (MC-4) mouse osteoblastic cells and primary calvarial osteoblasts with PTH for 30 min to 5 h followed by Western blot analysis showed 2- to 3-fold down-regulation of CARP-1 protein expression in a dose- and time-dependent manner compared to the respective vehicle treated control cells. H-89, a Protein Kinase A (PKA) inhibitor, suppressed PTH action on CARP-1 protein expression indicating PKA-dependent mechanism. PMA, a Protein Kinase C (PKC) agonist, mimicked PTH action, and the PKC inhibitor, GF109203X, partially blocked PTH-dependent downregulation of CARP-1, implying involvement of PKC. U0126, a Mitogen-Activated Protein Kinase (MAPK) Kinase (MEK) inhibitor, failed to interfere with CARP-1 suppression by PTH. In contrast, SB203580, p38 inhibitor, attenuated PTH down-regulation of CARP-1

  18. Kinetin improves IKBKAP mRNA splicing in patients with familial dysautonomia

    Science.gov (United States)

    Axelrod, Felicia B.; Liebes, Leonard; Gold-von Simson, Gabrielle; Mendoza, Sandra; Mull, James; Leyne, Maire; Norcliffe-Kaufmann, Lucy; Kaufmann, Horacio; Slaugenhaupt, Susan A.

    2011-01-01

    Familial dysautonomia (FD) is caused by an intronic splice mutation in the IKBKAP gene that leads to partial skipping of exon 20 and tissue-specific reduction in I-κ-B kinase complex associated protein/ elongation protein 1 (IKAP/ELP-1) expression. Kinetin (6-furfurylaminopurine) has been shown to improve splicing and increase wild-type IKBKAP mRNA and IKAP protein expression in FD cell lines and carriers. To determine if oral kinetin treatment could alter mRNA splicing in FD subjects and was tolerable, we administered kinetin to eight FD individuals homozygous for the splice mutation. Subjects received 23.5 mg/Kg/day for 28 days. An increase in wild-type IKBKAP mRNA expression in leukocytes was noted after eight days in six of eight individuals; after 28 days the mean increase as compared to baseline was significant (p=0.002). We have demonstrated that kinetin is tolerable in this medically fragile population. Not only did kinetin produce the desired effect on splicing in FD patients, but also that effect appears to improve with time despite lack of dose change. This is the first report of a drug that produces in vivo mRNA splicing changes in individuals with FD and supports future long-term trials to determine if kinetin will prove therapeutic in FD patients. PMID:21775922

  19. Misregulation of Alternative Splicing in a Mouse Model of Rett Syndrome.

    Directory of Open Access Journals (Sweden)

    Ronghui Li

    2016-06-01

    Full Text Available Mutations in the human MECP2 gene cause Rett syndrome (RTT, a severe neurodevelopmental disorder that predominantly affects girls. Despite decades of work, the molecular function of MeCP2 is not fully understood. Here we report a systematic identification of MeCP2-interacting proteins in the mouse brain. In addition to transcription regulators, we found that MeCP2 physically interacts with several modulators of RNA splicing, including LEDGF and DHX9. These interactions are disrupted by RTT causing mutations, suggesting that they may play a role in RTT pathogenesis. Consistent with the idea, deep RNA sequencing revealed misregulation of hundreds of splicing events in the cortex of Mecp2 knockout mice. To reveal the functional consequence of altered RNA splicing due to the loss of MeCP2, we focused on the regulation of the splicing of the flip/flop exon of Gria2 and other AMPAR genes. We found a significant splicing shift in the flip/flop exon toward the flop inclusion, leading to a faster decay in the AMPAR gated current and altered synaptic transmission. In summary, our study identified direct physical interaction between MeCP2 and splicing factors, a novel MeCP2 target gene, and established functional connection between a specific RNA splicing change and synaptic phenotypes in RTT mice. These results not only help our understanding of the molecular function of MeCP2, but also reveal potential drug targets for future therapies.

  20. Human intronless genes: Functional groups, associated diseases, evolution, and mRNA processing in absence of splicing

    International Nuclear Information System (INIS)

    Grzybowska, Ewa A.

    2012-01-01

    Highlights: ► Functional characteristics of intronless genes (IGs). ► Diseases associated with IGs. ► Origin and evolution of IGs. ► mRNA processing without splicing. -- Abstract: Intronless genes (IGs) constitute approximately 3% of the human genome. Human IGs are essentially different in evolution and functionality from the IGs of unicellular eukaryotes, which represent the majority in their genomes. Functional analysis of IGs has revealed a massive over-representation of signal transduction genes and genes encoding regulatory proteins important for growth, proliferation, and development. IGs also often display tissue-specific expression, usually in the nervous system and testis. These characteristics translate into IG-associated diseases, mainly neuropathies, developmental disorders, and cancer. IGs represent recent additions to the genome, created mostly by retroposition of processed mRNAs with retained functionality. Processing, nuclear export, and translation of these mRNAs should be hampered dramatically by the lack of splice factors, which normally tightly cover mature transcripts and govern their fate. However, natural IGs manage to maintain satisfactory expression levels. Different mechanisms by which IGs solve the problem of mRNA processing and nuclear export are discussed here, along with their possible impact on reporter studies.

  1. New Insights into Functional Roles of the Polypyrimidine Tract-Binding Protein

    Directory of Open Access Journals (Sweden)

    Maria Grazia Romanelli

    2013-11-01

    Full Text Available Polypyrimidine Tract Binding Protein (PTB is an intensely studied RNA binding protein involved in several post-transcriptional regulatory events of gene expression. Initially described as a pre-mRNA splicing regulator, PTB is now widely accepted as a multifunctional protein shuttling between nucleus and cytoplasm. Accordingly, PTB can interact with selected RNA targets, structural elements and proteins. There is increasing evidence that PTB and its paralog PTBP2 play a major role as repressors of alternatively spliced exons, whose transcription is tissue-regulated. In addition to alternative splicing, PTB is involved in almost all steps of mRNA metabolism, including polyadenylation, mRNA stability and initiation of protein translation. Furthermore, it is well established that PTB recruitment in internal ribosome entry site (IRES activates the translation of picornaviral and cellular proteins. Detailed studies of the structural properties of PTB have contributed to our understanding of the mechanism of RNA binding by RNA Recognition Motif (RRM domains. In the present review, we will describe the structural properties of PTB, its paralogs and co-factors, the role in post-transcriptional regulation and actions in cell differentiation and pathogenesis. Defining the multifunctional roles of PTB will contribute to the understanding of key regulatory events in gene expression.

  2. CIR, a corepressor of CBF1, binds to PAP-1 and effects alternative splicing

    International Nuclear Information System (INIS)

    Maita, Hiroshi; Kitaura, Hirotake; Ariga, Hiroyoshi; Iguchi-Ariga, Sanae M.M.

    2005-01-01

    We have reported that PAP-1, a product of a causative gene for autosomal retinitis pigmentosa, plays a role in splicing. In this study, CIR, a protein originally identified as a CBF1-interacting protein and reported to act as a transcriptional corepressor, was identified as a PAP-1 binding protein and its function as a splicing factor was investigated. In addition to a basic lysine and acidic serine-rich (BA) domain and a zinc knuckle-like motif, CIR has an arginine/serine dipeptide repeat (RS) domain in its C terminal region. The RS domain has been reported to be present in the superfamily of SR proteins, which are involved in splicing reactions. We generated CIR mutants with deletions of each BA and RS domain and studied their subcellular localizations and interactions with PAP-1 and other SR proteins, including SC35, SF2/ASF, and U2AF 35 . CIR was found to interact with U2AF 35 through the BA domain, with SC35 and SF2/ASF through the RS domain, and with PAP-1 outside the BA domain in vivo and in vitro. CIR was found to be colocalized with SC35 and PAP-1 in nuclear speckles. Then the effect of CIR on splicing was investigated using the E1a minigene as a reporter in HeLa cells. Ectopic expression of CIR with the E1a minigene changed the ratio of spliced isoforms of E1a that were produced by alternative selection of 5'-splice sites. These results indicate that CIR is a member of the family of SR-related proteins and that CIR plays a role in splicing regulation

  3. CLIP-seq analysis of multi-mapped reads discovers novel functional RNA regulatory sites in the human transcriptome.

    Science.gov (United States)

    Zhang, Zijun; Xing, Yi

    2017-09-19

    Crosslinking or RNA immunoprecipitation followed by sequencing (CLIP-seq or RIP-seq) allows transcriptome-wide discovery of RNA regulatory sites. As CLIP-seq/RIP-seq reads are short, existing computational tools focus on uniquely mapped reads, while reads mapped to multiple loci are discarded. We present CLAM (CLIP-seq Analysis of Multi-mapped reads). CLAM uses an expectation-maximization algorithm to assign multi-mapped reads and calls peaks combining uniquely and multi-mapped reads. To demonstrate the utility of CLAM, we applied it to a wide range of public CLIP-seq/RIP-seq datasets involving numerous splicing factors, microRNAs and m6A RNA methylation. CLAM recovered a large number of novel RNA regulatory sites inaccessible by uniquely mapped reads. The functional significance of these sites was demonstrated by consensus motif patterns and association with alternative splicing (splicing factors), transcript abundance (AGO2) and mRNA half-life (m6A). CLAM provides a useful tool to discover novel protein-RNA interactions and RNA modification sites from CLIP-seq and RIP-seq data, and reveals the significant contribution of repetitive elements to the RNA regulatory landscape of the human transcriptome. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  4. SplicePlot: a utility for visualizing splicing quantitative trait loci.

    Science.gov (United States)

    Wu, Eric; Nance, Tracy; Montgomery, Stephen B

    2014-04-01

    RNA sequencing has provided unprecedented resolution of alternative splicing and splicing quantitative trait loci (sQTL). However, there are few tools available for visualizing the genotype-dependent effects of splicing at a population level. SplicePlot is a simple command line utility that produces intuitive visualization of sQTLs and their effects. SplicePlot takes mapped RNA sequencing reads in BAM format and genotype data in VCF format as input and outputs publication-quality Sashimi plots, hive plots and structure plots, enabling better investigation and understanding of the role of genetics on alternative splicing and transcript structure. Source code and detailed documentation are available at http://montgomerylab.stanford.edu/spliceplot/index.html under Resources and at Github. SplicePlot is implemented in Python and is supported on Linux and Mac OS. A VirtualBox virtual machine running Ubuntu with SplicePlot already installed is also available.

  5. A novel processing system of sterol regulatory element-binding protein-1c regulated by polyunsaturated fatty acid.

    Science.gov (United States)

    Nakakuki, Masanori; Kawano, Hiroyuki; Notsu, Tatsuto; Imada, Kazunori; Mizuguchi, Kiyoshi; Shimano, Hitoshi

    2014-05-01

    The proteolytic cascade is the key step in transactivation of sterol regulatory element-binding proteins (SREBPs), a transcriptional factor of lipid synthesis. Proteolysis of SREBP-2 is strictly regulated by sterols, but that of SREBP-1c was not strongly sterol-regulated, but inhibited by polyunsaturated fatty acids (PUFAs). In this study, the proteolytic processing of SREBP-1 and -2 was examined by transfection studies of cDNA-encoding mutants in which all the known cleavage sites were disrupted. In cultured cells, sterol-regulated SREBP-2 processing was completely eliminated by mutation of cleavage sites. In contrast, the corresponding SREBP-1c mutants as well as wild type exhibited large amounts of cleaved products in the nuclear extracts from culture cells and murine liver in vivo. The nuclear form of the mutant SREBP-1c was induced by delipidated condition and suppressed by eicosapentaenoic acid, an n-3 PUFA, but not by sterols. This novel processing mechanism was affected by neither SREBP cleavage-activating protein (SCAP) nor insulin-induced gene (Insig)-1, unlike SREBP-2, but abolished by a serine protease inhibitor. Through analysis of deletion mutant, a site-2 protease recognition sequence (DRSR) was identified to be involved in this novel processing. These findings suggest that SREBP-1c cleavage could be subjected to a novel PUFA-regulated cleavage system in addition to the sterol-regulatory SCAP/Insig system.

  6. Loss of Pnn expression attenuates expression levels of SR family splicing factors and modulates alternative pre-mRNA splicing in vivo

    International Nuclear Information System (INIS)

    Chiu Yali; Ouyang Pin

    2006-01-01

    SR and SR-related proteins have been implicated as trans-acting factors that play an important role in splice selection and are involved at specific stages of spliceosome formation. A well-established property of SR protein splicing factors is their ability to influence selection of alternative splice sites in a concentration-dependent manner. Identification of molecules that regulate SR family protein expression is therefore of vital importance in RNA biology. Here we report that depletion of Pnn expression, a SR-related protein with functions involved in pre-mRNA splicing and mRNA export, induces reduced expression of a subset of cellular proteins, especially that of SR family proteins, including SC35, SRm300, SRp55, and SRp40, but not that of other nuclear proteins, such as p53, Mdm2, and ki67. Knocking down Pnn expression was achieved in vitro by siRNA transfection. Expression levels of SR and SR-related proteins in Pnn-depleted cells as compared to those in control cells were evaluated by immunofluorescent staining and Western blot with specific antibodies. In addition, we also demonstrate that loss of Pnn expression could modulate splice site selection of model reporter gene in vivo. Our finding is significant in terms of regulation of SR protein cellular concentration because it reveals that Pnn may play a general role in the control of the cellular amount of family SR proteins through down-regulation of its own expression, thereby providing us with a better understanding of the cellular mechanism by which Pnn fulfills its biological function

  7. Capacity of columns with splice imperfections

    International Nuclear Information System (INIS)

    Popov, E.P.; Stephen, R.M.

    1977-01-01

    To study the behavior of spliced columns subjected to tensile forces simulating situations which may develop in an earthquake, all of the spliced specimens were tested to failure in tension after first having been subjected to large compressive loads. The results of these tests indicate that the lack of perfect contact at compression splices of columns may not be important, provided that the gaps are shimmed and welding is used to maintain the sections in alignment

  8. Phosphorylation of protein kinase A (PKA) regulatory subunit RIα by protein kinase G (PKG) primes PKA for catalytic activity in cells.

    Science.gov (United States)

    Haushalter, Kristofer J; Casteel, Darren E; Raffeiner, Andrea; Stefan, Eduard; Patel, Hemal H; Taylor, Susan S

    2018-03-23

    cAMP-dependent protein kinase (PKAc) is a pivotal signaling protein in eukaryotic cells. PKAc has two well-characterized regulatory subunit proteins, RI and RII (each having α and β isoforms), which keep the PKAc catalytic subunit in a catalytically inactive state until activation by cAMP. Previous reports showed that the RIα regulatory subunit is phosphorylated by cGMP-dependent protein kinase (PKG) in vitro , whereupon phosphorylated RIα no longer inhibits PKAc at normal (1:1) stoichiometric ratios. However, the significance of this phosphorylation as a mechanism for activating type I PKA holoenzymes has not been fully explored, especially in cellular systems. In this study, we further examined the potential of RIα phosphorylation to regulate physiologically relevant "desensitization" of PKAc activity. First, the serine 101 site of RIα was validated as a target of PKGIα phosphorylation both in vitro and in cells. Analysis of a phosphomimetic substitution in RIα (S101E) showed that modification of this site increases PKAc activity in vitro and in cells, even without cAMP stimulation. Numerous techniques were used to show that although Ser 101 variants of RIα can bind PKAc, the modified linker region of the S101E mutant has a significantly reduced affinity for the PKAc active site. These findings suggest that RIα phosphorylation may be a novel mechanism to circumvent the requirement of cAMP stimulus to activate type I PKA in cells. We have thus proposed a model to explain how PKG phosphorylation of RIα creates a "sensitized intermediate" state that is in effect primed to trigger PKAc activity.

  9. Evidence for the interaction of the regulatory protein Ki-1/57 with p53 and its interacting proteins

    International Nuclear Information System (INIS)

    Nery, Flavia C.; Rui, Edmilson; Kuniyoshi, Tais M.; Kobarg, Joerg

    2006-01-01

    Ki-1/57 is a cytoplasmic and nuclear phospho-protein of 57 kDa and interacts with the adaptor protein RACK1, the transcription factor MEF2C, and the chromatin remodeling factor CHD3, suggesting that it might be involved in the regulation of transcription. Here, we describe yeast two-hybrid studies that identified a total of 11 proteins interacting with Ki-1/57, all of which interact or are functionally associated with p53 or other members of the p53 family of proteins. We further found that Ki-1/57 is able to interact with p53 itself in the yeast two-hybrid system when the interaction was tested directly. This interaction could be confirmed by pull down assays with purified proteins in vitro and by reciprocal co-immunoprecipitation assays from the human Hodgkin analogous lymphoma cell line L540. Furthermore, we found that the phosphorylation of p53 by PKC abolishes its interaction with Ki-1/57 in vitro

  10. Alternative Splicing in Neurogenesis and Brain Development.

    Science.gov (United States)

    Su, Chun-Hao; D, Dhananjaya; Tarn, Woan-Yuh

    2018-01-01

    Alternative splicing of precursor mRNA is an important mechanism that increases transcriptomic and proteomic diversity and also post-transcriptionally regulates mRNA levels. Alternative splicing occurs at high frequency in brain tissues and contributes to every step of nervous system development, including cell-fate decisions, neuronal migration, axon guidance, and synaptogenesis. Genetic manipulation and RNA sequencing have provided insights into the molecular mechanisms underlying the effects of alternative splicing in stem cell self-renewal and neuronal fate specification. Timely expression and perhaps post-translational modification of neuron-specific splicing regulators play important roles in neuronal development. Alternative splicing of many key transcription regulators or epigenetic factors reprograms the transcriptome and hence contributes to stem cell fate determination. During neuronal differentiation, alternative splicing also modulates signaling activity, centriolar dynamics, and metabolic pathways. Moreover, alternative splicing impacts cortical lamination and neuronal development and function. In this review, we focus on recent progress toward understanding the contributions of alternative splicing to neurogenesis and brain development, which has shed light on how splicing defects may cause brain disorders and diseases.

  11. Alternative Splicing in Neurogenesis and Brain Development

    Directory of Open Access Journals (Sweden)

    Chun-Hao Su

    2018-02-01

    Full Text Available Alternative splicing of precursor mRNA is an important mechanism that increases transcriptomic and proteomic diversity and also post-transcriptionally regulates mRNA levels. Alternative splicing occurs at high frequency in brain tissues and contributes to every step of nervous system development, including cell-fate decisions, neuronal migration, axon guidance, and synaptogenesis. Genetic manipulation and RNA sequencing have provided insights into the molecular mechanisms underlying the effects of alternative splicing in stem cell self-renewal and neuronal fate specification. Timely expression and perhaps post-translational modification of neuron-specific splicing regulators play important roles in neuronal development. Alternative splicing of many key transcription regulators or epigenetic factors reprograms the transcriptome and hence contributes to stem cell fate determination. During neuronal differentiation, alternative splicing also modulates signaling activity, centriolar dynamics, and metabolic pathways. Moreover, alternative splicing impacts cortical lamination and neuronal development and function. In this review, we focus on recent progress toward understanding the contributions of alternative splicing to neurogenesis and brain development, which has shed light on how splicing defects may cause brain disorders and diseases.

  12. Distinct responses of protein turnover regulatory pathways in hypoxia- and semistarvation-induced muscle atrophy

    NARCIS (Netherlands)

    de Theije, Chiel C.; Langen, Ramon C. J.; Lamers, Wouter H.; Schols, Annemie M. W. J.; Köhler, S. Eleonore

    2013-01-01

    The balance of muscle protein synthesis and degradation determines skeletal muscle mass. We hypothesized that hypoxia-induced muscle atrophy and alterations in the regulation of muscle protein turnover include a hypoxia-specific component, in addition to the observed effects of reduction in food

  13. Guanine nucleotide regulatory protein co-purifies with the D2-dopamine receptor

    International Nuclear Information System (INIS)

    Senogles, S.E.; Caron, M.G.

