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Sample records for premessenger rna binding

  1. Cancer-Associated Perturbations in Alternative Pre-messenger RNA Splicing.

    Science.gov (United States)

    Shkreta, Lulzim; Bell, Brendan; Revil, Timothée; Venables, Julian P; Prinos, Panagiotis; Elela, Sherif Abou; Chabot, Benoit

    2013-01-01

    For most of our 25,000 genes, the removal of introns by pre-messenger RNA (pre-mRNA) splicing represents an essential step toward the production of functional messenger RNAs (mRNAs). Alternative splicing of a single pre-mRNA results in the production of different mRNAs. Although complex organisms use alternative splicing to expand protein function and phenotypic diversity, patterns of alternative splicing are often altered in cancer cells. Alternative splicing contributes to tumorigenesis by producing splice isoforms that can stimulate cell proliferation and cell migration or induce resistance to apoptosis and anticancer agents. Cancer-specific changes in splicing profiles can occur through mutations that are affecting splice sites and splicing control elements, and also by alterations in the expression of proteins that control splicing decisions. Recent progress in global approaches that interrogate splicing diversity should help to obtain specific splicing signatures for cancer types. The development of innovative approaches for annotating and reprogramming splicing events will more fully establish the essential contribution of alternative splicing to the biology of cancer and will hopefully provide novel targets and anticancer strategies. Metazoan genes are usually made up of several exons interrupted by introns. The introns are removed from the pre-mRNA by RNA splicing. In conjunction with other maturation steps, such as capping and polyadenylation, the spliced mRNA is then transported to the cytoplasm to be translated into a functional protein. The basic mechanism of splicing requires accurate recognition of each extremity of each intron by the spliceosome. Introns are identified by the binding of U1 snRNP to the 5' splice site and the U2AF65/U2AF35 complex to the 3' splice site. Following these interactions, other proteins and snRNPs are recruited to generate the complete spliceosomal complex needed to excise the intron. While many introns are constitutively

  2. 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.

  3. The Cleavage and Polyadenylation Specificity Factor 6 (CPSF6) Subunit of the Capsid-recruited Pre-messenger RNA Cleavage Factor I (CFIm) Complex Mediates HIV-1 Integration into Genes.

    Science.gov (United States)

    Rasheedi, Sheeba; Shun, Ming-Chieh; Serrao, Erik; Sowd, Gregory A; Qian, Juan; Hao, Caili; Dasgupta, Twishasri; Engelman, Alan N; Skowronski, Jacek

    2016-05-27

    HIV-1 favors integration into active genes and gene-enriched regions of host cell chromosomes, thus maximizing the probability of provirus expression immediately after integration. This requires cleavage and polyadenylation specificity factor 6 (CPSF6), a cellular protein involved in pre-mRNA 3' end processing that binds HIV-1 capsid and connects HIV-1 preintegration complexes to intranuclear trafficking pathways that link integration to transcriptionally active chromatin. CPSF6 together with CPSF5 and CPSF7 are known subunits of the cleavage factor I (CFIm) 3' end processing complex; however, CPSF6 could participate in additional protein complexes. The molecular mechanisms underpinning the role of CPSF6 in HIV-1 infection remain to be defined. Here, we show that a majority of cellular CPSF6 is incorporated into the CFIm complex. HIV-1 capsid recruits CFIm in a CPSF6-dependent manner, which suggests that the CFIm complex mediates the known effects of CPSF6 in HIV-1 infection. To dissect the roles of CPSF6 and other CFIm complex subunits in HIV-1 infection, we analyzed virologic and integration site targeting properties of a CPSF6 variant with mutations that prevent its incorporation into CFIm We show, somewhat surprisingly, that CPSF6 incorporation into CFIm is not required for its ability to direct preferential HIV-1 integration into genes. The CPSF5 and CPSF7 subunits appear to have only a minor, if any, role in this process even though they appear to facilitate CPSF6 binding to capsid. Thus, CPSF6 alone controls the key molecular interactions that specify HIV-1 preintegration complex trafficking to active chromatin.

  4. Engineering RNA-binding proteins for biology

    OpenAIRE

    Chen,Yu; Varani, Gabriele

    2013-01-01

    RNA-binding proteins play essential roles in the regulation of gene expression. Many have modular structures and combine relatively few common domains in various arrangements to recognize RNA sequences and/or structures. Recent progress in engineering the specificity of the PUF class RNA-binding proteins has shown that RNA-binding domains may be combined with various effector or functional domains to regulate the metabolism of targeted RNAs. Designer RNA-binding proteins with tailored sequenc...