    1986-01-01

    The D 2 -dopamine receptor from bovine anterior pituitary was purified ∼1000 fold by affinity chromatography on CMOS-Sepharose. Reconstitution of the affinity-purified receptor into phospholipid vesicles revealed the presence of high and low affinity agonist sites as detected by N-n-propylnorapomorphine (NPA) competition experiments with 3 H-spiperone. High affinity agonist binding could be converted to the low affinity form by guanine nucleotides, indicating the presence of an endogenous guanine nucleotide binding protein (N protein) in the affinity-purified D 2 receptor preparations. Furthermore, this preparation contained an agonist-sensitive GTPase activity which was stimulated 2-3 fold over basal by 10 μM NPA. 35 S-GTPγS binding to these preparations revealed a stoichiometry of 0.4-0.7 mole N protein/mole receptor, suggesting the N protein may be specifically coupled with the purified D 2 -dopamine receptor and not present as a contaminant. Pertussis toxin treatment of the affinity purified receptor preparations prevented high affinity agonist binding, as well as agonist stimulation of the GTPase activity, presumably by inactivating the associated N protein. Pertussis toxin lead to the ADP-ribosylation of a protein of 39-40K on SDS-PAGE. These findings indicate that an endogenous N protein, N/sub i/ or N/sub o/, co-purifies with the D 2 -dopamine receptor which may reflect a precoupling of this receptor with an N protein within the membranes

  14. In situ detection of a heat-shock regulatory element binding protein using a soluble short synthetic enhancer sequence

    Energy Technology Data Exchange (ETDEWEB)

    Harel-Bellan, A; Brini, A T; Farrar, W L [National Cancer Institute, Frederick, MD (USA); Ferris, D K [Program Resources, Inc., Frederick, MD (USA); Robin, P [Institut Gustave Roussy, Villejuif (France)

    1989-06-12

    In various studies, enhancer binding proteins have been successfully absorbed out by competing sequences inserted into plasmids, resulting in the inhibition of the plasmid expression. Theoretically, such a result could be achieved using synthetic enhancer sequences not inserted into plasmids. In this study, a double stranded DNA sequence corresponding to the human heat shock regulatory element was chemically synthesized. By in vitro retardation assays, the synthetic sequence was shown to bind specifically a protein in extracts from the human T cell line Jurkat. When the synthetic enhancer was electroporated into Jurkat cells, not only the enhancer was shown to remain undegraded into the cells for up to 2 days, but also its was shown to bind intracellularly a protein. The binding was specific and was modulated upon heat shock. Furthermore, the binding protein was shown to be of the expected molecular weight by UV crosslinking. However, when the synthetic enhancer element was co-electroporated with an HSP 70-CAT reporter construct, the expression of the reporter plasmid was consistently enhanced in the presence of the exogenous synthetic enhancer.

  15. Splicing analysis of 14 BRCA1 missense variants classifies nine variants as pathogenic

    DEFF Research Database (Denmark)

    Ahlborn, Lise B; Dandanell, Mette; Steffensen, Ane Y

    2015-01-01

    by functional analysis at the protein level. Results from a validated mini-gene splicing assay indicated that nine BRCA1 variants resulted in splicing aberrations leading to truncated transcripts and thus can be considered pathogenic (c.4987A>T/p.Met1663Leu, c.4988T>A/p.Met1663Lys, c.5072C>T/p.Thr1691Ile, c......Pathogenic germline mutations in the BRCA1 gene predispose carriers to early onset breast and ovarian cancer. Clinical genetic screening of BRCA1 often reveals variants with uncertain clinical significance, complicating patient and family management. Therefore, functional examinations are urgently...... needed to classify whether these uncertain variants are pathogenic or benign. In this study, we investigated 14 BRCA1 variants by in silico splicing analysis and mini-gene splicing assay. All 14 alterations were missense variants located within the BRCT domain of BRCA1 and had previously been examined...

  16. Analysis and prediction of gene splice sites in four Aspergillus genomes

    DEFF Research Database (Denmark)

    Wang, Kai; Ussery, David; Brunak, Søren

    2009-01-01

    Several Aspergillus fungal genomic sequences have been published, with many more in progress. Obviously, it is essential to have high-quality, consistently annotated sets of proteins from each of the genomes, in order to make meaningful comparisons. We have developed a dedicated, publicly available......, splice site prediction program called NetAspGene, for the genus Aspergillus. Gene sequences from Aspergillus fumigatus, the most common mould pathogen, were used to build and test our model. Compared to many animals and plants, Aspergillus contains smaller introns; thus we have applied a larger window...... better splice site prediction than other available tools. NetAspGene will be very helpful for the study in Aspergillus splice sites and especially in alternative splicing. A webpage for NetAspGene is publicly available at http://www.cbs.dtu.dk/services/NetAspGene....

  17. The group II intron maturase: a reverse transcriptase and splicing factor go hand in hand.

    Science.gov (United States)

    Zhao, Chen; Pyle, Anna Marie

    2017-12-01

    The splicing of group II introns in vivo requires the assistance of a multifunctional intron encoded protein (IEP, or maturase). Each IEP is also a reverse-transcriptase enzyme that enables group II introns to behave as mobile genetic elements. During splicing or retro-transposition, each group II intron forms a tight, specific complex with its own encoded IEP, resulting in a highly reactive holoenzyme. This review focuses on the structural basis for IEP function, as revealed by recent crystal structures of an IEP reverse transcriptase domain and cryo-EM structures of an IEP-intron complex. These structures explain how the same IEP scaffold is utilized for intron recognition, splicing and reverse transcription, while providing a physical basis for understanding the evolutionary transformation of the IEP into the eukaryotic splicing factor Prp8. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Fast rate of evolution in alternatively spliced coding regions of mammalian genes

    Directory of Open Access Journals (Sweden)

    Nurtdinov Ramil N

    2006-04-01

    Full Text Available Abstract Background At least half of mammalian genes are alternatively spliced. Alternative isoforms are often genome-specific and it has been suggested that alternative splicing is one of the major mechanisms for generating protein diversity in the course of evolution. Another way of looking at alternative splicing is to consider sequence evolution of constitutive and alternative regions of protein-coding genes. Indeed, it turns out that constitutive and alternative regions evolve in different ways. Results A set of 3029 orthologous pairs of human and mouse alternatively spliced genes was considered. The rate of nonsynonymous substitutions (dN, the rate of synonymous substitutions (dS, and their ratio (ω = dN/dS appear to be significantly higher in alternatively spliced coding regions compared to constitutive regions. When N-terminal, internal and C-terminal alternatives are analysed separately, C-terminal alternatives appear to make the main contribution to the observed difference. The effects become even more pronounced in a subset of fast evolving genes. Conclusion These results provide evidence of weaker purifying selection and/or stronger positive selection in alternative regions and thus one more confirmation of accelerated evolution in alternative regions. This study corroborates the theory that alternative splicing serves as a testing ground for molecular evolution.

  19. Motile hepatocellular carcinoma cells preferentially secret sugar metabolism regulatory proteins via exosomes.

    Science.gov (United States)

    Zhang, Jing; Lu, Shaohua; Zhou, Ye; Meng, Kun; Chen, Zhipeng; Cui, Yizhi; Shi, Yunfeng; Wang, Tong; He, Qing-Yu

    2017-07-01

    Exosomes are deliverers of critically functional proteins, capable of transforming target cells in numerous cancers, including hepatocellular carcinoma (HCC). We hypothesize that the motility of HCC cells can be featured by comparative proteome of exosomes. Hence, we performed the super-SILAC-based MS analysis on the exosomes secreted by three human HCC cell lines, including the non-motile Hep3B cell, and the motile 97H and LM3 cells. More than 1400 exosomal proteins were confidently quantified in each MS analysis with highly biological reproducibility. We justified that 469 and 443 exosomal proteins represented differentially expressed proteins (DEPs) in the 97H/Hep3B and LM3/Hep3B comparisons, respectively. These DEPs focused on sugar metabolism-centric canonical pathways per ingenuity pathway analysis, which was consistent with the gene ontology analysis on biological process enrichment. These pathways included glycolysis I, gluconeogenesis I and pentose phosphate pathways; and the DEPs enriched in these pathways could form a tightly connected network. By analyzing the relative abundance of proteins and translating mRNAs, we found significantly positive correlation between exosomes and cells. The involved exosomal proteins were again focusing on sugar metabolism. In conclusion, motile HCC cells tend to preferentially export more sugar metabolism-associated proteins via exosomes that differentiate them from non-motile HCC cells. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Identification of a novel Leucine-rich repeat protein and candidate PP1 regulatory subunit expressed in developing spermatids

    Directory of Open Access Journals (Sweden)

    Sperry Ann O

    2008-01-01

    . TLRR is homologous to a class of regulatory subunits for PP1, a central phosphatase in the reversible phosphorylation of proteins that is key to modulation of many intracellular processes. TLRR may serve to target this important signaling molecule near the nucleus of developing spermatids in order to control the cellular rearrangements of spermiogenesis.

  1. Selection on Coding and Regulatory Variation Maintains Individuality in Major Urinary Protein Scent Marks in Wild Mice.

    Directory of Open Access Journals (Sweden)

    Michael J Sheehan

    2016-03-01

    Full Text Available Recognition of individuals by scent is widespread across animal taxa. Though animals can often discriminate chemical blends based on many compounds, recent work shows that specific protein pheromones are necessary and sufficient for individual recognition via scent marks in mice. The genetic nature of individuality in scent marks (e.g. coding versus regulatory variation and the evolutionary processes that maintain diversity are poorly understood. The individual signatures in scent marks of house mice are the protein products of a group of highly similar paralogs in the major urinary protein (Mup gene family. Using the offspring of wild-caught mice, we examine individuality in the major urinary protein (MUP scent marks at the DNA, RNA and protein levels. We show that individuality arises through a combination of variation at amino acid coding sites and differential transcription of central Mup genes across individuals, and we identify eSNPs in promoters. There is no evidence of post-transcriptional processes influencing phenotypic diversity as transcripts accurately predict the relative abundance of proteins in urine samples. The match between transcripts and urine samples taken six months earlier also emphasizes that the proportional relationships across central MUP isoforms in urine is stable. Balancing selection maintains coding variants at moderate frequencies, though pheromone diversity appears limited by interactions with vomeronasal receptors. We find that differential transcription of the central Mup paralogs within and between individuals significantly increases the individuality of pheromone blends. Balancing selection on gene regulation allows for increased individuality via combinatorial diversity in a limited number of pheromones.

  2. SplicingTypesAnno: annotating and quantifying alternative splicing events for RNA-Seq data.

    Science.gov (United States)

    Sun, Xiaoyong; Zuo, Fenghua; Ru, Yuanbin; Guo, Jiqiang; Yan, Xiaoyan; Sablok, Gaurav

    2015-04-01

    Alternative splicing plays a key role in the regulation of the central dogma. Four major types of alternative splicing have been classified as intron retention, exon skipping, alternative 5 splice sites or alternative donor sites, and alternative 3 splice sites or alternative acceptor sites. A few algorithms have been developed to detect splice junctions from RNA-Seq reads. However, there are few tools targeting at the major alternative splicing types at the exon/intron level. This type of analysis may reveal subtle, yet important events of alternative splicing, and thus help gain deeper understanding of the mechanism of alternative splicing. This paper describes a user-friendly R package, extracting, annotating and analyzing alternative splicing types for sequence alignment files from RNA-Seq. SplicingTypesAnno can: (1) provide annotation for major alternative splicing at exon/intron level. By comparing the annotation from GTF/GFF file, it identifies the novel alternative splicing sites; (2) offer a convenient two-level analysis: genome-scale annotation for users with high performance computing environment, and gene-scale annotation for users with personal computers; (3) generate a user-friendly web report and additional BED files for IGV visualization. SplicingTypesAnno is a user-friendly R package for extracting, annotating and analyzing alternative splicing types at exon/intron level for sequence alignment files from RNA-Seq. It is publically available at https://sourceforge.net/projects/splicingtypes/files/ or http://genome.sdau.edu.cn/research/software/SplicingTypesAnno.html. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  3. Assessment of orthologous splicing isoforms in human and mouse orthologous genes

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    Horner David S

    2010-10-01

    Full Text Available Abstract Background Recent discoveries have highlighted the fact that alternative splicing and alternative transcripts are the rule, rather than the exception, in metazoan genes. Since multiple transcript and protein variants expressed by the same gene are, by definition, structurally distinct and need not to be functionally equivalent, the concept of gene orthology should be extended to the transcript level in order to describe evolutionary relationships between structurally similar transcript variants. In other words, the identification of true orthology relationships between gene products now should progress beyond primary sequence and "splicing orthology", consisting in ancestrally shared exon-intron structures, is required to define orthologous isoforms at transcript level. Results As a starting step in this direction, in this work we performed a large scale human- mouse gene comparison with a twofold goal: first, to assess if and to which extent traditional gene annotations such as RefSeq capture genuine splicing orthology; second, to provide a more detailed annotation and quantification of true human-mouse orthologous transcripts defined as transcripts of orthologous genes exhibiting the same splicing patterns. Conclusions We observed an identical exon/intron structure for 32% of human and mouse orthologous genes. This figure increases to 87% using less stringent criteria for gene structure similarity, thus implying that for about 13% of the human RefSeq annotated genes (and about 25% of the corresponding transcripts we could not identify any mouse transcript showing sufficient similarity to be confidently assigned as a splicing ortholog. Our data suggest that current gene and transcript data may still be rather incomplete - with several splicing variants still unknown. The observation that alternative splicing produces large numbers of alternative transcripts and proteins, some of them conserved across species and others truly species

  4. Regulatory motifs for CREB-binding protein and Nfe2l2 transcription factors in the upstream enhancer of the mitochondrial uncoupling protein 1 gene.

    Science.gov (United States)

    Rim, Jong S; Kozak, Leslie P

    2002-09-13

    Thermogenesis against cold exposure in mammals occurs in brown adipose tissue (BAT) through mitochondrial uncoupling protein (UCP1). Expression of the Ucp1 gene is unique in brown adipocytes and is regulated tightly. The 5'-flanking region of the mouse Ucp1 gene contains cis-acting elements including PPRE, TRE, and four half-site cAMP-responsive elements (CRE) with BAT-specific enhancer elements. In the course of analyzing how these half-site CREs are involved in Ucp1 expression, we found that a DNA regulatory element for NF-E2 overlaps CRE2. Electrophoretic mobility shift assay and competition assays with the CRE2 element indicates that nuclear proteins from BAT, inguinal fat, and retroperitoneal fat tissue interact with the CRE2 motif (CGTCA) in a specific manner. A supershift assay using an antibody against the CRE-binding protein (CREB) shows specific affinity to the complex from CRE2 and nuclear extract of BAT. Additionally, Western blot analysis for phospho-CREB/ATF1 shows an increase in phosphorylation of CREB/ATF1 in HIB-1B cells after norepinephrine treatment. Transient transfection assay using luciferase reporter constructs also indicates that the two half-site CREs are involved in transcriptional regulation of Ucp1 in response to norepinephrine and cAMP. We also show that a second DNA regulatory element for NF-E2 is located upstream of the CRE2 region. This element, which is found in a similar location in the 5'-flanking region of the human and rodent Ucp1 genes, shows specific binding to rat and human NF-E2 by electrophoretic mobility shift assay with nuclear extracts from brown fat. Co-transfections with an Nfe2l2 expression vector and a luciferase reporter construct of the Ucp1 enhancer region provide additional evidence that Nfe2l2 is involved in the regulation of Ucp1 by cAMP-mediated signaling.

  5. A novel CARD containing splice-isoform of CIITA regulates nitric oxide synthesis in dendritic cells.

    Science.gov (United States)

    Huang, Dachuan; Lim, Sylvia; Chua, Rong Yuan Ray; Shi, Hong; Ng, Mah Lee; Wong, Siew Heng

    2010-03-01

    MHC class II expression is controlled mainly at transcriptional level by class II transactivator (CIITA), which is a non-DNA binding coactivator and serves as a master control factor for MHC class II genes expression. Here, we describe the function of a novel splice-isoform of CIITA, DC-expressed caspase inhibitory isoform of CIITA (or DC-CASPIC), and we show that the expression of DCCASPIC in DC is upregulated upon lipopolysaccharides (LPS) induction. DC-CASPIC localizes to mitochondria, and protein-protein interaction study demonstrates that DC-CASPIC interacts with caspases and inhibits its activity in DC. Consistently, DC-CASPIC suppresses caspases-induced degradation of nitric oxide synthase-2 (NOS2) and subsequently promotes the synthesis of nitric oxide (NO). NO is an essential regulatory molecule that modulates the capability of DC in stimulating T cell proliferation/activation in vitro; hence, overexpression of DC-CASPIC in DC enhances this stimulation. Collectively, our findings reveal that DC-CASPIC is a key molecule that regulates caspases activity and NO synthesis in DC.

  6. Phosphorylation inhibits DNA-binding of alternatively spliced aryl hydrocarbon receptor nuclear translocator

    International Nuclear Information System (INIS)

    Kewley, Robyn J.; Whitelaw, Murray L.

    2005-01-01

    The basic helix-loop-helix/PER-ARNT-SIM homology (bHLH/PAS) transcription factor ARNT (aryl hydrocarbon receptor nuclear translocator) is a key component of various pathways which induce the transcription of cytochrome P450 and hypoxia response genes. ARNT can be alternatively spliced to express Alt ARNT, containing an additional 15 amino acids immediately N-terminal to the DNA-binding basic region. Here, we show that ARNT and Alt ARNT proteins are differentially phosphorylated by protein kinase CKII in vitro. Phosphorylation had an inhibitory effect on DNA-binding to an E-box probe by Alt ARNT, but not ARNT, homodimers. This inhibitory phosphorylation occurs through Ser77. Moreover, a point mutant, Alt ARNT S77A, shows increased activity on an E-box reporter gene, consistent with Ser77 being a regulatory site in vivo. In contrast, DNA binding by an Alt ARNT/dioxin receptor heterodimer to the xenobiotic response element is not inhibited by phosphorylation with CKII, nor does Alt ARNT S77A behave differently from wild type Alt ARNT in the context of a dioxin receptor heterodimer

  7. Coordination of Hepatitis C Virus Assembly by Distinct Regulatory Regions in Nonstructural Protein 5A.

    Directory of Open Access Journals (Sweden)

    Margarita Zayas

    2016-01-01

    Full Text Available Hepatitis C virus (HCV nonstructural protein (NS5A is a RNA-binding protein composed of a N-terminal membrane anchor, a structured domain I (DI and two intrinsically disordered domains (DII and DIII interacting with viral and cellular proteins. While DI and DII are essential for RNA replication, DIII is required for assembly. How these processes are orchestrated by NS5A is poorly understood. In this study, we identified a highly conserved basic cluster (BC at the N-terminus of DIII that is critical for particle assembly. We generated BC mutants and compared them with mutants that are blocked at different stages of the assembly process: a NS5A serine cluster (SC mutant blocked in NS5A-core interaction and a mutant lacking the envelope glycoproteins (ΔE1E2. We found that BC mutations did not affect core-NS5A interaction, but strongly impaired core-RNA association as well as virus particle envelopment. Moreover, BC mutations impaired RNA-NS5A interaction arguing that the BC might be required for loading of core protein with viral RNA. Interestingly, RNA-core interaction was also reduced with the ΔE1E2 mutant, suggesting that nucleocapsid formation and envelopment are coupled. These findings argue for two NS5A DIII determinants regulating assembly at distinct, but closely linked steps: (i SC-dependent recruitment of replication complexes to core protein and (ii BC-dependent RNA genome delivery to core protein, triggering encapsidation that is tightly coupled to particle envelopment. These results provide a striking example how a single viral protein exerts multiple functions to coordinate the steps from RNA replication to the assembly of infectious virus particles.

  8. Purification and binding analysis of the nitrogen fixation regulatory NifA protein from Azospirillum brasilense

    Directory of Open Access Journals (Sweden)

    L.M.P. Passaglia

    1998-11-01

    Full Text Available NifA protein activates transcription of nitrogen fixation operons by the alternative sigma54 holoenzyme form of RNA polymerase. This protein binds to a well-defined upstream activator sequence (UAS located at the -200/-100 position of nif promoters with the consensus motif TGT-N10-ACA. NifA of Azospirillum brasilense was purified in the form of a glutathione-S-transferase (GST-NifA fusion protein and proteolytic release of GST yielded inactive and partially soluble NifA. However, the purified NifA was able to induce the production of specific anti-A. brasilense NifA-antiserum that recognized NifA from A. brasilense but not from K. pneumoniae. Both GST-NifA and NifA expressed from the E. coli tac promoter are able to activate transcription from the nifHDK promoter but only in an A. brasilense background. In order to investigate the mechanism that regulates NifA binding capacity we have used E. coli total protein extracts expressing A. brasilense nifA in mobility shift assays. DNA fragments carrying the two overlapping, wild-type or mutated UAS motifs present in the nifH promoter region revealed a retarded band of related size. These data show that the binding activity present in the C-terminal domain of A. brasilense NifA protein is still functional even in the presence of oxygen.

  9. MAPA distinguishes genotype-specific variability of highly similar regulatory protein isoforms in potato tuber.

    Science.gov (United States)

    Hoehenwarter, Wolfgang; Larhlimi, Abdelhalim; Hummel, Jan; Egelhofer, Volker; Selbig, Joachim; van Dongen, Joost T; Wienkoop, Stefanie; Weckwerth, Wolfram

    2011-07-01

    Mass Accuracy Precursor Alignment is a fast and flexible method for comparative proteome analysis that allows the comparison of unprecedented numbers of shotgun proteomics analyses on a personal computer in a matter of hours. We compared 183 LC-MS analyses and more than 2 million MS/MS spectra and could define and separate the proteomic phenotypes of field grown tubers of 12 tetraploid cultivars of the crop plant Solanum tuberosum. Protein isoforms of patatin as well as other major gene families such as lipoxygenase and cysteine protease inhibitor that regulate tuber development were found to be the primary source of variability between the cultivars. This suggests that differentially expressed protein isoforms modulate genotype specific tuber development and the plant phenotype. We properly assigned the measured abundance of tryptic peptides to different protein isoforms that share extensive stretches of primary structure and thus inferred their abundance. Peptides unique to different protein isoforms were used to classify the remaining peptides assigned to the entire subset of isoforms based on a common abundance profile using multivariate statistical procedures. We identified nearly 4000 proteins which we used for quantitative functional annotation making this the most extensive study of the tuber proteome to date.

  10. Identification of alternative splice variants in Aspergillus flavus through comparison of multiple tandem MS search algorithms

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    Chang Kung-Yen

    2011-07-01

    Full Text Available Abstract Background Database searching is the most frequently used approach for automated peptide assignment and protein inference of tandem mass spectra. The results, however, depend on the sequences in target databases and on search algorithms. Recently by using an alternative splicing database, we identified more proteins than with the annotated proteins in Aspergillus flavus. In this study, we aimed at finding a greater number of eligible splice variants based on newly available transcript sequences and the latest genome annotation. The improved database was then used to compare four search algorithms: Mascot, OMSSA, X! Tandem, and InsPecT. Results The updated alternative splicing database predicted 15833 putative protein variants, 61% more than the previous results. There was transcript evidence for 50% of the updated genes compared to the previous 35% coverage. Database searches were conducted using the same set of spectral data, search parameters, and protein database but with different algorithms. The false discovery rates of the peptide-spectrum matches were estimated Conclusions We were able to detect dozens of new peptides using the improved alternative splicing database with the recently updated annotation of the A. flavus genome. Unlike the identifications of the peptides and the RefSeq proteins, large variations existed between the putative splice variants identified by different algorithms. 12 candidates of putative isoforms were reported based on the consensus peptide-spectrum matches. This suggests that applications of multiple search engines effectively reduced the possible false positive results and validated the protein identifications from tandem mass spectra using an alternative splicing database.

  11. Impairment of interferon regulatory factor-3 activation by hepatitis C virus core protein basic amino acid region 1.

    Science.gov (United States)

    Inoue, Kazuaki; Tsukiyama-Kohara, Kyoko; Matsuda, Chiho; Yoneyama, Mitsutoshi; Fujita, Takashi; Kuge, Shusuke; Yoshiba, Makoto; Kohara, Michinori

    2012-11-30

    Interferon regulatory factor-3 (IRF-3), a key transcriptional factor in the type I interferon system, is frequently impaired by hepatitis C virus (HCV), in order to establish persistent infection. However, the exact mechanism by which the virus establishes persistent infection has not been fully understood yet. The present study aimed to investigate the effects of various HCV proteins on IRF-3 activation, and elucidate the underlying mechanisms. To achieve this, full-length HCV and HCV subgenomic constructs corresponding to structural and each of the nonstructural proteins were transiently transfected into HepG2 cells. IFN-β induction, plaque formation, and IRF-3 dimerization were elicited by Newcastle disease virus (NDV) infection. The expressions of IRF-3 homodimer and its monomer, Ser386-phosphorylated IRF-3, and HCV core protein were detected by immunofluorescence and western blotting. IFN-β mRNA expression was quantified by real-time PCR (RT-PCR), and IRF-3 activity was measured by the levels of IRF-3 dimerization and phosphorylation, induced by NDV infection or polyriboinosinic:polyribocytidylic acid [poly(I:C)]. Switching of the expression of the complete HCV genome as well as the core proteins, E1, E2, and NS2, suppressed IFN-β mRNA levels and IRF-3 dimerization, induced by NDV infection. Our study revealed a crucial region of the HCV core protein, basic amino acid region 1 (BR1), to inhibit IRF-3 dimerization as well as its phosphorylation induced by NDV infection and poly (I:C), thus interfering with IRF-3 activation. Therefore, our study suggests that rescue of the IRF-3 pathway impairment may be an effective treatment for HCV infection. Copyright © 2012 Elsevier Inc. All rights reserved.

  12. Phosphorylation and mRNA splicing of collapsin response mediator protein-2 determine inhibition of rho-associated protein kinase (ROCK) II function in carcinoma cell migration and invasion

    DEFF Research Database (Denmark)

    Morgan-Fisher, Marie; Couchman, John R; Yoneda, Atsuko

    2013-01-01

    The Rho-associated protein kinases (ROCK I and II) are central regulators of important cellular processes such as migration and invasion downstream of the GTP-Rho. Recently, we reported collapsin response mediator protein (CRMP)-2 as an endogenous ROCK II inhibitor. To reveal how the CRMP-2-ROCK II......, the presented data show that CRMP-2-dependent regulation of ROCK II activity is mediated through interaction of the CRMP-2L N terminus with the ROCK II catalytic domain as well as by GSK3-dependent phosphorylation of CRMP-2....

  13. Uncovering packaging features of co-regulated modules based on human protein interaction and transcriptional regulatory networks

    Directory of Open Access Journals (Sweden)

    He Weiming

    2010-07-01

    Full Text Available Abstract Background Network co-regulated modules are believed to have the functionality of packaging multiple biological entities, and can thus be assumed to coordinate many biological functions in their network neighbouring regions. Results Here, we weighted edges of a human protein interaction network and a transcriptional regulatory network to construct an integrated network, and introduce a probabilistic model and a bipartite graph framework to exploit human co-regulated modules and uncover their specific features in packaging different biological entities (genes, protein complexes or metabolic pathways. Finally, we identified 96 human co-regulated modules based on this method, and evaluate its effectiveness by comparing it with four other methods. Conclusions Dysfunctions in co-regulated interactions often occur in the development of cancer. Therefore, we focussed on an example co-regulated module and found that it could integrate a number of cancer-related genes. This was extended to causal dysfunctions of some complexes maintained by several physically interacting proteins, thus coordinating several metabolic pathways that directly underlie cancer.

  14. pH-Regulatory Proteins as Potential Targets in Breast Cancer

    DEFF Research Database (Denmark)

    Andersen, Anne Poder

    tissues. The focus of the present PhD study is on understanding the mechanisms through which pH-regulatory transporters are regulated by the breast tumor microenvironment, and how these transporters in turn favor cancer progression. In Paper I, we summarized the recent knowledge on the dynamic...... exhibit distinct spatial organization during 3D growth of MCF-7 and MDA-MB-231 breast cancer cells. By pharmacological inhibition and stable shRNA-mediated knockdown, we addressed the specific contributions of the transporters to spheroid growth and show that the specific transporters contribute to breast...... development in the more complex in vivo setting. In Paper III, we found that chemical induction of breast cancer in NBCn1 knockout (KO) mice is delayed and tumor growth rates reduced, compared to those in wildtype (WT) mice, demonstrating a causal link between NBCn1 and breast cancer development. In Paper IV...

  15. Evolution of a G protein-coupled receptor response by mutations in regulatory network interactions

    DEFF Research Database (Denmark)

    Di Roberto, Raphaël B; Chang, Belinda; Trusina, Ala

    2016-01-01

    All cellular functions depend on the concerted action of multiple proteins organized in complex networks. To understand how selection acts on protein networks, we used the yeast mating receptor Ste2, a pheromone-activated G protein-coupled receptor, as a model system. In Saccharomyces cerevisiae......, Ste2 is a hub in a network of interactions controlling both signal transduction and signal suppression. Through laboratory evolution, we obtained 21 mutant receptors sensitive to the pheromone of a related yeast species and investigated the molecular mechanisms behind this newfound sensitivity. While...... demonstrate that a new receptor-ligand pair can evolve through network-altering mutations independently of receptor-ligand binding, and suggest a potential role for such mutations in disease....

  16. Integrative analysis of RNA, translation, and protein levels reveals distinct regulatory variation across humans.

    Science.gov (United States)

    Cenik, Can; Cenik, Elif Sarinay; Byeon, Gun W; Grubert, Fabian; Candille, Sophie I; Spacek, Damek; Alsallakh, Bilal; Tilgner, Hagen; Araya, Carlos L; Tang, Hua; Ricci, Emiliano; Snyder, Michael P

    2015-11-01

    Elucidating the consequences of genetic differences between humans is essential for understanding phenotypic diversity and personalized medicine. Although variation in RNA levels, transcription factor binding, and chromatin have been explored, little is known about global variation in translation and its genetic determinants. We used ribosome profiling, RNA sequencing, and mass spectrometry to perform an integrated analysis in lymphoblastoid cell lines from a diverse group of individuals. We find significant differences in RNA, translation, and protein levels suggesting diverse mechanisms of personalized gene expression control. Combined analysis of RNA expression and ribosome occupancy improves the identification of individual protein level differences. Finally, we identify genetic differences that specifically modulate ribosome occupancy--many of these differences lie close to start codons and upstream ORFs. Our results reveal a new level of gene expression variation among humans and indicate that genetic variants can cause changes in protein levels through effects on translation. © 2015 Cenik et al.; Published by Cold Spring Harbor Laboratory Press.

  17. Moonlighting microtubule-associated proteins: regulatory functions by day and pathological functions at night.

    Science.gov (United States)

    Oláh, J; Tőkési, N; Lehotzky, A; Orosz, F; Ovádi, J

    2013-11-01

    The sensing, integrating, and coordinating features of the eukaryotic cells are achieved by the complex ultrastructural arrays and multifarious functions of the cytoskeletal network. Cytoskeleton comprises fibrous protein networks of microtubules, actin, and intermediate filaments. These filamentous polymer structures are highly dynamic and undergo constant and rapid reorganization during cellular processes. The microtubular system plays a crucial role in the brain, as it is involved in an enormous number of cellular events including cell differentiation and pathological inclusion formation. These multifarious functions of microtubules can be achieved by their decoration with proteins/enzymes that exert specific effects on the dynamics and organization of the cytoskeleton and mediate distinct functions due to their moonlighting features. This mini-review focuses on two aspects of the microtubule cytoskeleton. On the one hand, we describe the heteroassociation of tubulin/microtubules with metabolic enzymes, which in addition to their catalytic activities stabilize microtubule structures via their cross-linking functions. On the other hand, we focus on the recently identified moonlighting tubulin polymerization promoting protein, TPPP/p25. TPPP/p25 is a microtubule-associated protein and it displays distinct physiological or pathological (aberrant) functions; thus it is a prototype of Neomorphic Moonlighting Proteins. The expression of TPPP/p25 is finely controlled in the human brain; this protein is indispensable for the development of projections of oligodendrocytes that are responsible for the ensheathment of axons. The nonphysiological, higher or lower TPPP/p25 level leads to distinct CNS diseases. Mechanisms contributing to the control of microtubule stability and dynamics by metabolic enzymes and TPPP/p25 will be discussed. Copyright © 2013 Wiley Periodicals, Inc.

  18. Interplay of the modified nucleotide phosphoadenosine 5'-phosphosulfate (PAPS) with global regulatory proteins in Escherichia coli: modulation of cyclic AMP (cAMP)-dependent gene expression and interaction with the HupA regulatory protein.

    Science.gov (United States)

    Longo, Francesca; Motta, Sara; Mauri, Pierluigi; Landini, Paolo; Rossi, Elio

    2016-11-25

    In the bacterium Escherichia coli, some intermediates of the sulfate assimilation and cysteine biosynthesis pathway can act as signal molecules and modulate gene expression. In addition to sensing and utilization of sulphur sources, these signaling mechanisms also impact more global cell processes, such as resistance to antimicrobial agents and biofilm formation. In a recent work, we have shown that inactivation of the cysH gene, encoding phosphoadenosine-phosphosulfate (PAPS) reductase, and the consequent increase in intracellular PAPS concentration, strongly affect production of several cell surface-associated structures, enhancing surface adhesion and cell aggregation. In order to identify the molecular mechanism relaying intracellular PAPS concentration to regulation of cell surface-associated structures, we looked for mutations able to suppress the effects of cysH inactivation. We found that mutations in the adenylate cyclase-encoding cyaA gene abolished the effects of PAPS accumulation; consistent with this result, cyclic AMP (cAMP)-dependent gene expression appears to be increased in the cysH mutant. Experiments aimed at the direct identification of proteins interacting with either CysC or CysH, i.e. the PAPS-related proteins APS kinase and PAPS reductase, allowed us to identify several regulators, namely, CspC, CspE, HNS and HupA. Protein-protein interaction between HupA and CysH was confirmed by a bacterial two hybrid system, and inactivation of the hupA gene enhanced the effects of the cysH mutation in terms of production of cell surface-associated factors. Our results indicate that PAPS can modulate different regulatory systems, providing evidence that this molecule acts as a global signal molecule in E. coli. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  19. Alternative Splicing Control of Abiotic Stress Responses.

    Science.gov (United States)

    Laloum, Tom; Martín, Guiomar; Duque, Paula

    2018-02-01

    Alternative splicing, which generates multiple transcripts from the same gene, is an important modulator of gene expression that can increase proteome diversity and regulate mRNA levels. In plants, this post-transcriptional mechanism is markedly induced in response to environmental stress, and recent studies have identified alternative splicing events that allow rapid adjustment of the abundance and function of key stress-response components. In agreement, plant mutants defective in splicing factors are severely impaired in their response to abiotic stress. Notably, mounting evidence indicates that alternative splicing regulates stress responses largely by targeting the abscisic acid (ABA) pathway. We review here current understanding of post-transcriptional control of plant stress tolerance via alternative splicing and discuss research challenges for the near future. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Multi-level Strategy for Identifying Proteasome-Catalyzed Spliced Epitopes Targeted by CD8+ T Cells during Bacterial Infection

    Directory of Open Access Journals (Sweden)

    Anouk C.M. Platteel

    2017-08-01

    Full Text Available Proteasome-catalyzed peptide splicing (PCPS generates peptides that are presented by MHC class I molecules, but because their identification is challenging, the immunological relevance of spliced peptides remains unclear. Here, we developed a reverse immunology-based multi-level approach to identify proteasome-generated spliced epitopes. Applying this strategy to a murine Listeria monocytogenes infection model, we identified two spliced epitopes within the secreted bacterial phospholipase PlcB that primed antigen-specific CD8+ T cells in L. monocytogenes-infected mice. While reacting to the spliced epitopes, these CD8+ T cells failed to recognize the non-spliced peptide parts in the context of their natural flanking sequences. Thus, we here show that PCPS expands the CD8+ T cell response against L. monocytogenes by exposing spliced epitopes on the cell surface. Moreover, our multi-level strategy opens up opportunities to systematically investigate proteins for spliced epitope candidates and thus strategies for immunotherapies or vaccine design.

  1. A Gene Gun-mediated Nonviral RNA trans-splicing Strategy for Col7a1 Repair

    Directory of Open Access Journals (Sweden)

    Patricia Peking

    2016-01-01

    Full Text Available RNA trans-splicing represents an auspicious option for the correction of genetic mutations at RNA level. Mutations within COL7A1 causing strong reduction or absence of type VII collagen are associated with the severe skin blistering disease dystrophic epidermolysis bullosa. The human COL7A1 mRNA constitutes a suitable target for this RNA therapy approach, as only a portion of the almost 9 kb transcript has to be delivered into the target cells. Here, we have proven the feasibility of 5′ trans-splicing into the Col7a1 mRNA in vitro and in vivo. We designed a 5′ RNA trans-splicing molecule, capable of replacing Col7a1 exons 1–15 and verified it in a fluorescence-based trans-splicing model system. Specific and efficient Col7a1 trans-splicing was confirmed in murine keratinocytes. To analyze trans-splicing in vivo, we used gene gun delivery of a minicircle expressing a FLAG-tagged 5′ RNA trans-splicing molecule into the skin of wild-type mice. Histological and immunofluorescence analysis of bombarded skin sections revealed vector delivery and expression within dermis and epidermis. Furthermore, we have detected trans-spliced type VII collagen protein using FLAG-tag antibodies. In conclusion, we describe a novel in vivo nonviral RNA therapy approach to restore type VII collagen expression for causative treatment of dystrophic epidermolysis bullosa.

  2. Malignant Tregs express low molecular splice forms of FOXP3 in Sézary syndrome

    DEFF Research Database (Denmark)

    Krejsgaard, T; Gjerdrum, L M; Ralfkiaer, E

    2008-01-01

    Sézary syndrome (SS) is an aggressive variant of cutaneous T-cell lymphoma. During disease progression, immunodeficiency develops; however, the underlying molecular and cellular mechanisms are not fully understood. Here, we study the regulatory T cell (Treg) function and the expression of FOXP3...... in SS. We demonstrate that malignant T cells in 8 of 15 patients stain positive with an anti-FOXP3 antibody. Western blotting analysis shows expression of two low molecular splice forms of FOXP3, but not of wild-type (wt) FOXP3. The malignant T cells produce interleukin-10 and TGF-beta and suppress...... the growth of non-malignant T cells. The Treg phenotype and the production of suppressive cytokines are driven by aberrant activation of Jak3 independent of the FOXP3 splice forms. In contrast to wt FOXP3, the low molecular splice forms of FOXP3 have no inhibitory effect on nuclear factor-kappaB (NF...

  3. High qualitative and quantitative conservation of alternative splicing in Caenorhabditis elegans and Caenorhabditis briggsae

    DEFF Research Database (Denmark)

    Rukov, Jakob Lewin; Irimia, Manuel; Mørk, Søren

    2007-01-01

    Alternative splicing (AS) is an important contributor to proteome diversity and is regarded as an explanatory factor for the relatively low number of human genes compared with less complex animals. To assess the evolutionary conservation of AS and its developmental regulation, we have investigated...... the qualitative and quantitative expression of 21 orthologous alternative splice events through the development of 2 nematode species separated by 85-110 Myr of evolutionary time. We demonstrate that most of these alternative splice events present in Caenorhabditis elegans are conserved in Caenorhabditis briggsae....... Moreover, we find that relative isoform expression levels vary significantly during development for 78% of the AS events and that this quantitative variation is highly conserved between the 2 species. Our results suggest that AS is generally tightly regulated through development and that the regulatory...

  4. Splicing of phenylalanine hydroxylase (PAH) exon 11 is vulnerable - Molecular pathology of mutations in PAH exon 11

    DEFF Research Database (Denmark)

    Heintz, Caroline; Dobrowolski, Steven F.; Andersen, Henriette Skovgaard

    2012-01-01

    as a vulnerable exon and used patient derived lymphoblast cell lines and PAH minigenes to study the molecular defect that impacted pre-mRNA processing. We showed that the c.1144T>C and c.1066-3C>T mutations cause exon 11 skipping, while the c.1139C>T mutation is neutral or slightly beneficial. The c.1144T......In about 20-30% of phenylketonuria (PKU) patients, phenylalanine (Phe) levels can be controlled by cofactor 6R-tetrahydrobiopterin (BH(4)) administration. The phenylalanine hydroxylase (PAH) genotype has a predictive value concerning BH(4)-response and therefore a correct assessment of the mutation...... molecular pathology is important. Mutations that disturb the splicing of exons (e.g. interplay between splice site strength and regulatory sequences like exon splicing enhancers (ESEs)/exon splicing silencers (ESSs)) may cause different severity of PKU. In this study, we identified PAH exon 11...

  5. Identification of Novel Targets of the Human Cell Cycle Regulatory Protein Cdc34

    Science.gov (United States)

    1999-07-01

    centrifugal elutriation, with a purity of -80% as shown by micro - ethyl acetate and separated on thin-layer chromatography plates (Whatman, scopic...Spain. (2) Servicio Bioquimica, Hosp. La Paz. Madrid. ICER protein is elevated in mHR6b-/- (murine Rad6B) fibroblasts Spain. and in human cells

  6. Conservation of protein abundance patterns reveals the regulatory architecture of the EGFR-MAPK pathway

    Energy Technology Data Exchange (ETDEWEB)

    Shi, T.; Niepel, M.; McDermott, J. E.; Gao, Y.; Nicora, C. D.; Chrisler, W. B.; Markillie, L. M.; Petyuk, V. A.; Smith, R. D.; Rodland, K. D.; Sorger, P. K.; Qian, W. -J.; Wiley, H. S.

    2016-07-12

    It is not known whether cancer cells generally show quantitative differences in the expression of signaling pathway proteins that could dysregulate signal transduction. To explore this issue, we first defined the primary components of the EGF-MAPK pathway in normal human mammary epithelial cells, identifying 16 core proteins and 10 feedback regulators. We then quantified their absolute abundance across a panel of normal and cancer cell lines. We found that core pathway proteins were expressed at very similar levels across all cell types. In contrast, the EGFR and transcriptionally controlled feedback regulators were expressed at highly variable levels. The absolute abundance of most core pathway proteins was between 50,000- 70,000 copies per cell, but the adaptors SOS1, SOS2, and GAB1 were found at far lower levels (2,000-5,000 per cell). MAPK signaling showed saturation in all cells between 3,000-10,000 occupied EGFR, consistent with the idea that low adaptor levels limit signaling. Our results suggest that the core MAPK pathway is essentially invariant across different cell types, with cell- specific differences in signaling likely due to variable levels of feedback regulators. The low abundance of adaptors relative to the EGFR could be responsible for previous observation of saturable signaling, endocytosis, and high affinity EGFR.