  5. Plant RNA binding proteins for control of RNA virus infection

    OpenAIRE

    Huh, Sung Un; Paek, Kyung-Hee

    2013-01-01

    Plant RNA viruses have effective strategies to infect host plants through either direct or indirect interactions with various host proteins, thus suppressing the host immune system. When plant RNA viruses enter host cells exposed RNAs of viruses are recognized by the host immune system through processes such as siRNA-dependent silencing. Interestingly, some host RNA binding proteins have been involved in the inhibition of RNA virus replication, movement, and translation through RNA-specific b...

  6. An RNA motif that binds ATP

    Science.gov (United States)

    Sassanfar, M.; Szostak, J. W.

    1993-01-01

    RNAs that contain specific high-affinity binding sites for small molecule ligands immobilized on a solid support are present at a frequency of roughly one in 10(10)-10(11) in pools of random sequence RNA molecules. Here we describe a new in vitro selection procedure designed to ensure the isolation of RNAs that bind the ligand of interest in solution as well as on a solid support. We have used this method to isolate a remarkably small RNA motif that binds ATP, a substrate in numerous biological reactions and the universal biological high-energy intermediate. The selected ATP-binding RNAs contain a consensus sequence, embedded in a common secondary structure. The binding properties of ATP analogues and modified RNAs show that the binding interaction is characterized by a large number of close contacts between the ATP and RNA, and by a change in the conformation of the RNA.

  7. Computational Prediction of RNA-Binding Proteins and Binding Sites

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    Jingna Si

    2015-11-01

    Full Text Available Proteins and RNA interaction have vital roles in many cellular processes such as protein synthesis, sequence encoding, RNA transfer, and gene regulation at the transcriptional and post-transcriptional levels. Approximately 6%–8% of all proteins are RNA-binding proteins (RBPs. Distinguishing these RBPs or their binding residues is a major aim of structural biology. Previously, a number of experimental methods were developed for the determination of protein–RNA interactions. However, these experimental methods are expensive, time-consuming, and labor-intensive. Alternatively, researchers have developed many computational approaches to predict RBPs and protein–RNA binding sites, by combining various machine learning methods and abundant sequence and/or structural features. There are three kinds of computational approaches, which are prediction from protein sequence, prediction from protein structure, and protein-RNA docking. In this paper, we review all existing studies of predictions of RNA-binding sites and RBPs and complexes, including data sets used in different approaches, sequence and structural features used in several predictors, prediction method classifications, performance comparisons, evaluation methods, and future directions.

  8. Computational Prediction of RNA-Binding Proteins and Binding Sites.

    Science.gov (United States)

    Si, Jingna; Cui, Jing; Cheng, Jin; Wu, Rongling

    2015-01-01

    Proteins and RNA interaction have vital roles in many cellular processes such as protein synthesis, sequence encoding, RNA transfer, and gene regulation at the transcriptional and post-transcriptional levels. Approximately 6%-8% of all proteins are RNA-binding proteins (RBPs). Distinguishing these RBPs or their binding residues is a major aim of structural biology. Previously, a number of experimental methods were developed for the determination of protein-RNA interactions. However, these experimental methods are expensive, time-consuming, and labor-intensive. Alternatively, researchers have developed many computational approaches to predict RBPs and protein-RNA binding sites, by combining various machine learning methods and abundant sequence and/or structural features. There are three kinds of computational approaches, which are prediction from protein sequence, prediction from protein structure, and protein-RNA docking. In this paper, we review all existing studies of predictions of RNA-binding sites and RBPs and complexes, including data sets used in different approaches, sequence and structural features used in several predictors, prediction method classifications, performance comparisons, evaluation methods, and future directions.

  9. CAG trinucleotide RNA repeats interact with RNA-binding proteins

    Energy Technology Data Exchange (ETDEWEB)

    McLaughlin, B.A.; Eberwine, J.; Spencer, C. [Univ. of Pennsylvania, Philadelphia, PA (United States)

    1996-09-01

    Genes associated with several neurological diseases are characterized by the presence of an abnormally long trinucleotide repeat sequence. By way of example, Huntington`s disease (HD), is characterized by selective neuronal degeneration associated with the expansion of a polyglutamine-encoding CAG tract. Normally, this CAG tract is comprised of 11-34 repeats, but in HD it is expanded to >37 repeats in affected individuals. The mechanism by which CAG repeats cause neuronal degeneration is unknown, but it has been speculated that the expansion primarily causes abnormal protein functioning, which in turn causes HD pathology. Other mechanisms, however, have not been ruled out. Interactions between RNA and RNA-binding proteins have previously been shown to play a role in the expression of several eukaryotic genes. Herein, we report the association of cytoplasmic proteins with normal length and extended CAG repeats, using gel shift and LJV crosslinking assays. Cytoplasmic protein extracts from several rat brain regions, including the striatum and cortex, sites of neuronal degeneration in HD, contain a 63-kD RNA-binding protein that specifically interacts with these CAG-repeat sequences. These protein-RNA interactions are dependent on the length of the CAG repeat, with longer repeats binding substantially more protein. Two CAG repeat-binding proteins are present in human cortex and striatum; one comigrates with the rat protein at 63 kD, while the other migrates at 49 kD. These data suggest mechanisms by which RNA-binding proteins may be involved in the pathological course of trinucleotide repeat-associated neurological diseases. 47 refs., 5 figs.