  7. Determinants of RNA binding and translational repression by the Bicaudal-C regulatory protein.

    Science.gov (United States)

    Zhang, Yan; Park, Sookhee; Blaser, Susanne; Sheets, Michael D

    2014-03-14

    Bicaudal-C (Bic-C) RNA binding proteins function as important translational repressors in multiple biological contexts within metazoans. However, their RNA binding sites are unknown. We recently demonstrated that Bic-C functions in spatially regulated translational repression of the xCR1 mRNA during Xenopus development. This repression contributes to normal development by confining the xCR1 protein, a regulator of key signaling pathways, to specific cells of the embryo. In this report, we combined biochemical approaches with in vivo mRNA reporter assays to define the minimal Bic-C target site within the xCR1 mRNA. This 32-nucleotide Bic-C target site is predicted to fold into a stem-loop secondary structure. Mutational analyses provided evidence that this stem-loop structure is important for Bic-C binding. The Bic-C target site was sufficient for Bic-C mediated repression in vivo. Thus, we describe the first RNA binding site for a Bic-C protein. This identification provides an important step toward understanding the mechanisms by which evolutionarily conserved Bic-C proteins control cellular function in metazoans.

  8. Expression of cytoskeleton regulatory protein Mena in human hepatocellular carcinoma and its prognostic significance.

    Science.gov (United States)

    Hu, Kunpeng; Wang, Jiani; Yao, Zhicheng; Liu, Bo; Lin, Yuan; Liu, Lei; Xu, Lihua

    2014-05-01

    The molecular mechanisms of the development and progression of hepatocellular carcinoma (HCC) are poorly understood. The main objective of this study was to analyze the expression of Enabled [mammalian Ena (Mena)] protein and its clinical significance in human HCC. The Mena expression was examined at mRNA and protein levels by real-time quantitative polymerase chain reaction and Western blotting analysis in ten paired HCC tissues and the adjacent normal tissues. The expression of Mena protein in 81 specimens of HCC tissues was determined by immunohistochemistry. Associations of Mena expression with the clinicopathological features were analyzed, and prognosis of HCC patients was evaluated. The result shows the expression of Mena mRNA and protein was higher in HCC than in the adjacent normal tissues in ten paired samples. Mena was mainly accumulated in the cytoplasm of tumor cells and over-expressed in 40.74% (33/81) patients by immunohistochemical staining. Over-expression of Mena was significantly associated with poor cellular differentiation (P = 0.025), advanced tumor stage (P = 0.003) and worse disease-free survival (DFS, P Mena is an independent prognostic factor for DFS in multivariate analysis (HR 2.309, 95% CI 1.104-4.828; P = 0.026). Mena is up-regulated in HCC and associated with tumor differentiation and clinical stage. Mena may be an independent prognostic marker for DFS of HCC patients.

  9. Upstream ORF affects MYCN translation depending on exon 1b alternative splicing

    International Nuclear Information System (INIS)

    Besançon, Roger; Puisieux, Alain; Valsesia-Wittmann, Sandrine; Locher, Clara; Delloye-Bourgeois, Céline; Furhman, Lydie; Tutrone, Giovani; Bertrand, Christophe; Jallas, Anne-Catherine; Garin, Elisabeth

    2009-01-01

    The MYCN gene is transcribed into two major mRNAs: one full-length (MYCN) and one exon 1b-spliced (MYCN Δ1b ) mRNA. But nothing is known about their respective ability to translate the MYCN protein. Plasmids were prepared to enable translation from the upstream (uORF) and major ORF of the two MYCN transcripts. Translation was studied after transfection in neuroblastoma SH-EP cell line. Impact of the upstream AUG on translation was evaluated after directed mutagenesis. Functional study with the two MYCN mRNAs was conducted by a cell viability assay. Existence of a new protein encoded by the MYCN Δ1b uORF was explored by designing a rabbit polyclonal antibody against a specific epitope of this protein. Both are translated, but higher levels of protein were seen with MYCN Δ1b mRNA. An upstream ORF was shown to have positive cis-regulatory activity on translation from MYCN but not from MYCN Δ1b mRNA. In transfected SH-EP neuroblastoma cells, high MYCN dosage obtained with MYCN Δ1b mRNA translation induces an antiapoptotic effect after serum deprivation that was not observed with low MYCN expression obtained with MYCN mRNA. Here, we showed that MYCNOT: MYCN Overlap Transcript, a new protein of unknown function is translated from the upstream AUG of MYCN Δ1b mRNA. Existence of upstream ORF in MYCN transcripts leads to a new level of MYCN regulation. The resulting MYCN dosage has a weak but significant anti-apoptotic activity after intrinsic apoptosis induction

  10. WrpA Is an Atypical Flavodoxin Family Protein under Regulatory Control of the Brucella abortus General Stress Response System.

    Science.gov (United States)

    Herrou, Julien; Czyż, Daniel M; Willett, Jonathan W; Kim, Hye-Sook; Chhor, Gekleng; Babnigg, Gyorgy; Kim, Youngchang; Crosson, Sean

    2016-04-01

    The general stress response (GSR) system of the intracellular pathogen Brucella abortus controls the transcription of approximately 100 genes in response to a range of stress cues. The core genetic regulatory components of the GSR are required for B. abortus survival under nonoptimal growth conditions in vitro and for maintenance of chronic infection in an in vivo mouse model. The functions of the majority of the genes in the GSR transcriptional regulon remain undefined. bab1_1070 is among the most highly regulated genes in this regulon: its transcription is activated 20- to 30-fold by the GSR system under oxidative conditions in vitro. We have solved crystal structures of Bab1_1070 and demonstrate that it forms a homotetrameric complex that resembles those of WrbA-type NADH:quinone oxidoreductases, which are members of the flavodoxin protein family. However, B. abortus WrbA-related protein (WrpA) does not bind flavin cofactors with a high affinity and does not function as an NADH:quinone oxidoreductase in vitro. Soaking crystals with flavin mononucleotide (FMN) revealed a likely low-affinity binding site adjacent to the canonical WrbA flavin binding site. Deletion of wrpA (ΔwrpA) does not compromise cell survival under acute oxidative stress in vitro or attenuate infection in cell-based or mouse models. However, a ΔwrpA strain does elicit increased splenomegaly in a mouse model, suggesting that WrpA modulates B. abortus interaction with its mammalian host. Despite high structural homology with canonical WrbA proteins, we propose that B. abortus WrpA represents a functionally distinct member of the diverse flavodoxin family. Brucella abortus is an etiological agent of brucellosis, which is among the most common zoonotic diseases worldwide. The general stress response (GSR) regulatory system of B. abortus controls the transcription of approximately 100 genes and is required for maintenance of chronic infection in a murine model; the majority of GSR-regulated genes

  11. Characterization of TTN Novex Splicing Variants across Species and the Role of RBM20 in Novex-Specific Exon Splicing

    Directory of Open Access Journals (Sweden)

    Zhilong Chen

    2018-02-01

    Full Text Available Titin (TTN is a major disease-causing gene in cardiac muscle. Titin (TTN contains 363 exons in human encoding various sizes of TTN protein due to alternative splicing regulated mainly by RNA binding motif 20 (RBM20. Three isoforms of TTN protein are produced by mutually exclusive exons 45 (Novex 1, 46 (Novex 2, and 48 (Novex 3. Alternatively splicing in Novex isoforms across species and whether Novex isoforms are associated with heart disease remains completely unknown. Cross-species exon comparison with the mVISTA online tool revealed that exon 45 is more highly conserved across all species than exons 46 and 48. Importantly, a conserved region between exons 47 and 48 across species was revealed for the first time. Reverse transcript polymerase chain reaction (RT-PCR and DNA sequencing confirmed a new exon named as 48′ in Novex 3. In addition, with primer pairs for Novex 1, a new truncated form preserving introns 44 and 45 was discovered. We discovered that Novex 2 is not expressed in the pig, mouse, and rat with Novex 2 primer pairs. Unexpectedly, three truncated forms were identified. One TTN variant with intron 46 retention is mainly expressed in the human and frog heart, another variant with co-expression of exons 45 and 46 exists predominantly in chicken and frog heart, and a third with retention of introns 45 and 46 is mainly expressed in pig, mouse, rat, and chicken. Using Rbm20 knockout rat heart, we revealed that RBM20 is not a splicing regulator of Novex variants. Furthermore, the expression levels of Novex variants in human hearts with cardiomyopathies suggested that Novexes 2 and 3 could be associated with dilated cardiomyopathy (DCM and/or arrhythmogenic right ventricular cardiomyopathy (ARVC. Taken together, our study reveals that splicing diversity of Novex exons across species and Novex variants might play a role in cardiomyopathy.

  12. Spliced

    DEFF Research Database (Denmark)

    Addison, Courtney Page

    2017-01-01

    Human gene therapy (HGT) aims to cure disease by inserting or editing the DNA of patients with genetic conditions. Since foundational genetic techniques came into use in the 1970s, the field has developed to the point that now three therapies have market approval, and over 1800 clinical trials have...

  13. ZmbZIP60 mRNA is spliced in maize in response to ER stress

    Directory of Open Access Journals (Sweden)

    Li Yanjie

    2012-03-01

    Full Text Available Abstract Background Adverse environmental conditions produce ER stress and elicit the unfolded protein response (UPR in plants. Plants are reported to have two "arms" of the ER stress signaling pathway-one arm involving membrane-bound transcription factors and the other involving a membrane-associated RNA splicing factor, IRE1. IRE1 in yeast to mammals recognizes a conserved twin loop structure in the target RNA. Results A segment of the mRNA encoding ZmbZIP60 in maize can be folded into a twin loop structure, and in response to ER stress this mRNA is spliced, excising a 20b intron. Splicing converts the predicted protein from a membrane-associated transcription factor to one that is targeted to the nucleus. Splicing of ZmbZIP60 can be elicited in maize seedlings by ER stress agents such as dithiothreitol (DTT or tunicamycin (TM or by heat treatment. Younger, rather than older seedlings display a more robust splicing response as do younger parts of leaf, along a developmental gradient in a leaf. The molecular signature of an ER stress response in plants includes the upregulation of Binding Protein (BIP genes. Maize has numerous BIP-like genes, and ER stress was found to upregulate one of these, ZmBIPb. Conclusions The splicing of ZmbZIP60 mRNA is an indicator of ER stress in maize seedlings resulting from adverse environmental conditions such as heat stress. ZmbZIP60 mRNA splicing in maize leads predictively to the formation of active bZIP transcription factor targeted to the nucleus to upregulate stress response genes. Among the genes upregulated by ER stress in maize is one of 22 BIP-like genes, ZmBIPb.

  14. regSNPs-splicing: a tool for prioritizing synonymous single-nucleotide substitution.

    Science.gov (United States)

    Zhang, Xinjun; Li, Meng; Lin, Hai; Rao, Xi; Feng, Weixing; Yang, Yuedong; Mort, Matthew; Cooper, David N; Wang, Yue; Wang, Yadong; Wells, Clark; Zhou, Yaoqi; Liu, Yunlong

    2017-09-01

    While synonymous single-nucleotide variants (sSNVs) have largely been unstudied, since they do not alter protein sequence, mounting evidence suggests that they may affect RNA conformation, splicing, and the stability of nascent-mRNAs to promote various diseases. Accurately prioritizing deleterious sSNVs from a pool of neutral ones can significantly improve our ability of selecting functional genetic variants identified from various genome-sequencing projects, and, therefore, advance our understanding of disease etiology. In this study, we develop a computational algorithm to prioritize sSNVs based on their impact on mRNA splicing and protein function. In addition to genomic features that potentially affect splicing regulation, our proposed algorithm also includes dozens structural features that characterize the functions of alternatively spliced exons on protein function. Our systematical evaluation on thousands of sSNVs suggests that several structural features, including intrinsic disorder protein scores, solvent accessible surface areas, protein secondary structures, and known and predicted protein family domains, show significant differences between disease-causing and neutral sSNVs. Our result suggests that the protein structure features offer an added dimension of information while distinguishing disease-causing and neutral synonymous variants. The inclusion of structural features increases the predictive accuracy for functional sSNV prioritization.

  15. Structural and dynamic characterization of eukaryotic gene regulatory protein domains in solution

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Andrew Loyd [Univ. of California, Berkeley, CA (United States). Dept. of Chemistry

    1996-05-01

    Solution NMR was primarily used to characterize structure and dynamics in two different eukaryotic protein systems: the δ-Al-ε activation domain from c-jun and the Drosophila RNA-binding protein Sex-lethal. The second system is the Drosophila Sex-lethal (Sxl) protein, an RNA-binding protein which is the ``master switch`` in sex determination. Sxl contains two adjacent RNA-binding domains (RBDs) of the RNP consensus-type. The NMR spectrum of the second RBD (Sxl-RBD2) was assigned using multidimensional heteronuclear NMR, and an intermediate-resolution family of structures was calculated from primarily NOE distance restraints. The overall fold was determined to be similar to other RBDs: a βαβ-βαβ pattern of secondary structure, with the two helices packed against a 4-stranded anti-parallel β-sheet. In addition 15N T1, T2, and 15N/1H NOE relaxation measurements were carried out to characterize the backbone dynamics of Sxl-RBD2 in solution. RNA corresponding to the polypyrimidine tract of transformer pre-mRNA was generated and titrated into 3 different Sxl-RBD protein constructs. Combining Sxl-RBD1+2 (bht RBDs) with this RNA formed a specific, high affinity protein/RNA complex that is amenable to further NMR characterization. The backbone 1H, 13C, and 15N resonances of Sxl-RBD1+2 were assigned using a triple-resonance approach, and 15N relaxation experiments were carried out to characterize the backbone dynamics of this complex. The changes in chemical shift in Sxl-RBD1+2 upon binding RNA are observed using Sxl-RBD2 as a substitute for unbound Sxl-RBD1+2. This allowed the binding interface to be qualitatively mapped for the second domain.

  16. Transcriptional analysis of the jamaicamide gene cluster from the marine cyanobacterium Lyngbya majuscula and identification of possible regulatory proteins

    Directory of Open Access Journals (Sweden)

    Dorrestein Pieter C

    2009-12-01

    Full Text Available Abstract Background The marine cyanobacterium Lyngbya majuscula is a prolific producer of bioactive secondary metabolites. Although biosynthetic gene clusters encoding several of these compounds have been identified, little is known about how these clusters of genes are transcribed or regulated, and techniques targeting genetic manipulation in Lyngbya strains have not yet been developed. We conducted transcriptional analyses of the jamaicamide gene cluster from a Jamaican strain of Lyngbya majuscula, and isolated proteins that could be involved in jamaicamide regulation. Results An unusually long untranslated leader region of approximately 840 bp is located between the jamaicamide transcription start site (TSS and gene cluster start codon. All of the intergenic regions between the pathway ORFs were transcribed into RNA in RT-PCR experiments; however, a promoter prediction program indicated the possible presence of promoters in multiple intergenic regions. Because the functionality of these promoters could not be verified in vivo, we used a reporter gene assay in E. coli to show that several of these intergenic regions, as well as the primary promoter preceding the TSS, are capable of driving β-galactosidase production. A protein pulldown assay was also used to isolate proteins that may regulate the jamaicamide pathway. Pulldown experiments using the intergenic region upstream of jamA as a DNA probe isolated two proteins that were identified by LC-MS/MS. By BLAST analysis, one of these had close sequence identity to a regulatory protein in another cyanobacterial species. Protein comparisons suggest a possible correlation between secondary metabolism regulation and light dependent complementary chromatic adaptation. Electromobility shift assays were used to evaluate binding of the recombinant proteins to the jamaicamide promoter region. Conclusion Insights into natural product regulation in cyanobacteria are of significant value to drug discovery

  17. The evolutionary landscape of intergenic trans-splicing events in insects

    Science.gov (United States)

    Kong, Yimeng; Zhou, Hongxia; Yu, Yao; Chen, Longxian; Hao, Pei; Li, Xuan

    2015-01-01

    To explore the landscape of intergenic trans-splicing events and characterize their functions and evolutionary dynamics, we conduct a mega-data study of a phylogeny containing eight species across five orders of class Insecta, a model system spanning 400 million years of evolution. A total of 1,627 trans-splicing events involving 2,199 genes are identified, accounting for 1.58% of the total genes. Homology analysis reveals that mod(mdg4)-like trans-splicing is the only conserved event that is consistently observed in multiple species across two orders, which represents a unique case of functional diversification involving trans-splicing. Thus, evolutionarily its potential for generating proteins with novel function is not broadly utilized by insects. Furthermore, 146 non-mod trans-spliced transcripts are found to resemble canonical genes from different species. Trans-splicing preserving the function of ‘breakup' genes may serve as a general mechanism for relaxing the constraints on gene structure, with profound implications for the evolution of genes and genomes. PMID:26521696

  18. In Vitro and In Vivo Modulation of Alternative Splicing by the Biguanide Metformin

    Directory of Open Access Journals (Sweden)

    Delphine Laustriat

    2015-01-01

    Full Text Available Major physiological changes are governed by alternative splicing of RNA, and its misregulation may lead to specific diseases. With the use of a genome-wide approach, we show here that this splicing step can be modified by medication and demonstrate the effects of the biguanide metformin, on alternative splicing. The mechanism of action involves AMPK activation and downregulation of the RBM3 RNA-binding protein. The effects of metformin treatment were tested on myotonic dystrophy type I (DM1, a multisystemic disease considered to be a spliceopathy. We show that this drug promotes a corrective effect on several splicing defects associated with DM1 in derivatives of human embryonic stem cells carrying the causal mutation of DM1 as well as in primary myoblasts derived from patients. The biological effects of metformin were shown to be compatible with typical therapeutic dosages in a clinical investigation involving diabetic patients. The drug appears to act as a modifier of alternative splicing of a subset of genes and may therefore have novel therapeutic potential for many more diseases besides those directly linked to defective alternative splicing.

  19. Expanding the action of duplex RNAs into the nucleus: redirecting alternative splicing

    Science.gov (United States)

    Liu, Jing; Hu, Jiaxin; Corey, David R.

    2012-01-01

    Double-stranded RNAs are powerful agents for silencing gene expression in the cytoplasm of mammalian cells. The potential for duplex RNAs to control expression in the nucleus has received less attention. Here, we investigate the ability of small RNAs to redirect splicing. We identify RNAs targeting an aberrant splice site that restore splicing and production of functional protein. RNAs can target sequences within exons or introns and affect the inclusion of exons within SMN2 and dystrophin, genes responsible for spinal muscular atrophy and Duchenne muscular dystrophy, respectively. Duplex RNAs recruit argonaute 2 (AGO2) to pre-mRNA transcripts and altered splicing requires AGO2 expression. AGO2 promotes transcript cleavage in the cytoplasm, but recruitment of AGO2 to pre-mRNAs does not reduce transcript levels, exposing a difference between cytoplasmic and nuclear pathways. Involvement of AGO2 in splicing, a classical nuclear process, reinforces the conclusion from studies of RNA-mediated transcriptional silencing that RNAi pathways can be adapted to function in the mammalian nucleus. These data provide a new strategy for controlling splicing and expand the reach of small RNAs within the nucleus of mammalian cells. PMID:21948593

  20. Characterization of an apparently synonymous F5 mutation causing aberrant splicing and factor V deficiency.

    Science.gov (United States)

    Nuzzo, F; Bulato, C; Nielsen, B I; Lee, K; Wielders, S J; Simioni, P; Key, N S; Castoldi, E

    2015-03-01

    Coagulation factor V (FV) deficiency is a rare autosomal recessive bleeding disorder. We investigated a patient with severe FV deficiency (FV:C mutation in exon 4 (c.578G>C, p.Cys193Ser), predicting the abolition of a conserved disulphide bridge, and an apparently synonymous variant in exon 8 (c.1281C>G). The observation that half of the patient's F5 mRNA lacked the last 18 nucleotides of exon 8 prompted us to re-evaluate the c.1281C>G variant for its possible effects on splicing. Bioinformatics sequence analysis predicted that this transversion would activate a cryptic donor splice site and abolish an exonic splicing enhancer. Characterization in a F5 minigene model confirmed that the c.1281C>G variant was responsible for the patient's splicing defect, which could be partially corrected by a mutation-specific morpholino antisense oligonucleotide. The aberrantly spliced F5 mRNA, whose stability was similar to that of the normal mRNA, encoded a putative FV mutant lacking amino acids 427-432. Expression in COS-1 cells indicated that the mutant protein is poorly secreted and not functional. In conclusion, the c.1281C>G mutation, which was predicted to be translationally silent and hence neutral, causes FV deficiency by impairing pre-mRNA splicing. This finding underscores the importance of cDNA analysis for the correct assessment of exonic mutations. © 2014 John Wiley & Sons Ltd.

  1. Rhythmic Behavior Is Controlled by the SRm160 Splicing Factor in Drosophila melanogaster.

    Science.gov (United States)

    Beckwith, Esteban J; Hernando, Carlos E; Polcowñuk, Sofía; Bertolin, Agustina P; Mancini, Estefania; Ceriani, M Fernanda; Yanovsky, Marcelo J

    2017-10-01

    Circadian clocks organize the metabolism, physiology, and behavior of organisms throughout the day-night cycle by controlling daily rhythms in gene expression at the transcriptional and post-transcriptional levels. While many transcription factors underlying circadian oscillations are known, the splicing factors that modulate these rhythms remain largely unexplored. A genome-wide assessment of the alterations of gene expression in a null mutant of the alternative splicing regulator SR-related matrix protein of 160 kDa (SRm160) revealed the extent to which alternative splicing impacts on behavior-related genes. We show that SRm160 affects gene expression in pacemaker neurons of the Drosophila brain to ensure proper oscillations of the molecular clock. A reduced level of SRm160 in adult pacemaker neurons impairs circadian rhythms in locomotor behavior, and this phenotype is caused, at least in part, by a marked reduction in period ( per ) levels. Moreover, rhythmic accumulation of the neuropeptide PIGMENT DISPERSING FACTOR in the dorsal projections of these neurons is abolished after SRm160 depletion. The lack of rhythmicity in SRm160-downregulated flies is reversed by a fully spliced per construct, but not by an extra copy of the endogenous locus, showing that SRm160 positively regulates per levels in a splicing-dependent manner. Our findings highlight the significant effect of alternative splicing on the nervous system and particularly on brain function in an in vivo model. Copyright © 2017 by the Genetics Society of America.

  2. Sterol regulatory element binding protein and dietary lipid regulation of fatty acid synthesis in the mammary epithelium.

    Science.gov (United States)

    Rudolph, Michael C; Monks, Jenifer; Burns, Valerie; Phistry, Meridee; Marians, Russell; Foote, Monica R; Bauman, Dale E; Anderson, Steven M; Neville, Margaret C

    2010-12-01

    The lactating mammary gland synthesizes large amounts of triglyceride from fatty acids derived from the blood and from de novo lipogenesis. The latter is significantly increased at parturition and decreased when additional dietary fatty acids become available. To begin to understand the molecular regulation of de novo lipogenesis, we tested the hypothesis that the transcription factor sterol regulatory element binding factor (SREBF)-1c is a primary regulator of this system. Expression of Srebf1c mRNA and six of its known target genes increased ≥2.5-fold at parturition. However, Srebf1c-null mice showed only minor deficiencies in lipid synthesis during lactation, possibly due to compensation by Srebf1a expression. To abrogate the function of both isoforms of Srebf1, we bred mice to obtain a mammary epithelial cell-specific deletion of SREBF cleavage-activating protein (SCAP), the SREBF escort protein. These dams showed a significant lactation deficiency, and expression of mRNA for fatty acid synthase (Fasn), insulin-induced gene 1 (Insig1), mitochondrial citrate transporter (Slc25a1), and stearoyl-CoA desaturase 2 (Scd2) was reduced threefold or more; however, the mRNA levels of acetyl-CoA carboxylase-1α (Acaca) and ATP citrate lyase (Acly) were unchanged. Furthermore, a 46% fat diet significantly decreased de novo fatty acid synthesis and reduced the protein levels of ACACA, ACLY, and FASN significantly, with no change in their mRNA levels. These data lead us to conclude that two modes of regulation exist to control fatty acid synthesis in the mammary gland of the lactating mouse: the well-known SREBF1 system and a novel mechanism that acts at the posttranscriptional level in the presence of SCAP deletion and high-fat feeding to alter enzyme protein.

  3. Position dependence of the rous sarcoma virus negative regulator of splicing element reflects proximity to a 5' splice site

    International Nuclear Information System (INIS)

    Wang Yuedi; McNally, Mark T.

    2003-01-01

    Rous sarcoma virus (RSV) requires incomplete splicing of its viral transcripts to maintain efficient replication. A splicing inhibitor element, the negative regulator of splicing (NRS), is located near the 5' end of the RNA but the significance of this positioning is not known. In a heterologous intron the NRS functions optimally when positioned close to the authentic 5' splice site. This observation led us to investigate the basis of the position dependence. Four explanations were put forth and stressed the role of three major elements involved in splicing, the 3' splice site, the 5' splice site, and the 5' end cap structure. NRS function was unrelated to its position relative to the 3' splice site or the cap structure and appeared to depend on its position relative to the authentic 5' splice site. We conclude that position dependence may reflect distance constraints necessary for competition of the NRS with the authentic 5' splice site for pairing with the 3' splice sites

  4. Exploring the regulatory role of isocitrate dehydrogenase mutant protein on glioma stem cell proliferation.

    Science.gov (United States)

    Lu, H-C; Ma, J; Zhuang, Z; Qiu, F; Cheng, H-L; Shi, J-X

    2016-08-01

    Glioma is the most lethal form of cancer that originates mostly from the brain and less frequently from the spine. Glioma is characterized by abnormal regulation of glial cell differentiation. The severity of the glioma was found to be relaxed in isocitrate dehydrogenase 1 (IDH1) mutant. The present study focused on histological discrimination and regulation of cancer stem cell between IDH1 mutant and in non-IDH1 mutant glioma tissue. Histology, immunohistochemistry and Western blotting techniques are used to analyze the glioma nature and variation in glioma stem cells that differ between IDH1 mutant and in non-IDH1 mutant glioma tissue. The aggressive form of non-IDH1 mutant glioma shows abnormal cellular histological variation with prominent larger nucleus along with abnormal clustering of cells. The longer survival form of IDH1 mutant glioma has a control over glioma stem cell proliferation. Immunohistochemistry with stem cell markers, CD133 and EGFRvIII are used to demonstrate that the IDH1 mutant glioma shows limited dependence on cancer stem cells and it shows marked apoptotic signals in TUNEL assay to regulate abnormal cells. The non-IDH1 mutant glioma failed to regulate misbehaving cells and it promotes cancer stem cell proliferation. Our finding supports that the IDH1 mutant glioma has a regulatory role in glioma stem cells and their survival.

  5. Drosophila Protein Kinase CK2: Genetics, Regulatory Complexity and Emerging Roles during Development

    Directory of Open Access Journals (Sweden)

    Mohna Bandyopadhyay

    2016-12-01

    Full Text Available CK2 is a Ser/Thr protein kinase that is highly conserved amongst all eukaryotes. It is a well-known oncogenic kinase that regulates vital cell autonomous functions and animal development. Genetic studies in the fruit fly Drosophila are providing unique insights into the roles of CK2 in cell signaling, embryogenesis, organogenesis, neurogenesis, and the circadian clock, and are revealing hitherto unknown complexities in CK2 functions and regulation. Here, we review Drosophila CK2 with respect to its structure, subunit diversity, potential mechanisms of regulation, developmental abnormalities linked to mutations in the gene encoding CK2 subunits, and emerging roles in multiple aspects of eye development. We examine the Drosophila CK2 “interaction map” and the eye-specific “transcriptome” databases, which raise the prospect that this protein kinase has many additional targets in the developing eye. We discuss the possibility that CK2 functions during early retinal neurogenesis in Drosophila and mammals bear greater similarity than has been recognized, and that this conservation may extend to other developmental programs. Together, these studies underscore the immense power of the Drosophila model organism to provide new insights and avenues to further investigate developmentally relevant targets of this protein kinase.

  6. The splicing of tiny introns of Paramecium is controlled by MAGO.

    Science.gov (United States)

    Contreras, Julia; Begley, Victoria; Marsella, Laura; Villalobo, Eduardo

    2018-07-15

    The exon junction complex (EJC) is a key element of the splicing machinery. The EJC core is composed of eIF4A3, MAGO, Y14 and MLN51. Few accessory proteins, such as CWC22 or UPF3, bind transiently to the EJC. The EJC has been implicated in the control of the splicing of long introns. To ascertain whether the EJC controls the splicing of short introns, we used Paramecium tetraurelia as a model organism, since it has thousands of very tiny introns. To elucidate whether EJC affects intron splicing in P. tetraurelia, we searched for EJC protein-coding genes, and silenced those genes coding for eIF4A3, MAGO and CWC22. We found that P. tetraurelia likely assembles an active EJC with only three of the core proteins, since MLN51 is lacking. Silencing of eIF4A3 or CWC22 genes, but not that of MAGO, caused lethality. Silencing of the MAGO gene caused either an increase, decrease, or no change in intron retention levels of some intron-containing mRNAs used as reporters. We suggest that a fine-tuning expression of EJC genes is required for steady intron removal in P. tetraurelia. Taking into consideration our results and those published by others, we conclude that the EJC controls splicing independently of the intron size. Copyright © 2018 Elsevier B.V. All rights reserved.

  7. Optimization of Peptide Nucleic Acid Antisense Oligonucleotides for Local and Systemic Dystrophin Splice Correction in the mdx Mouse

    Science.gov (United States)

    Yin, HaiFang; Betts, Corinne; Saleh, Amer F; Ivanova, Gabriela D; Lee, Hyunil; Seow, Yiqi; Kim, Dalsoo; Gait, Michael J; Wood, Matthew JA

    2010-01-01

    Antisense oligonucleotides (AOs) have the capacity to alter the processing of pre-mRNA transcripts in order to correct the function of aberrant disease-related genes. Duchenne muscular dystrophy (DMD) is a fatal X-linked muscle degenerative disease that arises from mutations in the DMD gene leading to an absence of dystrophin protein. AOs have been shown to restore the expression of functional dystrophin via splice correction by intramuscular and systemic delivery in animal models of DMD and in DMD patients via intramuscular administration. Major challenges in developing this splice correction therapy are to optimize AO chemistry and to develop more effective systemic AO delivery. Peptide nucleic acid (PNA) AOs are an alternative AO chemistry with favorable in vivo biochemical properties and splice correcting abilities. Here, we show long-term splice correction of the DMD gene in mdx mice following intramuscular PNA delivery and effective splice correction in aged mdx mice. Further, we report detailed optimization of systemic PNA delivery dose regimens and PNA AO lengths to yield splice correction, with 25-mer PNA AOs providing the greatest splice correcting efficacy, restoring dystrophin protein in multiple peripheral muscle groups. PNA AOs therefore provide an attractive candidate AO chemistry for DMD exon skipping therapy. PMID:20068555

  8. Regulatory proteins (inhibitors or activators) affect estimates of Msub(r) of enzymes and receptors by radiation inactivation

    International Nuclear Information System (INIS)

    Potier, M.; Giroux, S.

    1985-01-01

    The radiation-inactivation method allows the determination of the Msub(r) of enzymes and receptors by monitoring the decay of biological activity as a function of absorbed dose. The presence of regulatory or effector proteins (inhibitors or activators) associated with an enzyme or receptor, or released in the preparation after tissue homogenization, may affect the decay of biological activity. How the activity is affected, however, will depend on the type of inhibition (competitive or non-competitive), the inhibitor or activator concentration, the dissociation constant of the enzyme-effector system, and the effector Msub(r) relative to that of the enzyme. Since little is known on how effector proteins influence radiation inactivation of enzymes and receptors, we have considered a theoretical model in an effort to provide a framework for the interpretation of experimentally obtained data. Our model predicts that competitive and non-competitive inhibitors of enzymes could be distinguished by analysing irradiated samples with various substrate concentrations. Inhibitors will decrease whereas activators will increase the apparent target size of enzymes or receptors. (author)

  9. Regulation of steroid 5-{alpha} reductase type 2 (Srd5a2) by sterol regulatory element binding proteins and statin

    Energy Technology Data Exchange (ETDEWEB)

    Seo, Young-Kyo [Department of Molecular Biology and Biochemistry, 3244 McGaugh Hall, University of California, UC Irvine, Irvine, CA 92697-3900 (United States); Zhu, Bing [Department of Pathology, University of Texas Medical Branch, Galveston, TX 77555-0144 (United States); Jeon, Tae-Il [Department of Molecular Biology and Biochemistry, 3244 McGaugh Hall, University of California, UC Irvine, Irvine, CA 92697-3900 (United States); Osborne, Timothy F., E-mail: tfosborn@uci.edu [Department of Molecular Biology and Biochemistry, 3244 McGaugh Hall, University of California, UC Irvine, Irvine, CA 92697-3900 (United States)

    2009-11-01

    In this study, we show that sterol regulatory element binding proteins (SREBPs) regulate expression of Srd5a2, an enzyme that catalyzes the irreversible conversion of testosterone to dihydroxytestosterone in the male reproductive tract and is highly expressed in androgen-sensitive tissues such as the prostate and skin. We show that Srd5a2 is induced in livers and prostate from mice fed a chow diet supplemented with lovastatin plus ezitimibe (L/E), which increases the activity of nuclear SREBP-2. The three fold increase in Srd5a2 mRNA mediated by L/E treatment was accompanied by the induction of SREBP-2 binding to the Srd5a2 promoter detected by a ChIP-chip assay in liver. We identified a SREBP-2 responsive region within the first 300 upstream bases of the mouse Srd5a2 promoter by co-transfection assays which contain a site that bound SREBP-2 in vitro by an EMSA. Srd5a2 protein was also induced in cells over-expressing SREBP-2 in culture. The induction of Srd5a2 through SREBP-2 provides a mechanistic explanation for why even though statin therapy is effective in reducing cholesterol levels in treating hypercholesterolemia it does not compromise androgen production in clinical studies.

  10. Protein Arginine Methyltransferase 5 Inhibition Upregulates Foxp3+ Regulatory T Cells Frequency and Function during the Ulcerative Colitis

    Directory of Open Access Journals (Sweden)

    Yingxia Zheng

    2017-05-01

    Full Text Available Ulcerative colitis (UC pathogenesis is related to imbalance of immune responses, and the equilibrium between inflammatory T cells and Foxp3+ regulatory T cells (Tregs plays an important role in the intestinal homeostasis. Protein arginine methyltransferases (PRMTs regulate chromatin remodeling and gene expression. Here, we investigated whether inhibition of PRMTs affects colitis pathogenesis in mice and inflammatory bowel disease patients and further explored the underlying mechanisms. In this study, we found that protein arginine N-methyltransferase inhibitor 1 (AMI-1 treatments increased Tregs frequency, function, and reduced colitis incidence. Adoptive transfer of AMI-1-treated Tregs could reduce the colitis incidence. Colitis was associated with increased local PRMT5 expression, which was inhibited by AMI-1 treatment. Additionally, PRMT5 knockdown T cells produced a better response to TGFβ and promoted Tregs differentiation through decreased DNA methyltransferase 1 (DNMT1 expression. PRMT5 also enhanced H3K27me3 and DNMT1 binding to Foxp3 promoter, which restricted Tregs differentiation. Furthermore, PRMT5 knockdown led to decreased Foxp3 promoter methylation during Tregs induction. PRMT5 expression had a negative relationship with Tregs in UC patients, knockdown of PRMT5 expression increased Tregs frequency and decreased TNFα, IL-6, and IL-13 levels. Our study outlines a novel regulation of PRMT5 on Tregs development and function. Strategies to decrease PRMT5 expression might have therapeutic potential to control UC.

  11. Regulation of steroid 5-α reductase type 2 (Srd5a2) by sterol regulatory element binding proteins and statin

    International Nuclear Information System (INIS)

    Seo, Young-Kyo; Zhu, Bing; Jeon, Tae-Il; Osborne, Timothy F.

    2009-01-01

    In this study, we show that sterol regulatory element binding proteins (SREBPs) regulate expression of Srd5a2, an enzyme that catalyzes the irreversible conversion of testosterone to dihydroxytestosterone in the male reproductive tract and is highly expressed in androgen-sensitive tissues such as the prostate and skin. We show that Srd5a2 is induced in livers and prostate from mice fed a chow diet supplemented with lovastatin plus ezitimibe (L/E), which increases the activity of nuclear SREBP-2. The three fold increase in Srd5a2 mRNA mediated by L/E treatment was accompanied by the induction of SREBP-2 binding to the Srd5a2 promoter detected by a ChIP-chip assay in liver. We identified a SREBP-2 responsive region within the first 300 upstream bases of the mouse Srd5a2 promoter by co-transfection assays which contain a site that bound SREBP-2 in vitro by an EMSA. Srd5a2 protein was also induced in cells over-expressing SREBP-2 in culture. The induction of Srd5a2 through SREBP-2 provides a mechanistic explanation for why even though statin therapy is effective in reducing cholesterol levels in treating hypercholesterolemia it does not compromise androgen production in clinical studies.

  12. Glycogen Synthase Kinase 3 Inactivation Induces Cell Senescence through Sterol Regulatory Element Binding Protein 1-Mediated Lipogenesis in Chang Cells.

    Science.gov (United States)

    Kim, You-Mie; Song, Insun; Seo, Yong-Hak; Yoon, Gyesoon

    2013-12-01

    Enhanced lipogenesis plays a critical role in cell senescence via induction of expression of the mature form of sterol regulatory element binding protein 1 (SREBP1), which contributes to an increase in organellar mass, one of the indicators of senescence. We investigated the molecular mechanisms by which signaling molecules control SREBP1-mediated lipogenesis and senescence. We developed cellular models for stress-induced senescence, by exposing Chang cells, which are immortalized human liver cells, to subcytotoxic concentrations (200 µM) of deferoxamine (DFO) and H2O2. In this model of stress-induced cell senescence using DFO and H2O2, the phosphorylation profile of glycogen synthase kinase 3α (GSK3α) and β corresponded closely to the expression profile of the mature form of SREBP-1 protein. Inhibition of GSK3 with a subcytotoxic concentration of the selective GSK3 inhibitor SB415286 significantly increased mature SREBP1 expression, as well as lipogenesis and organellar mass. In addition, GSK3 inhibition was sufficient to induce senescence in Chang cells. Suppression of GSK3 expression with siRNAs specific to GSK3α and β also increased mature SREBP1 expression and induced senescence. Finally, blocking lipogenesis with fatty acid synthase inhibitors (cerulenin and C75) and siRNA-mediated silencing of SREBP1 and ATP citrate lyase (ACL) significantly attenuated GSK3 inhibition-induced senescence. GSK3 inactivation is an important upstream event that induces SREBP1-mediated lipogenesis and consequent cell senescence.

  13. Structural basis for regulation of rhizobial nodulation and symbiosis gene expression by the regulatory protein NolR.

    Science.gov (United States)

    Lee, Soon Goo; Krishnan, Hari B; Jez, Joseph M

    2014-04-29

    The symbiosis between rhizobial microbes and host plants involves the coordinated expression of multiple genes, which leads to nodule formation and nitrogen fixation. As part of the transcriptional machinery for nodulation and symbiosis across a range of Rhizobium, NolR serves as a global regulatory protein. Here, we present the X-ray crystal structures of NolR in the unliganded form and complexed with two different 22-base pair (bp) double-stranded operator sequences (oligos AT and AA). Structural and biochemical analysis of NolR reveals protein-DNA interactions with an asymmetric operator site and defines a mechanism for conformational switching of a key residue (Gln56) to accommodate variation in target DNA sequences from diverse rhizobial genes for nodulation and symbiosis. This conformational switching alters the energetic contributions to DNA binding without changes in affinity for the target sequence. Two possible models for the role of NolR in the regulation of different nodulation and symbiosis genes are proposed. To our knowledge, these studies provide the first structural insight on the regulation of genes involved in the agriculturally and ecologically important symbiosis of microbes and plants that leads to nodule formation and nitrogen fixation.

  14. Bioinformatic analysis of microRNA biogenesis and function related proteins in eleven animal genomes.

    Science.gov (United States)

    Liu, Xiuying; Luo, GuanZheng; Bai, Xiujuan; Wang, Xiu-Jie

    2009-10-01

    MicroRNAs are approximately 22 nt long small non-coding RNAs that play important regulatory roles in eukaryotes. The biogenesis and functional processes of microRNAs require the participation of many proteins, of which, the well studied ones are Dicer, Drosha, Argonaute and Exportin 5. To systematically study these four protein families, we screened 11 animal genomes to search for genes encoding above mentioned proteins, and identified some new members for each family. Domain analysis results revealed that most proteins within the same family share identical or similar domains. Alternative spliced transcript variants were found for some proteins. We also examined the expression patterns of these proteins in different human tissues and identified other proteins that could potentially interact with these proteins. These findings provided systematic information on the four key proteins involved in microRNA biogenesis and functional pathways in animals, and will shed light on further functional studies of these proteins.