  10. PRBP: Prediction of RNA-Binding Proteins Using a Random Forest Algorithm Combined with an RNA-Binding Residue Predictor.

    Science.gov (United States)

    Ma, Xin; Guo, Jing; Xiao, Ke; Sun, Xiao

    2015-01-01

    The prediction of RNA-binding proteins is an incredibly challenging problem in computational biology. Although great progress has been made using various machine learning approaches with numerous features, the problem is still far from being solved. In this study, we attempt to predict RNA-binding proteins directly from amino acid sequences. A novel approach, PRBP predicts RNA-binding proteins using the information of predicted RNA-binding residues in conjunction with a random forest based method. For a given protein, we first predict its RNA-binding residues and then judge whether the protein binds RNA or not based on information from that prediction. If the protein cannot be identified by the information associated with its predicted RNA-binding residues, then a novel random forest predictor is used to determine if the query protein is a RNA-binding protein. We incorporated features of evolutionary information combined with physicochemical features (EIPP) and amino acid composition feature to establish the random forest predictor. Feature analysis showed that EIPP contributed the most to the prediction of RNA-binding proteins. The results also showed that the information from the RNA-binding residue prediction improved the overall performance of our RNA-binding protein prediction. It is anticipated that the PRBP method will become a useful tool for identifying RNA-binding proteins. A PRBP Web server implementation is freely available at http://www.cbi.seu.edu.cn/PRBP/.

  11. DNA and RNA Quadruplex-Binding Proteins

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    Václav Brázda

    2014-09-01

    Full Text Available Four-stranded DNA structures were structurally characterized in vitro by NMR, X-ray and Circular Dichroism spectroscopy in detail. Among the different types of quadruplexes (i-Motifs, minor groove quadruplexes, G-quadruplexes, etc., the best described are G-quadruplexes which are featured by Hoogsteen base-paring. Sequences with the potential to form quadruplexes are widely present in genome of all organisms. They are found often in repetitive sequences such as telomeric ones, and also in promoter regions and 5' non-coding sequences. Recently, many proteins with binding affinity to G-quadruplexes have been identified. One of the initially portrayed G-rich regions, the human telomeric sequence (TTAGGGn, is recognized by many proteins which can modulate telomerase activity. Sequences with the potential to form G-quadruplexes are often located in promoter regions of various oncogenes. The NHE III1 region of the c-MYC promoter has been shown to interact with nucleolin protein as well as other G-quadruplex-binding proteins. A number of G-rich sequences are also present in promoter region of estrogen receptor alpha. In addition to DNA quadruplexes, RNA quadruplexes, which are critical in translational regulation, have also been predicted and observed. For example, the RNA quadruplex formation in telomere-repeat-containing RNA is involved in interaction with TRF2 (telomere repeat binding factor 2 and plays key role in telomere regulation. All these fundamental examples suggest the importance of quadruplex structures in cell processes and their understanding may provide better insight into aging and disease development.

  12. Binding of ampholine to transfer RNA.

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    Galante, E; Caravaggio, T; Righetti, P G

    1976-09-06

    The melting temperature of isoaccepting tRNAfMet is affected by Ampholine. The plot of Tm versus the logarithm of Ampholine concentration shows clearly an increasing effect of Ampholine when the pH changes from 7.4 to 4.2. This result is interpreted as binding of Ampholine to the nucleic acid. The effects of Ampholine have been compared with those of soidum, magnesium and tetraethylene pentamine. Ampholine carrier ampholytes at pH 4.2 bind to tRNA with the same affinity as magnesium; at higher pH values they are less active. An hypothesis for the mechanism of action of Ampholine on nucleic acids during isoelectric focusing is proposed.