  15. Antioxidant and regulatory role of mitochondrial uncoupling protein UCP2 in pancreatic beta-cells

    Czech Academy of Sciences Publication Activity Database

    Ježek, Petr; Olejár, Tomáš; Smolková, Katarína; Ježek, Jan; Dlasková, Andrea; Plecitá-Hlavatá, Lydie; Zelenka, Jaroslav; Špaček, Tomáš; Engstová, Hana; Reguera Pajuelo, David; Jabůrek, Martin

    2014-01-01

    Roč. 63, Suppl.1 (2014), S73-S91 ISSN 0862-8408 R&D Projects: GA ČR(CZ) GAP302/10/0346; GA ČR(CZ) GAP305/12/1247; GA ČR(CZ) GPP304/10/P204; GA MŠk(CZ) EE2.3.30.0025 Institutional support: RVO:67985823 Keywords : mitochondria * uncoupling protein UCP2 * pancreatic beta-cells * reactive oxygen species * glucose-stimulated insulin secretion Subject RIV: EA - Cell Biology Impact factor: 1.293, year: 2014

  16. Periostin shows increased evolutionary plasticity in its alternatively spliced region

    Directory of Open Access Journals (Sweden)

    Hoersch Sebastian

    2010-01-01

    Full Text Available Abstract Background Periostin (POSTN is a secreted extracellular matrix protein of poorly defined function that has been related to bone and heart development as well as to cancer. In human and mouse, it is known to undergo alternative splicing in its C-terminal region, which is devoid of known protein domains. Differential expression of periostin, sometimes of specific splicing isoforms, is observed in a broad range of human cancers, including breast, pancreatic, and colon cancer. Here, we combine genomic and transcriptomic sequence data from vertebrate organisms to study the evolution of periostin and particularly of its C-terminal region. Results We found that the C-terminal part of periostin is markedly more variable among vertebrates than the rest of periostin in terms of exon count, length, and splicing pattern, which we interpret as a consequence of neofunctionalization after the split between periostin and its paralog transforming growth factor, beta-induced (TGFBI. We also defined periostin's sequential 13-amino acid repeat units - well conserved in teleost fish, but more obscure in higher vertebrates - whose secondary structure is predicted to be consecutive beta strands. We suggest that these beta strands may mediate binding interactions with other proteins through an extended beta-zipper in a manner similar to the way repeat units in bacterial cell wall proteins have been reported to bind human fibronectin. Conclusions Our results, obtained with the help of the increasingly large collection of complete vertebrate genomes, document the evolutionary plasticity of periostin's C-terminal region, and for the first time suggest a basis for its functional role.

  17. Altered expression of the cell cycle regulatory protein cyclin D1 in the rat dentate gyrus after adrenalectomy-induced granular cell lass

    NARCIS (Netherlands)

    Postigo, JA; Van der Werf, YD; Korf, J; Krugers, HJ

    1998-01-01

    The loss of dentate gyrus (DG) granular cells after removal of the rat adrenal glands (ADX) is mediated by a process that is apoptotic in nature. The present study was initiated to compare changes in the immunocytochemical distribution of the cell-cycle regulatory protein cyclin D1, which has been

  18. Sterol regulatory element binding protein 2 overexpression is associated with reduced adipogenesis and ectopic fat accumulation in transgenic spontaneously hypertensive rats

    Czech Academy of Sciences Publication Activity Database

    Landa, Vladimír; Zídek, Václav; Mlejnek, Petr; Šimáková, Miroslava; Šilhavý, Jan; Trnovská, J.; Kazdová, L.; Pravenec, Michal

    2014-01-01

    Roč. 63, č. 5 (2014), s. 587-590 ISSN 0862-8408 R&D Projects: GA MŠk(CZ) LH12061 Institutional support: RVO:67985823 Keywords : sterol regulatory element binding protein 2 * transgenic * spontaneously hypertensive rat * lipid metabolism Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.293, year: 2014

  19. A widespread amino acid polymorphism at codon 905 of the glycogen-associated regulatory subunit of protein phosphatase-1 is associated with insulin resistance and hypersecretion of insulin

    DEFF Research Database (Denmark)

    Hansen, L; Hansen, T; Vestergaard, H

    1995-01-01

    The regulatory G-subunit of the glycogen-associated form of protein phosphatase 1 (PP1) plays a crucial part in muscle tissue glycogen synthesis and breakdown. As impaired insulin stimulated glycogen synthesis in peripheral tissues is considered to be a pathogenic factor in subsets of non-insulin...

  20. A novel Snf2 protein maintains trans-generational regulatory states established by paramutation in maize.

    Directory of Open Access Journals (Sweden)

    Christopher J Hale

    2007-10-01

    Full Text Available Paramutations represent heritable epigenetic alterations that cause departures from Mendelian inheritance. While the mechanism responsible is largely unknown, recent results in both mouse and maize suggest paramutations are correlated with RNA molecules capable of affecting changes in gene expression patterns. In maize, multiple required to maintain repression (rmr loci stabilize these paramutant states. Here we show rmr1 encodes a novel Snf2 protein that affects both small RNA accumulation and cytosine methylation of a proximal transposon fragment at the Pl1-Rhoades allele. However, these cytosine methylation differences do not define the various epigenetic states associated with paramutations. Pedigree analyses also show RMR1 does not mediate the allelic interactions that typically establish paramutations. Strikingly, our mutant analyses show that Pl1-Rhoades RNA transcript levels are altered independently of transcription rates, implicating a post-transcriptional level of RMR1 action. These results suggest the RNA component of maize paramutation maintains small heterochromatic-like domains that can affect, via the activity of a Snf2 protein, the stability of nascent transcripts from adjacent genes by way of a cotranscriptional repression process. These findings highlight a mechanism by which alleles of endogenous loci can acquire novel expression patterns that are meiotically transmissible.

  1. Novel functions for the endocytic regulatory proteins MICAL-L1 and EHD1 in mitosis.

    Science.gov (United States)

    Reinecke, James B; Katafiasz, Dawn; Naslavsky, Naava; Caplan, Steve

    2015-01-01

    During interphase, recycling endosomes mediate the transport of internalized cargo back to the plasma membrane. However, in mitotic cells, recycling endosomes are essential for the completion of cytokinesis, the last phase of mitosis that promotes the physical separation the two daughter cells. Despite recent advances, our understanding of the molecular determinants that regulate recycling endosome dynamics during cytokinesis remains incomplete. We have previously demonstrated that Molecule Interacting with CasL Like-1 (MICAL-L1) and C-terminal Eps15 Homology Domain protein 1 (EHD1) coordinately regulate receptor transport from tubular recycling endosomes during interphase. However, their potential roles in controlling cytokinesis had not been addressed. In this study, we show that MICAL-L1 and EHD1 regulate mitosis. Depletion of either protein resulted in increased numbers of bi-nucleated cells. We provide evidence that bi-nucleation in MICAL-L1- and EHD1-depleted cells is a consequence of impaired recycling endosome transport during late cytokinesis. However, depletion of MICAL-L1, but not EHD1, resulted in aberrant chromosome alignment and lagging chromosomes, suggesting an EHD1-independent function for MICAL-L1 earlier in mitosis. Moreover, we provide evidence that MICAL-L1 and EHD1 differentially influence microtubule dynamics during early and late mitosis. Collectively, our new data suggest several unanticipated roles for MICAL-L1 and EHD1 during the cell cycle. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  2. Splicing pattern - ASTRA | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available List Contact us ASTRA Splicing pattern Data detail Data name Splicing pattern DOI 10.18908/lsdba.nbdc00371-0...04 Description of data contents The patterns of alternative splicing/transcriptional initiation Data file Fi...le name: astra_splicing_pattern.zip File URL: ftp://ftp.biosciencedbc.jp/archive/astra/LATEST/astra_splicing_patt...ogodb/view/astra_splicing_pattern#en Data acquisition method For the five organisms (H. sapiens, M. musculus...apping data into bit arrays, detection of splicing patterns and distribution to t

  3. Perilipin-mediated lipid droplet formation in adipocytes promotes sterol regulatory element-binding protein-1 processing and triacylglyceride accumulation.

    Directory of Open Access Journals (Sweden)

    Yu Takahashi

    Full Text Available Sterol regulatory element-binding protein-1 (SREBP-1 has been thought to be a critical factor that assists adipogenesis. During adipogenesis SREBP-1 stimulates lipogenic gene expression, and peroxisome proliferator-activated receptor γ (PPARγ enhances perilipin (plin gene expression, resulting in generating lipid droplets (LDs to store triacylglycerol (TAG in adipocytes. Plin coats adipocyte LDs and protects them from lipolysis. Here we show in white adipose tissue (WAT of plin-/- mice that nuclear active SREBP-1 and its target gene expression, but not nuclear SREBP-2, significantly decreased on attenuated LD formation. When plin-/- mouse embryonic fibroblasts (MEFs differentiated into adipocytes, attenuated LDs were formed and nuclear SREBP-1 decreased, but enforced plin expression restored them to their original state. Since LDs are largely derived from the endoplasmic reticulum (ER, alterations in the ER cholesterol content were investigated during adipogenesis of 3T3-L1 cells. The ER cholesterol greatly reduced in differentiated adipocytes. The ER cholesterol level in plin-/- WAT was significantly higher than that of wild-type mice, suggesting that increased LD formation caused a change in ER environment along with a decrease in cholesterol. When GFP-SREBP-1 fusion proteins were exogenously expressed in 3T3-L1 cells, a mutant protein lacking the S1P cleavage site was poorly processed during adipogenesis, providing evidence of the increased canonical pathway for SREBP processing in which SREBP-1 is activated by two cleavage enzymes in the Golgi. Therefore, LD biogenesis may create the ER microenvironment favorable for SREBP-1 activation. We describe the novel interplay between LD formation and SREBP-1 activation through a positive feedback loop.

  4. Infantile 4-tert-octylphenol exposure transiently inhibits rat ovarian steroidogenesis and steroidogenic acute regulatory protein (StAR) expression

    International Nuclear Information System (INIS)

    Myllymaeki, S.A.; Karjalainen, M.; Haavisto, T.E.; Toppari, J.; Paranko, J.

    2005-01-01

    Phenolic compounds, such as 4-tert-octylphenol (OP), have been shown to interfere with rat ovarian steroidogenesis. However, little is known about steroidogenic effects of infantile OP exposure on immature ovary. The aim of the present study was to investigate the effects of infantile OP exposure on plasma FSH, LH, estradiol, and progesterone levels in 14-day-old female rats. The effect on ovarian steroidogenic acute regulatory protein (StAR) and FSH receptor (FSHr) expression was analyzed by Western blotting. Ex vivo analysis was carried out for follicular estradiol, progesterone, testosterone, and cAMP production. Sprague-Dawley rats were given OP (0, 10, 50, or 100 mg/kg) subcutaneously on postnatal days 6, 8, 10, and 12. On postnatal day 14, plasma FSH was decreased and progesterone increased significantly at a dose of 100 mg OP/kg. In addition, the highest OP dose advanced the time of vaginal opening in puberty. OP had no effect on infantile LH and estradiol levels or ovarian FSHr content. Ovarian StAR protein content and ex vivo hormone and cAMP production were decreased at all OP doses compared to controls. However, hormone levels recovered independent on FSH and even increased above the control level during a prolonged culture. On postnatal day 35, no statistically significant differences were seen between control and OP-exposed animals in plasma FSH, LH, estradiol, and progesterone levels, or in ovarian StAR protein content. The results indicate that the effect of OP on the infantile ovary is reversible, while more permanent effects in the hypothalamus and pituitary, as described earlier, are involved in the reduction of circulating FSH levels and premature vaginal opening

  5. The function of the RNA-binding protein TEL1 in moss reveals ancient regulatory mechanisms of shoot development.

    Science.gov (United States)

    Vivancos, Julien; Spinner, Lara; Mazubert, Christelle; Charlot, Florence; Paquet, Nicolas; Thareau, Vincent; Dron, Michel; Nogué, Fabien; Charon, Céline

    2012-03-01

    The shoot represents the basic body plan in land plants. It consists of a repeated structure composed of stems and leaves. Whereas vascular plants generate a shoot in their diploid phase, non-vascular plants such as mosses form a shoot (called the gametophore) in their haploid generation. The evolution of regulatory mechanisms or genetic networks used in the development of these two kinds of shoots is unclear. TERMINAL EAR1-like genes have been involved in diploid shoot development in vascular plants. Here, we show that disruption of PpTEL1 from the moss Physcomitrella patens, causes reduced protonema growth and gametophore initiation, as well as defects in gametophore development. Leafy shoots formed on ΔTEL1 mutants exhibit shorter stems with more leaves per shoot, suggesting an accelerated leaf initiation (shortened plastochron), a phenotype shared with the Poaceae vascular plants TE1 and PLA2/LHD2 mutants. Moreover, the positive correlation between plastochron length and leaf size observed in ΔTEL1 mutants suggests a conserved compensatory mechanism correlating leaf growth and leaf initiation rate that would minimize overall changes in plant biomass. The RNA-binding protein encoded by PpTEL1 contains two N-terminus RNA-recognition motifs, and a third C-terminus non-canonical RRM, specific to TEL proteins. Removal of the PpTEL1 C-terminus (including this third RRM) or only 16-18 amino acids within it seriously impairs PpTEL1 function, suggesting a critical role for this third RRM. These results show a conserved function of the RNA-binding PpTEL1 protein in the regulation of shoot development, from early ancestors to vascular plants, that depends on the third TEL-specific RRM.

  6. Spliced XBP1 promotes macrophage survival and autophagy by interacting with Beclin-1

    Energy Technology Data Exchange (ETDEWEB)

    Tian, Ping-Ge [Southern Medical University, Guangzhou, Guangdong 510515 (China); Jiang, Zhi-Xin [Centre Laboratory, The 305th Hospital of the People' s Liberation Army, Beijing 100017 (China); Li, Jian-Hua [Department of Geriatric Cardiology, Chinese PLA General Hosptial, Beijing 100853 (China); Zhou, Zhe, E-mail: zhouzhe76@126.com [Laboratory of Biotechnology, Beijing Institute of Radiation Medicine, Beijing 100850 (China); Zhang, Qing-Hua, E-mail: 1056055170@qq.com [Department of Cardiology, The 305th Hospital of the People' s Liberation Army, Beijing 100017 (China)

    2015-08-07

    Macrophage autophagy plays an important role in the development of atherosclerosis, but the precise mechanism mediating this process is unclear. The potential role of the X-box binding protein 1 (XBP1), a crucial transduction factor that is involved in endoplasmic reticulum stress and the unfolded protein response, in bone marrow-derived macrophage autophagy is unknown. This study mainly explores the roles of XBP1 mRNA splicing in bone marrow-derived macrophage autophagy. The present study shows that the transient overexpression of spliced XBP1 via adenovirus-mediated gene transfer induces autophagy and promotes proliferation in bone marrow-derived macrophages via the down-regulation of Beclin-1, but that the sustained overexpression of spliced XBP1 leads to apoptosis. When XBP1 is down-regulated in bone marrow-derived macrophages using siRNA, rapamycin-induced autophagosome formation is ablated. Furthermore, we have detected the overexpression of XBP1 in areas of atherosclerotic plaques in the arteries of ApoE−/− mice. These results demonstrate that XBP1 mRNA splicing plays an important role in maintaining the function of bone marrow-derived macrophages and provide new insight into the study and treatment of atherosclerosis. - Highlights: • XBP1 was up-regulated in atherosclerotic plaques of ApoE−/− mice. • Transient spliced XBP1 overexpression induced macrophages autophagy via Beclin-1. • Sustained spliced XBP1 overexpression triggered macrophages apoptosis. • Spliced XBP1 plays a key role in maintaining the macrophages survival.

  7. Multiple regulatory roles of the mouse transmembrane adaptor protein NTAL in gene transcription and mast cell physiology.

    Directory of Open Access Journals (Sweden)

    Iva Polakovicova

    Full Text Available Non-T cell activation linker (NTAL; also called LAB or LAT2 is a transmembrane adaptor protein that is expressed in a subset of hematopoietic cells, including mast cells. There are conflicting reports on the role of NTAL in the high affinity immunoglobulin E receptor (FcεRI signaling. Studies carried out on mast cells derived from mice with NTAL knock out (KO and wild type mice suggested that NTAL is a negative regulator of FcεRI signaling, while experiments with RNAi-mediated NTAL knockdown (KD in human mast cells and rat basophilic leukemia cells suggested its positive regulatory role. To determine whether different methodologies of NTAL ablation (KO vs KD have different physiological consequences, we compared under well defined conditions FcεRI-mediated signaling events in mouse bone marrow-derived mast cells (BMMCs with NTAL KO or KD. BMMCs with both NTAL KO and KD exhibited enhanced degranulation, calcium mobilization, chemotaxis, tyrosine phosphorylation of LAT and ERK, and depolymerization of filamentous actin. These data provide clear evidence that NTAL is a negative regulator of FcεRI activation events in murine BMMCs, independently of possible compensatory developmental alterations. To gain further insight into the role of NTAL in mast cells, we examined the transcriptome profiles of resting and antigen-activated NTAL KO, NTAL KD, and corresponding control BMMCs. Through this analysis we identified several genes that were differentially regulated in nonactivated and antigen-activated NTAL-deficient cells, when compared to the corresponding control cells. Some of the genes seem to be involved in regulation of cholesterol-dependent events in antigen-mediated chemotaxis. The combined data indicate multiple regulatory roles of NTAL in gene expression and mast cell physiology.

  8. The Endocytic Recycling Regulatory Protein EHD1 Is Required for Ocular Lens Development

    Science.gov (United States)

    Arya, Priyanka; Rainey, Mark A.; Bhattacharyya, Sohinee; Mohapatra, Bhopal; George, Manju; Kuracha, Murali R; Storck, Matthew D.; Band, Vimla; Govindarajan, Venkatesh; Band, Hamid

    2015-01-01

    The C-terminal Eps15 homology domain-containing (EHD) proteins play a key role in endocytic recycling, a fundamental cellular process that ensures the return of endocytosed membrane components and receptors back to the cell surface. To define the in vivo biological functions of EHD1, we have generated Ehd1 knockout mice and previously reported a requirement of EHD1 for spermatogenesis. Here, we show that approximately 56% of the Ehd1-null mice displayed gross ocular abnormalities, including anophthalmia, aphakia, microphthalmia and congenital cataracts. Histological characterization of ocular abnormalities showed pleiotropic defects that include a smaller or absent lens, persistence of lens stalk and hyaloid vasculature, and deformed optic cups. To test whether these profound ocular defects resulted from the loss of EHD1 in the lens or in non-lenticular tissues, we deleted the Ehd1 gene selectively in the presumptive lens ectoderm using Le-Cre. Conditional Ehd1 deletion in the lens resulted in developmental defects that included thin epithelial layers, small lenses and absence of corneal endothelium. Ehd1 deletion in the lens also resulted in reduced lens epithelial proliferation, survival and expression of junctional proteins E-cadherin and ZO-1. Finally, Le-Cre-mediated deletion of Ehd1 in the lens led to defects in corneal endothelial differentiation. Taken together, these data reveal a unique role for EHD1 in early lens development and suggest a previously unknown link between the endocytic recycling pathway and regulation of key developmental processes including proliferation, differentiation and morphogenesis. PMID:26455409

  9. Hepatitis C virus nonstructural protein-5A activates sterol regulatory element-binding protein-1c through transcription factor Sp1

    Energy Technology Data Exchange (ETDEWEB)

    Xiang, Zhonghua; Qiao, Ling; Zhou, Yan [Vaccine and Infectious Disease Organization, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5E3 (Canada); Babiuk, Lorne A. [University of Alberta, Edmonton, Alberta (Canada); Liu, Qiang, E-mail: qiang.liu@usask.ca [Vaccine and Infectious Disease Organization, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5E3 (Canada)

    2010-11-19

    Research highlights: {yields} A chimeric subgenomic HCV replicon expresses HCV-3a NS5A in an HCV-1b backbone. {yields} HCV-3a NS5A increases mature SREBP-1c protein level. {yields} HCV-3a NS5A activates SREBP-1c transcription. {yields} Domain II of HCV-3a NS5A is more effective in SREBP-1c promoter activation. {yields} Transcription factor Sp1 is required for SREBP-1c activation by HCV-3a NS5A. -- Abstract: Steatosis is an important clinical manifestation of hepatitis C virus (HCV) infection. The molecular mechanisms of HCV-associated steatosis are not well understood. Sterol regulatory element-binding protein-1c (SREBP-1c) is a key transcription factor which activates the transcription of lipogenic genes. Here we showed that the nuclear, mature SREBP-1c level increases in the nucleus of replicon cells expressing HCV-3a nonstructural protein-5A (NS5A). We further showed that HCV-3a NS5A up-regulates SREBP-1c transcription. Additional analysis showed that transcriptional factor Sp1 is involved in SREBP-1c activation by HCV-3a NS5A because inhibition of Sp1 activity by mithramycin A or a dominant-negative Sp1 construct abrogated SREBP-1c promoter activation by HCV-3a NS5A. In addition, chromatin immunoprecipitation (ChIP) assay demonstrated enhanced binding of Sp1 on the SREBP-1c promoter in HCV-3a NS5A replicon cells. These results showed that HCV-3a NS5A activates SREBP-1c transcription through Sp1. Taken together, our results suggest that HCV-3a NS5A is a contributing factor for steatosis caused by HCV-3a infection.

  10. Hepatitis C virus nonstructural protein-5A activates sterol regulatory element-binding protein-1c through transcription factor Sp1

    International Nuclear Information System (INIS)

    Xiang, Zhonghua; Qiao, Ling; Zhou, Yan; Babiuk, Lorne A.; Liu, Qiang

    2010-01-01

    Research highlights: → A chimeric subgenomic HCV replicon expresses HCV-3a NS5A in an HCV-1b backbone. → HCV-3a NS5A increases mature SREBP-1c protein level. → HCV-3a NS5A activates SREBP-1c transcription. → Domain II of HCV-3a NS5A is more effective in SREBP-1c promoter activation. → Transcription factor Sp1 is required for SREBP-1c activation by HCV-3a NS5A. -- Abstract: Steatosis is an important clinical manifestation of hepatitis C virus (HCV) infection. The molecular mechanisms of HCV-associated steatosis are not well understood. Sterol regulatory element-binding protein-1c (SREBP-1c) is a key transcription factor which activates the transcription of lipogenic genes. Here we showed that the nuclear, mature SREBP-1c level increases in the nucleus of replicon cells expressing HCV-3a nonstructural protein-5A (NS5A). We further showed that HCV-3a NS5A up-regulates SREBP-1c transcription. Additional analysis showed that transcriptional factor Sp1 is involved in SREBP-1c activation by HCV-3a NS5A because inhibition of Sp1 activity by mithramycin A or a dominant-negative Sp1 construct abrogated SREBP-1c promoter activation by HCV-3a NS5A. In addition, chromatin immunoprecipitation (ChIP) assay demonstrated enhanced binding of Sp1 on the SREBP-1c promoter in HCV-3a NS5A replicon cells. These results showed that HCV-3a NS5A activates SREBP-1c transcription through Sp1. Taken together, our results suggest that HCV-3a NS5A is a contributing factor for steatosis caused by HCV-3a infection.

  11. Antisense Oligonucleotides Promote Exon Inclusion and Correct the Common c.-32-13T>G GAA Splicing Variant in Pompe Disease

    Directory of Open Access Journals (Sweden)

    Erik van der Wal

    2017-06-01

    Full Text Available The most common variant causing Pompe disease is c.-32-13T>G (IVS1 in the acid α-glucosidase (GAA gene, which weakens the splice acceptor of GAA exon 2 and induces partial and complete exon 2 skipping. It also allows a low level of leaky wild-type splicing, leading to a childhood/adult phenotype. We hypothesized that cis-acting splicing motifs may exist that could be blocked using antisense oligonucleotides (AONs to promote exon inclusion. To test this, a screen was performed in patient-derived primary fibroblasts using a tiling array of U7 small nuclear RNA (snRNA-based AONs. This resulted in the identification of a splicing regulatory element in GAA intron 1. We designed phosphorodiamidate morpholino oligomer-based AONs to this element, and these promoted exon 2 inclusion and enhanced GAA enzyme activity to levels above the disease threshold. These results indicate that the common IVS1 GAA splicing variant in Pompe disease is subject to negative regulation, and inhibition of a splicing regulatory element using AONs is able to restore canonical GAA splicing and endogenous GAA enzyme activity.

  12. Yeast Interacting Proteins Database: YGR013W, YKL012W [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available tion U1 snRNP protein involved in splicing, interacts with the branchpoint-binding protein during the formation of the second commitm... PRP40 U1 snRNP protein involved in splicing, interacts with the branchpoint-binding protein during the form...ation of the second commitment complex Rows with this prey as prey (1) Rows with

  13. High-resolution temporal and regional mapping of MAPT expression and splicing in human brain development.

    Science.gov (United States)

    Hefti, Marco M; Farrell, Kurt; Kim, SoongHo; Bowles, Kathryn R; Fowkes, Mary E; Raj, Towfique; Crary, John F

    2018-01-01

    The microtubule associated protein tau plays a critical role in the pathogenesis of neurodegenerative disease. Recent studies suggest that tau also plays a role in disorders of neuronal connectivity, including epilepsy and post-traumatic stress disorder. Animal studies have shown that the MAPT gene, which codes for the tau protein, undergoes complex pre-mRNA alternative splicing to produce multiple isoforms during brain development. Human data, particularly on temporal and regional variation in tau splicing during development are however lacking. In this study, we present the first detailed examination of the temporal and regional sequence of MAPT alternative splicing in the developing human brain. We used a novel computational analysis of large transcriptomic datasets (total n = 502 patients), quantitative polymerase chain reaction (qPCR) and western blotting to examine tau expression and splicing in post-mortem human fetal, pediatric and adult brains. We found that MAPT exons 2 and 10 undergo abrupt shifts in expression during the perinatal period that are unique in the canonical human microtubule-associated protein family, while exon 3 showed small but significant temporal variation. Tau isoform expression may be a marker of neuronal maturation, temporally correlated with the onset of axonal growth. Immature brain regions such as the ganglionic eminence and rhombic lip had very low tau expression, but within more mature regions, there was little variation in tau expression or splicing. We thus demonstrate an abrupt, evolutionarily conserved shift in tau isoform expression during the human perinatal period that may be due to tau expression in maturing neurons. Alternative splicing of the MAPT pre-mRNA may play a vital role in normal brain development across multiple species and provides a basis for future investigations into the developmental and pathological functions of the tau protein.

  14. Molecular dynamics simulations of conformation changes of HIV-1 regulatory protein on graphene

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Daohui; Li, Libo; He, Daohang; Zhou, Jian, E-mail: jianzhou@scut.edu.cn

    2016-07-30

    Graphical abstract: Preferential adsorption of Vpr13-33 on graphene accompanied by early conformational change from α-helix to β-sheet structures was observed by molecular simulations. This work presents the molecular mechanism of graphene-induced peptide conformational alteration and sheds light on developing graphene-based materials to inhibit HIV. - Highlights: • Graphene induced early structural transition of Vpr13-33 is studied by MD simulations. • Both π-π stacking and hydrophobic interactions orchestrate the peptide adsorption. • Vpr has an increased propensity of β-sheet content on graphene surface. • To develop graphene-based materials to inhibit HIV is possible. - Abstract: The fragment of viral protein R (Vpr), Vpr13-33, plays an important role in regulating nuclear importing of HIV genes through channel formation in which it adopts a leucine-zipper-like alpha-helical conformation. A recent experimental study reported that helical Vpr13-33 would transform to β-sheet or random coil structures and aggregate on the surface of graphene or graphene oxide through hydrophobic interactions. Due to experimental limitations, however, there is still a considerable lack of understanding on the adsorption dynamics at the early stage of the conformational transition at water-graphene interface and the underlying driving force at molecular level. In this study, atomistic molecular dynamics simulations were used to explore the conformation transition phenomena. Vpr13-33 kept α-helical structure in solution, but changed to β-sheet structure when strongly adsorbed onto graphene. Preferential adsorption of Vpr13-33 on graphene is dominated by hydrophobic interactions. The cluster analysis identified the most significant populated conformation and the early stage of structure conversion from α-helical to β-sheet was found, but the full β-sheet propagation was not observed. Free energy landscape analysis further complemented the transformation analysis of

  15. Molecular dynamics simulations of conformation changes of HIV-1 regulatory protein on graphene

    International Nuclear Information System (INIS)

    Zhao, Daohui; Li, Libo; He, Daohang; Zhou, Jian

    2016-01-01

    Graphical abstract: Preferential adsorption of Vpr13-33 on graphene accompanied by early conformational change from α-helix to β-sheet structures was observed by molecular simulations. This work presents the molecular mechanism of graphene-induced peptide conformational alteration and sheds light on developing graphene-based materials to inhibit HIV. - Highlights: • Graphene induced early structural transition of Vpr13-33 is studied by MD simulations. • Both π-π stacking and hydrophobic interactions orchestrate the peptide adsorption. • Vpr has an increased propensity of β-sheet content on graphene surface. • To develop graphene-based materials to inhibit HIV is possible. - Abstract: The fragment of viral protein R (Vpr), Vpr13-33, plays an important role in regulating nuclear importing of HIV genes through channel formation in which it adopts a leucine-zipper-like alpha-helical conformation. A recent experimental study reported that helical Vpr13-33 would transform to β-sheet or random coil structures and aggregate on the surface of graphene or graphene oxide through hydrophobic interactions. Due to experimental limitations, however, there is still a considerable lack of understanding on the adsorption dynamics at the early stage of the conformational transition at water-graphene interface and the underlying driving force at molecular level. In this study, atomistic molecular dynamics simulations were used to explore the conformation transition phenomena. Vpr13-33 kept α-helical structure in solution, but changed to β-sheet structure when strongly adsorbed onto graphene. Preferential adsorption of Vpr13-33 on graphene is dominated by hydrophobic interactions. The cluster analysis identified the most significant populated conformation and the early stage of structure conversion from α-helical to β-sheet was found, but the full β-sheet propagation was not observed. Free energy landscape analysis further complemented the transformation analysis of

  16. Absence of residual structure in the intrinsically disordered regulatory protein CP12 in its reduced state

    Energy Technology Data Exchange (ETDEWEB)

    Launay, Hélène; Barré, Patrick [Laboratory of integrative Structural and Chemical Biology (iSCB), Centre de Recherche en Cancérologie de Marseille (CRCM), CNRS UMR 7258, INSERM U 1068, Institut Paoli-Calmettes, Aix-Marseille Universités, Marseille 13009 (France); Puppo, Carine [Aix-Marseille Université, Centre National de la Recherche Scientifique, UMR 7281, Laboratoire de Bioénergétique et Ingénierie des Protéines, 31 Chemin Joseph Aiguier, 13402, Marseille Cedex 20 (France); Manneville, Stéphanie [Laboratory of integrative Structural and Chemical Biology (iSCB), Centre de Recherche en Cancérologie de Marseille (CRCM), CNRS UMR 7258, INSERM U 1068, Institut Paoli-Calmettes, Aix-Marseille Universités, Marseille 13009 (France); Gontero, Brigitte [Aix-Marseille Université, Centre National de la Recherche Scientifique, UMR 7281, Laboratoire de Bioénergétique et Ingénierie des Protéines, 31 Chemin Joseph Aiguier, 13402, Marseille Cedex 20 (France); Receveur-Bréchot, Véronique, E-mail: veronique.brechot@inserm.fr [Laboratory of integrative Structural and Chemical Biology (iSCB), Centre de Recherche en Cancérologie de Marseille (CRCM), CNRS UMR 7258, INSERM U 1068, Institut Paoli-Calmettes, Aix-Marseille Universités, Marseille 13009 (France)

    2016-08-12

    The redox switch protein CP12 is a key player of the regulation of the Benson–Calvin cycle. Its oxidation state is controlled by the formation/dissociation of two intramolecular disulphide bridges during the day/night cycle. CP12 was known to be globally intrinsically disordered on a large scale in its reduced state, while being partly ordered in the oxidised state. By combining Nuclear Magnetic Resonance and Small Angle X-ray Scattering experiments, we showed that, contrary to secondary structure or disorder predictions, reduced CP12 is fully disordered, with no transient or local residual structure likely to be precursor of the structures identified in the oxidised active state and/or in the bound state with GAPDH or PRK. These results highlight the diversity of the mechanisms of regulation of conditionally disordered redox switches, and question the stability of oxidised CP12 scaffold. - Highlights: • CP12 is predicted to form two helices in its N-terminal sequence. • Reduced CP12 is disordered as a random coil according to SAXS. • Limited or no transient structures are observed in reduced CP12 by NMR.

  17. Whole Genome Shotgun Sequencing Shows Selection on Leptospira Regulatory Proteins during in vitro Culture Attenuation

    Science.gov (United States)

    Lehmann, Jason S.; Corey, Victoria C.; Ricaldi, Jessica N.; Vinetz, Joseph M.; Winzeler, Elizabeth A.; Matthias, Michael A.

    2016-01-01

    Leptospirosis is the most common zoonotic disease worldwide with an estimated 500,000 severe cases reported annually, and case fatality rates of 12–25%, due primarily to acute kidney and lung injuries. Despite its prevalence, the molecular mechanisms underlying leptospirosis pathogenesis remain poorly understood. To identify virulence-related genes in Leptospira interrogans, we delineated cumulative genome changes that occurred during serial in vitro passage of a highly virulent strain of L. interrogans serovar Lai into a nearly avirulent isogenic derivative. Comparison of protein coding and computationally predicted noncoding RNA (ncRNA) genes between these two polyclonal strains identified 15 nonsynonymous single nucleotide variant (nsSNV) alleles that increased in frequency and 19 that decreased, whereas no changes in allelic frequency were observed among the ncRNA genes. Some of the nsSNV alleles were in six genes shown previously to be transcriptionally upregulated during exposure to in vivo-like conditions. Five of these nsSNVs were in evolutionarily conserved positions in genes related to signal transduction and metabolism. Frequency changes of minor nsSNV alleles identified in this study likely contributed to the loss of virulence during serial in vitro culture. The identification of new virulence-associated genes should spur additional experimental inquiry into their potential role in Leptospira pathogenesis. PMID:26711524

  18. Absence of residual structure in the intrinsically disordered regulatory protein CP12 in its reduced state

    International Nuclear Information System (INIS)

    Launay, Hélène; Barré, Patrick; Puppo, Carine; Manneville, Stéphanie; Gontero, Brigitte; Receveur-Bréchot, Véronique

    2016-01-01

    The redox switch protein CP12 is a key player of the regulation of the Benson–Calvin cycle. Its oxidation state is controlled by the formation/dissociation of two intramolecular disulphide bridges during the day/night cycle. CP12 was known to be globally intrinsically disordered on a large scale in its reduced state, while being partly ordered in the oxidised state. By combining Nuclear Magnetic Resonance and Small Angle X-ray Scattering experiments, we showed that, contrary to secondary structure or disorder predictions, reduced CP12 is fully disordered, with no transient or local residual structure likely to be precursor of the structures identified in the oxidised active state and/or in the bound state with GAPDH or PRK. These results highlight the diversity of the mechanisms of regulation of conditionally disordered redox switches, and question the stability of oxidised CP12 scaffold. - Highlights: • CP12 is predicted to form two helices in its N-terminal sequence. • Reduced CP12 is disordered as a random coil according to SAXS. • Limited or no transient structures are observed in reduced CP12 by NMR.

  19. MAR binding protein SMAR1 favors IL-10 mediated regulatory T cell function in acute colitis

    International Nuclear Information System (INIS)

    Mirlekar, Bhalchandra; Patil, Sachin; Bopanna, Ramanamurthy; Chattopadhyay, Samit

    2015-01-01

    T reg cells are not only crucial for controlling immune responses to autoantigens but also prevent those directed towards commensal pathogens. Control of effector immune responses by T reg cells depend on their capacity to accumulate at inflammatory site and accordingly accommodate to inflammatory environment. Till date, the factors associated with maintaining these aspects of T reg phenotype is not understood properly. Here we have shown that a known nuclear matrix binding protein SMAR1 is selectively expressed more in colonic T reg cells and is required for their ability to accumulate at inflammatory site and to sustain high levels of Foxp3 and IL-10 expression during acute colitis. Elimination of anti-inflammatory subsets revealed a protective role for IL-10 producing T reg cells in SMAR1 −/− mice. Moreover, a combined action of Foxp3 and SMAR1 restricts effector cytokine production and enhance the production of IL-10 by colonic T reg cells that controls acute colitis. This data highlights a critical role of SMAR1 in maintaining T reg physiology during inflammatory disorders. - Highlights: • SMAR1 is essential to sustain high level of Foxp3 and IL-10 in T reg cells. • SMAR1 −/− T reg cells produce pro-inflammatory cytokine IL-17 leads to inflammation. • IL-10 administration can control the inflammation in SMAR1 −/− mice. • Both Foxp3 and SMAR1 maintain T reg phenotype that controls colitis

  20. Differential 3’ processing of specific transcripts expands regulatory and protein diversity across neuronal cell types

    Science.gov (United States)

    Jereb, Saša; Hwang, Hun-Way; Van Otterloo, Eric; Govek, Eve-Ellen; Fak, John J; Yuan, Yuan; Hatten, Mary E

    2018-01-01

    Alternative polyadenylation (APA) regulates mRNA translation, stability, and protein localization. However, it is unclear to what extent APA regulates these processes uniquely in specific cell types. Using a new technique, cTag-PAPERCLIP, we discovered significant differences in APA between the principal types of mouse cerebellar neurons, the Purkinje and granule cells, as well as between proliferating and differentiated granule cells. Transcripts that differed in APA in these comparisons were enriched in key neuronal functions and many differed in coding sequence in addition to 3’UTR length. We characterize Memo1, a transcript that shifted from expressing a short 3’UTR isoform to a longer one during granule cell differentiation. We show that Memo1 regulates granule cell precursor proliferation and that its long 3’UTR isoform is targeted by miR-124, contributing to its downregulation during development. Our findings provide insight into roles for APA in specific cell types and establish a platform for further functional studies. PMID:29578408

  1. Alternative splicing and differential gene expression in colon cancer detected by a whole genome exon array

    Directory of Open Access Journals (Sweden)

    Sugnet Charles

    2006-12-01

    Full Text Available Abstract Background Alternative splicing is a mechanism for increasing protein diversity by excluding or including exons during post-transcriptional processing. Alternatively spliced proteins are particularly relevant in oncology since they may contribute to the etiology of cancer, provide selective drug targets, or serve as a marker set for cancer diagnosis. While conventional identification of splice variants generally targets individual genes, we present here a new exon-centric array (GeneChip Human Exon 1.0 ST that allows genome-wide identification of differential splice variation, and concurrently provides a flexible and inclusive analysis of gene expression. Results We analyzed 20 paired tumor-normal colon cancer samples using a microarray designed to detect over one million putative exons that can be virtually assembled into potential gene-level transcripts according to various levels of prior supporting evidence. Analysis of high confidence (empirically supported transcripts identified 160 differentially expressed genes, with 42 genes occupying a network impacting cell proliferation and another twenty nine genes with unknown functions. A more speculative analysis, including transcripts based solely on computational prediction, produced another 160 differentially expressed genes, three-fourths of which have no previous annotation. We also present a comparison of gene signal estimations from the Exon 1.0 ST and the U133 Plus 2.0 arrays. Novel splicing events were predicted by experimental algorithms that compare the relative contribution of each exon to the cognate transcript intensity in each tissue. The resulting candidate splice variants were validated with RT-PCR. We found nine genes that were differentially spliced between colon tumors and normal colon tissues, several of which have not been previously implicated in cancer. Top scoring candidates from our analysis were also found to substantially overlap with EST-based bioinformatic

  2. MAR binding protein SMAR1 favors IL-10 mediated regulatory T cell function in acute colitis

    Energy Technology Data Exchange (ETDEWEB)

    Mirlekar, Bhalchandra; Patil, Sachin [Chromatin and Disease Biology Laboratory, National Centre for Cell Science, Ganeshkhind, Pune 411007 (India); Bopanna, Ramanamurthy [Experimental Animal Facility, National Centre for Cell Science, Ganeshkhind, Pune 411007 (India); Chattopadhyay, Samit, E-mail: samit@nccs.res.in [Chromatin and Disease Biology Laboratory, National Centre for Cell Science, Ganeshkhind, Pune 411007 (India)

    2015-08-21

    T{sub reg} cells are not only crucial for controlling immune responses to autoantigens but also prevent those directed towards commensal pathogens. Control of effector immune responses by T{sub reg} cells depend on their capacity to accumulate at inflammatory site and accordingly accommodate to inflammatory environment. Till date, the factors associated with maintaining these aspects of T{sub reg} phenotype is not understood properly. Here we have shown that a known nuclear matrix binding protein SMAR1 is selectively expressed more in colonic T{sub reg} cells and is required for their ability to accumulate at inflammatory site and to sustain high levels of Foxp3 and IL-10 expression during acute colitis. Elimination of anti-inflammatory subsets revealed a protective role for IL-10 producing T{sub reg} cells in SMAR1{sup −/−} mice. Moreover, a combined action of Foxp3 and SMAR1 restricts effector cytokine production and enhance the production of IL-10 by colonic T{sub reg} cells that controls acute colitis. This data highlights a critical role of SMAR1 in maintaining T{sub reg} physiology during inflammatory disorders. - Highlights: • SMAR1 is essential to sustain high level of Foxp3 and IL-10 in T{sub reg} cells. • SMAR1{sup −/−} T{sub reg} cells produce pro-inflammatory cytokine IL-17 leads to inflammation. • IL-10 administration can control the inflammation in SMAR1{sup −/−} mice. • Both Foxp3 and SMAR1 maintain T{sub reg} phenotype that controls colitis.

  3. The HTLV-1 Tax protein binding domain of cyclin-dependent kinase 4 (CDK4 includes the regulatory PSTAIRE helix

    Directory of Open Access Journals (Sweden)

    Grassmann Ralph

    2005-09-01

    Full Text Available Abstract Background The Tax oncoprotein of human T-cell leukemia virus type 1 (HTLV-1 is leukemogenic in transgenic mice and induces permanent T-cell growth in vitro. It is found in active CDK holoenzyme complexes from adult T-cell leukemia-derived cultures and stimulates the G1- to-S phase transition by activating the cyclin-dependent kinase (CDK CDK4. The Tax protein directly and specifically interacts with CDK4 and cyclin D2 and binding is required for enhanced CDK4 kinase activity. The protein-protein contact between Tax and the components of the cyclin D/CDK complexes increases the association of CDK4 and its positive regulatory subunit cyclin D and renders the complex resistant to p21CIP inhibition. Tax mutants affecting the N-terminus cannot bind cyclin D and CDK4. Results To analyze, whether the N-terminus of Tax is capable of CDK4-binding, in vitro binding -, pull down -, and mammalian two-hybrid analyses were performed. These experiments revealed that a segment of 40 amino acids is sufficient to interact with CDK4 and cyclin D2. To define a Tax-binding domain and analyze how Tax influences the kinase activity, a series of CDK4 deletion mutants was tested. Different assays revealed two regions which upon deletion consistently result in reduced binding activity. These were isolated and subjected to mammalian two-hybrid analysis to test their potential to interact with the Tax N-terminus. These experiments concurrently revealed binding at the N- and C-terminus of CDK4. The N-terminal segment contains the PSTAIRE helix, which is known to control the access of substrate to the active cleft of CDK4 and thus the kinase activity. Conclusion Since the N- and C-terminus of CDK4 are neighboring in the predicted three-dimensional protein structure, it is conceivable that they comprise a single binding domain, which interacts with the Tax N-terminus.

  4. Induction of tumor cell death through targeting tubulin and evoking dysregulation of cell cycle regulatory proteins by multifunctional cinnamaldehydes.

    Directory of Open Access Journals (Sweden)

    Amrita A Nagle

    Full Text Available Multifunctional trans-cinnamaldehyde (CA and its analogs display anti-cancer properties, with 2-benzoyloxycinnamaldehyde (BCA and 5-fluoro-2-hydroxycinnamaldehyde (FHCA being identified as the ortho-substituted analogs that possess potent anti-tumor activities. In this study, BCA, FHCA and a novel analog 5-fluoro-2-benzoyloxycinnamaldehyde (FBCA, were demonstrated to decrease growth and colony formation of human colon-derived HCT 116 and mammary-derived MCF-7 carcinoma cells under non-adhesive conditions. The 2-benzoyloxy and 5-fluoro substituents rendered FBCA more potent than BCA and equipotent to FHCA. The cellular events by which these cinnamaldehydes caused G(2/M phase arrest and halted proliferation of HCT 116 cells were thereby investigated. Lack of significant accumulation of mitosis marker phospho-histone H3 in cinnamaldehyde-treated cells indicated that the analogs arrested cells in G(2 phase. G(2 arrest was brought about partly by cinnamaldehyde-mediated depletion of cell cycle proteins involved in regulating G(2 to M transition and spindle assembly, namely cdk1, cdc25C, mad2, cdc20 and survivin. Cyclin B1 levels were found to be increased, which in the absence of active cdk1, would fail to drive cells into M phase. Concentrations of cinnamaldehydes that brought about dysregulation of levels of cell cycle proteins also caused tubulin aggregation, as evident from immunodetection of dose-dependent tubulin accumulation in the insoluble cell lysate fractions. In a cell-free system, reduced biotin-conjugated iodoacetamide (BIAM labeling of tubulin protein pretreated with cinnamaldehydes was indicative of drug interaction with the sulfhydryl groups in tubulin. In conclusion, cinnamaldehydes treatment at proapoptotic concentrations caused tubulin aggregation and dysegulation of cell cycle regulatory proteins cdk1 and cdc25C that contributed at least in part to arresting cells at G(2 phase, resulting in apoptotic cell death characterized by

  5. Induction of tumor cell death through targeting tubulin and evoking dysregulation of cell cycle regulatory proteins by multifunctional cinnamaldehydes.

    Science.gov (United States)

    Nagle, Amrita A; Gan, Fei-Fei; Jones, Gavin; So, Choon-Leng; Wells, Geoffrey; Chew, Eng-Hui

    2012-01-01

    Multifunctional trans-cinnamaldehyde (CA) and its analogs display anti-cancer properties, with 2-benzoyloxycinnamaldehyde (BCA) and 5-fluoro-2-hydroxycinnamaldehyde (FHCA) being identified as the ortho-substituted analogs that possess potent anti-tumor activities. In this study, BCA, FHCA and a novel analog 5-fluoro-2-benzoyloxycinnamaldehyde (FBCA), were demonstrated to decrease growth and colony formation of human colon-derived HCT 116 and mammary-derived MCF-7 carcinoma cells under non-adhesive conditions. The 2-benzoyloxy and 5-fluoro substituents rendered FBCA more potent than BCA and equipotent to FHCA. The cellular events by which these cinnamaldehydes caused G(2)/M phase arrest and halted proliferation of HCT 116 cells were thereby investigated. Lack of significant accumulation of mitosis marker phospho-histone H3 in cinnamaldehyde-treated cells indicated that the analogs arrested cells in G(2) phase. G(2) arrest was brought about partly by cinnamaldehyde-mediated depletion of cell cycle proteins involved in regulating G(2) to M transition and spindle assembly, namely cdk1, cdc25C, mad2, cdc20 and survivin. Cyclin B1 levels were found to be increased, which in the absence of active cdk1, would fail to drive cells into M phase. Concentrations of cinnamaldehydes that brought about dysregulation of levels of cell cycle proteins also caused tubulin aggregation, as evident from immunodetection of dose-dependent tubulin accumulation in the insoluble cell lysate fractions. In a cell-free system, reduced biotin-conjugated iodoacetamide (BIAM) labeling of tubulin protein pretreated with cinnamaldehydes was indicative of drug interaction with the sulfhydryl groups in tubulin. In conclusion, cinnamaldehydes treatment at proapoptotic concentrations caused tubulin aggregation and dysegulation of cell cycle regulatory proteins cdk1 and cdc25C that contributed at least in part to arresting cells at G(2) phase, resulting in apoptotic cell death characterized by emergence

  6. Differential HFE gene expression is regulated by alternative splicing in human tissues.

    Science.gov (United States)

    Martins, Rute; Silva, Bruno; Proença, Daniela; Faustino, Paula

    2011-03-03

    The pathophysiology of HFE-derived Hereditary Hemochromatosis and the function of HFE protein in iron homeostasis remain uncertain. Also, the role of alternative splicing in HFE gene expression regulation and the possible function of the corresponding protein isoforms are still unknown. The aim of this study was to gain insights into the physiological significance of these alternative HFE variants. Alternatively spliced HFE transcripts in diverse human tissues were identified by RT-PCR, cloning and sequencing. Total HFE transcripts, as well as two alternative splicing transcripts were quantified using a real-time PCR methodology. Intracellular localization, trafficking and protein association of GFP-tagged HFE protein variants were analysed in transiently transfected HepG2 cells by immunoprecipitation and immunofluorescence assays. Alternatively spliced HFE transcripts present both level- and tissue-specificity. Concerning the exon 2 skipping and intron 4 inclusion transcripts, the liver presents the lowest relative level, while duodenum presents one of the highest amounts. The protein resulting from exon 2 skipping transcript is unable to associate with β2M and TfR1 and reveals an ER retention. Conversely, the intron 4 inclusion transcript gives rise to a truncated, soluble protein (sHFE) that is mostly secreted by cells to the medium in association with β2M. HFE gene post-transcriptional regulation is clearly affected by a tissue-dependent alternative splicing mechanism. Among the corresponding proteins, a sHFE isoform stands out, which upon being secreted into the bloodstream, may act in remote tissues. It could be either an agonist or antagonist of the full length HFE, through hepcidin expression regulation in the liver or by controlling dietary iron absorption in the duodenum.

  7. Differential HFE gene expression is regulated by alternative splicing in human tissues.

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    Rute Martins

    Full Text Available BACKGROUND: The pathophysiology of HFE-derived Hereditary Hemochromatosis and the function of HFE protein in iron homeostasis remain uncertain. Also, the role of alternative splicing in HFE gene expression regulation and the possible function of the corresponding protein isoforms are still unknown. The aim of this study was to gain insights into the physiological significance of these alternative HFE variants. METHODOLOGY/PRINCIPAL FINDINGS: Alternatively spliced HFE transcripts in diverse human tissues were identified by RT-PCR, cloning and sequencing. Total HFE transcripts, as well as two alternative splicing transcripts were quantified using a real-time PCR methodology. Intracellular localization, trafficking and protein association of GFP-tagged HFE protein variants were analysed in transiently transfected HepG2 cells by immunoprecipitation and immunofluorescence assays. Alternatively spliced HFE transcripts present both level- and tissue-specificity. Concerning the exon 2 skipping and intron 4 inclusion transcripts, the liver presents the lowest relative level, while duodenum presents one of the highest amounts. The protein resulting from exon 2 skipping transcript is unable to associate with β2M and TfR1 and reveals an ER retention. Conversely, the intron 4 inclusion transcript gives rise to a truncated, soluble protein (sHFE that is mostly secreted by cells to the medium in association with β2M. CONCLUSIONS/SIGNIFICANCE: HFE gene post-transcriptional regulation is clearly affected by a tissue-dependent alternative splicing mechanism. Among the corresponding proteins, a sHFE isoform stands out, which upon being secreted into the bloodstream, may act in remote tissues. It could be either an agonist or antagonist of the full length HFE, through hepcidin expression regulation in the liver or by controlling dietary iron absorption in the duodenum.

  8. Thermopriming Triggers Splicing Memory in Arabidopsis

    KAUST Repository

    Ling, Yu

    2018-02-20

    Abiotic and biotic stresses limit crop productivity. Exposure to a non-lethal stress, referred to as priming, can allow plants to survive subsequent and otherwise lethal conditions; the priming effect persists even after a prolonged stress-free period. However, the molecular mechanisms underlying priming are not fully understood. Here, we investigated the molecular basis of heat shock memory and the role of priming in Arabidopsisthaliana. Comprehensive analysis of transcriptome-wide changes in gene expression and alternative splicing in primed and non-primed plants revealed that alternative splicing functions as a novel component of heat shock memory. We show that priming of plants with a non-lethal heat stress results in de-repression of splicing after a second exposure to heat stress. By contrast, non-primed plants showed significant repression of splicing. These observations link ‘splicing memory’ to the ability of plants to survive subsequent and otherwise lethal heat stress. This newly discovered priming-induced splicing memory may represent a general feature of heat stress responses in plants and other organisms as many of the key components of heat shock responses are conserved among eukaryotes. Furthermore, this finding could facilitate the development of novel approaches to improve plant survival under extreme heat stress.

  9. Glucokinase regulatory protein genetic variant interacts with omega-3 PUFA to influence insulin resistance and inflammation in metabolic syndrome.

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    Pablo Perez-Martinez

    Full Text Available Glucokinase Regulatory Protein (GCKR plays a central role regulating both hepatic triglyceride and glucose metabolism. Fatty acids are key metabolic regulators, which interact with genetic factors and influence glucose metabolism and other metabolic traits. Omega-3 polyunsaturated fatty acids (n-3 PUFA have been of considerable interest, due to their potential to reduce metabolic syndrome (MetS risk.To examine whether genetic variability at the GCKR gene locus was associated with the degree of insulin resistance, plasma concentrations of C-reactive protein (CRP and n-3 PUFA in MetS subjects.Homeostasis model assessment of insulin resistance (HOMA-IR, HOMA-B, plasma concentrations of C-peptide, CRP, fatty acid composition and the GCKR rs1260326-P446L polymorphism, were determined in a cross-sectional analysis of 379 subjects with MetS participating in the LIPGENE dietary cohort.Among subjects with n-3 PUFA levels below the population median, carriers of the common C/C genotype had higher plasma concentrations of fasting insulin (P = 0.019, C-peptide (P = 0.004, HOMA-IR (P = 0.008 and CRP (P = 0.032 as compared with subjects carrying the minor T-allele (Leu446. In contrast, homozygous C/C carriers with n-3 PUFA levels above the median showed lower plasma concentrations of fasting insulin, peptide C, HOMA-IR and CRP, as compared with individuals with the T-allele.We have demonstrated a significant interaction between the GCKR rs1260326-P446L polymorphism and plasma n-3 PUFA levels modulating insulin resistance and inflammatory markers in MetS subjects. Further studies are needed to confirm this gene-diet interaction in the general population and whether targeted dietary recommendations can prevent MetS in genetically susceptible individuals.ClinicalTrials.gov NCT00429195.

  10. Identification of Rbd2 as a candidate protease for sterol regulatory element binding protein (SREBP) cleavage in fission yeast

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jinsil; Ha, Hye-Jeong [Aging Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 34141 (Korea, Republic of); Kim, Sujin [Aging Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 34141 (Korea, Republic of); Department of Functional Genomics, University of Science and Technology (UST), 217 Gajeong-ro, Yuseong-gu, Daejeon 34113 (Korea, Republic of); Choi, Ah-Reum [Aging Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 34141 (Korea, Republic of); Lee, Sook-Jeong [Department of New Drug Discovery and Development, Chungnam National University, 99 Daehak-ro, Yuseong-gu, Daejeon 34134 (Korea, Republic of); Hoe, Kwang-Lae, E-mail: kwanghoe@cnu.ac.kr [Department of New Drug Discovery and Development, Chungnam National University, 99 Daehak-ro, Yuseong-gu, Daejeon 34134 (Korea, Republic of); Kim, Dong-Uk, E-mail: kimdongu@kribb.re.kr [Aging Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 34141 (Korea, Republic of)

    2015-12-25

    Lipid homeostasis in mammalian cells is regulated by sterol regulatory element-binding protein (SREBP) transcription factors that are activated through sequential cleavage by Golgi Site-1 and Site-2 proteases. Fission yeast SREBP, Sre1, engages a different mechanism involving the Golgi Dsc E3 ligase complex, but it is not clearly understood exactly how Sre1 is proteolytically cleaved and activated. In this study, we screened the Schizosaccharomyces pombe non-essential haploid deletion collection to identify missing components of the Sre1 cleavage machinery. Our screen identified an additional component of the SREBP pathway required for Sre1 proteolysis named rhomboid protein 2 (Rbd2). We show that an rbd2 deletion mutant fails to grow under hypoxic and hypoxia-mimetic conditions due to lack of Sre1 activity and that this growth phenotype is rescued by Sre1N, a cleaved active form of Sre1. We found that the growth inhibition phenotype under low oxygen conditions is specific to the strain with deletion of rbd2, not any other fission yeast rhomboid-encoding genes. Our study also identified conserved residues of Rbd2 that are required for Sre1 proteolytic cleavage. All together, our results suggest that Rbd2 is a functional SREBP protease with conserved residues required for Sre1 cleavage and provide an important piece of the puzzle to understand the mechanisms for Sre1 activation and the regulation of various biological and pathological processes involving SREBPs. - Highlights: • An rbd2-deleted yeast strain shows defects in growth in response to low oxygen levels. • rbd2-deficient cells fail to generate cleaved Sre1 (Sre1N) under hypoxic conditions. • Expression of Sre1N rescues the rbd2 deletion mutant growth phenotype. • Rbd2 contains conserved residues potentially critical for catalytic activity. • Mutation of the conserved Rbd2 catalytic residues leads to defects in Sre1 cleavage.

  11. Protein Kinase A Regulatory Subunit Isoforms Regulate Growth and Differentiation in Mucor circinelloides: Essential Role of PKAR4

    Science.gov (United States)

    Ocampo, J.; McCormack, B.; Navarro, E.; Moreno, S.; Garre, V.

    2012-01-01

    The protein kinase A (PKA) signaling pathway plays a role in regulating growth and differentiation in the dimorphic fungus Mucor circinelloides. PKA holoenzyme is comprised of two catalytic (C) and two regulatory (R) subunits. In M. circinelloides, four genes encode the PKAR1, PKAR2, PKAR3, and PKAR4 isoforms of R subunits. We have constructed null mutants and demonstrate that each isoform has a different role in growth and differentiation. The most striking finding is that pkaR4 is an essential gene, because only heterokaryons were obtained in knockout experiments. Heterokaryons with low levels of wild-type nuclei showed an impediment in the emission of the germ tube, suggesting a pivotal role of this gene in germ tube emergence. The remaining null strains showed different alterations in germ tube emergence, sporulation, and volume of the mother cell. The pkaR2 null mutant showed an accelerated germ tube emission and was the only mutant that germinated under anaerobic conditions when glycine was used as a nitrogen source, suggesting that pkaR2 participates in germ tube emergence by repressing it. From the measurement of the mRNA and protein levels of each isoform in the wild-type and knockout strains, it can be concluded that the expression of each subunit has its own mechanism of differential regulation. The PKAR1 and PKAR2 isoforms are posttranslationally modified by ubiquitylation, suggesting another regulation point in the specificity of the signal transduction. The results indicate that each R isoform has a different role in M. circinelloides physiology, controlling the dimorphism and contributing to the specificity of cyclic AMP (cAMP)-PKA pathway. PMID:22635921

  12. Characterization of novel StAR (steroidogenic acute regulatory protein mutations causing non-classic lipoid adrenal hyperplasia.

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    Christa E Flück

    Full Text Available CONTEXT: Steroidogenic acute regulatory protein (StAR is crucial for transport of cholesterol to mitochondria where biosynthesis of steroids is initiated. Loss of StAR function causes lipoid congenital adrenal hyperplasia (LCAH. OBJECTIVE: StAR gene mutations causing partial loss of function manifest atypical and may be mistaken as familial glucocorticoid deficiency. Only a few mutations have been reported. DESIGN: To report clinical, biochemical, genetic, protein structure and functional data on two novel StAR mutations, and to compare them with published literature. SETTING: Collaboration between the University Children's Hospital Bern, Switzerland, and the CIBERER, Hospital Vall d'Hebron, Autonomous University, Barcelona, Spain. PATIENTS: Two subjects of a non-consanguineous Caucasian family were studied. The 46,XX phenotypic normal female was diagnosed with adrenal insufficiency at the age of 10 months, had normal pubertal development and still has no signs of hypergonodatropic hypogonadism at 32 years of age. Her 46,XY brother was born with normal male external genitalia and was diagnosed with adrenal insufficiency at 14 months. Puberty was normal and no signs of hypergonadotropic hypogonadism are present at 29 years of age. RESULTS: StAR gene analysis revealed two novel compound heterozygote mutations T44HfsX3 and G221S. T44HfsX3 is a loss-of-function StAR mutation. G221S retains partial activity (∼30% and is therefore responsible for a milder, non-classic phenotype. G221S is located in the cholesterol binding pocket and seems to alter binding/release of cholesterol. CONCLUSIONS: StAR mutations located in the cholesterol binding pocket (V187M, R188C, R192C, G221D/S seem to cause non-classic lipoid CAH. Accuracy of genotype-phenotype prediction by in vitro testing may vary with the assays employed.

  13. Identification of interleukin-26 in the dromedary camel (Camelus dromedarius): Evidence of alternative splicing and isolation of novel splice variants.

    Science.gov (United States)

    Premraj, Avinash; Nautiyal, Binita; Aleyas, Abi G; Rasool, Thaha Jamal

    2015-10-01

    Interleukin-26 (IL-26) is a member of the IL-10 family of cytokines. Though conserved across vertebrates, the IL-26 gene is functionally inactivated in a few mammals like rat, mouse and horse. We report here the identification, isolation and cloning of the cDNA of IL-26 from the dromedary camel. The camel cDNA contains a 516 bp open reading frame encoding a 171 amino acid precursor protein, including a 21 amino acid signal peptide. Sequence analysis revealed high similarity with other mammalian IL-26 homologs and the conservation of IL-10 cytokine family domain structure including key amino acid residues. We also report the identification and cloning of four novel transcript variants produced by alternative splicing at the Exon 3-Exon 4 regions of the gene. Three of the alternative splice variants had premature termination codons and are predicted to code for truncated proteins. The transcript variant 4 (Tv4) having an insertion of an extra 120 bp nucleotides in the ORF was predicted to encode a full length protein product with 40 extra amino acid residues. The mRNA transcripts of all the variants were identified in lymph node, where as fewer variants were observed in other tissues like blood, liver and kidney. The expression of Tv2 and Tv3 were found to be up regulated in mitogen induced camel peripheral blood mononuclear cells. IL-26-Tv2 expression was also induced in camel fibroblast cells infected with Camel pox virus in-vitro. The identification of the transcript variants of IL-26 from the dromedary camel is the first report of alternative splicing for IL-26 in a species in which the gene has not been inactivated. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. A Model of Yeast Cell-Cycle Regulation Based on a Standard Component Modeling Strategy for Protein Regulatory Networks.

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    Teeraphan Laomettachit

    Full Text Available To understand the molecular mechanisms that regulate cell cycle progression in eukaryotes, a variety of mathematical modeling approaches have been employed, ranging from Boolean networks and differential equations to stochastic simulations. Each approach has its own characteristic strengths and weaknesses. In this paper, we propose a "standard component" modeling strategy that combines advantageous features of Boolean networks, differential equations and stochastic simulations in a framework that acknowledges the typical sorts of reactions found in protein regulatory networks. Applying th