  13. Native mitochondrial RNA-binding complexes in kinetoplastid RNA editing differ in guide RNA composition.

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    Madina, Bhaskara R; Kumar, Vikas; Metz, Richard; Mooers, Blaine H M; Bundschuh, Ralf; Cruz-Reyes, Jorge

    2014-07-01

    Mitochondrial mRNAs in kinetoplastids require extensive U-insertion/deletion editing that progresses 3'-to-5' in small blocks, each directed by a guide RNA (gRNA), and exhibits substrate and developmental stage-specificity by unsolved mechanisms. Here, we address compositionally related factors, collectively known as the mitochondrial RNA-binding complex 1 (MRB1) or gRNA-binding complex (GRBC), that contain gRNA, have a dynamic protein composition, and transiently associate with several mitochondrial factors including RNA editing core complexes (RECC) and ribosomes. MRB1 controls editing by still unknown mechanisms. We performed the first next-generation sequencing study of native subcomplexes of MRB1, immunoselected via either RNA helicase 2 (REH2), that binds RNA and associates with unwinding activity, or MRB3010, that affects an early editing step. The particles contain either REH2 or MRB3010 but share the core GAP1 and other proteins detected by RNA photo-crosslinking. Analyses of the first editing blocks indicate an enrichment of several initiating gRNAs in the MRB3010-purified complex. Our data also indicate fast evolution of mRNA 3' ends and strain-specific alternative 3' editing within 3' UTR or C-terminal protein-coding sequence that could impact mitochondrial physiology. Moreover, we found robust specific copurification of edited and pre-edited mRNAs, suggesting that these particles may bind both mRNA and gRNA editing substrates. We propose that multiple subcomplexes of MRB1 with different RNA/protein composition serve as a scaffold for specific assembly of editing substrates and RECC, thereby forming the editing holoenzyme. The MRB3010-subcomplex may promote early editing through its preferential recruitment of initiating gRNAs.

  14. Identification and characterization of anion binding sites in RNA

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    Kieft, Jeffrey S.; Chase, Elaine; Costantino, David A.; Golden, Barbara L. (Purdue); (Colorado)

    2010-05-24

    Although RNA molecules are highly negatively charged, anions have been observed bound to RNA in crystal structures. It has been proposed that anion binding sites found within isolated RNAs represent regions of the molecule that could be involved in intermolecular interactions, indicating potential contact points for negatively charged amino acids from proteins or phosphate groups from an RNA. Several types of anion binding sites have been cataloged based on available structures. However, currently there is no method for unambiguously assigning anions to crystallographic electron density, and this has precluded more detailed analysis of RNA-anion interaction motifs and their significance. We therefore soaked selenate into two different types of RNA crystals and used the anomalous signal from these anions to identify binding sites in these RNA molecules unambiguously. Examination of these sites and comparison with other suspected anion binding sites reveals features of anion binding motifs, and shows that selenate may be a useful tool for studying RNA-anion interactions.

  15. Analysis of RNA binding by the dengue virus NS5 RNA capping enzyme.

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    Brittney R Henderson

    Full Text Available Flaviviruses are small, capped positive sense RNA viruses that replicate in the cytoplasm of infected cells. Dengue virus and other related flaviviruses have evolved RNA capping enzymes to form the viral RNA cap structure that protects the viral genome and directs efficient viral polyprotein translation. The N-terminal domain of NS5 possesses the methyltransferase and guanylyltransferase activities necessary for forming mature RNA cap structures. The mechanism for flavivirus guanylyltransferase activity is currently unknown, and how the capping enzyme binds its diphosphorylated RNA substrate is important for deciphering how the flavivirus guanylyltransferase functions. In this report we examine how flavivirus NS5 N-terminal capping enzymes bind to the 5' end of the viral RNA using a fluorescence polarization-based RNA binding assay. We observed that the K(D for RNA binding is approximately 200 nM Dengue, Yellow Fever, and West Nile virus capping enzymes. Removal of one or both of the 5' phosphates reduces binding affinity, indicating that the terminal phosphates contribute significantly to binding. RNA binding affinity is negatively affected by the presence of GTP or ATP and positively affected by S-adensyl methoninine (SAM. Structural superpositioning of the dengue virus capping enzyme with the Vaccinia virus VP39 protein bound to RNA suggests how the flavivirus capping enzyme may bind RNA, and mutagenesis analysis of residues in the putative RNA binding site demonstrate that several basic residues are critical for RNA binding. Several mutants show differential binding to 5' di-, mono-, and un-phosphorylated RNAs. The mode of RNA binding appears similar to that found with other methyltransferase enzymes, and a discussion of diphosphorylated RNA binding is presented.

  16. The RNA-binding landscapes of two SR proteins reveal unique functions and binding to diverse RNA classes

    OpenAIRE

    Neugebauer, Karla M.; Änkö, Minna-Liisa; Müller-McNicoll, Michaela; Brandl, Holger; Henry, Ian; Gorup, Črtomir; Curk, Tomaž; Ule, Jernej

    2015-01-01

    Background The SR proteins comprise a family of essential, structurally related RNA binding proteins. The complexity of their RNA targets and specificity of RNA recognition in vivo is not well understood. Here we use iCLIP to globally analyze and compare the RNA binding properties of two SR proteins, SRSF3 and SRSF4, in murine cells. Results SRSF3 and SRSF4 binding sites mapped to largely non-overlapping target genes, and in vivo consensus binding motifs were distinct. Interactions with intro...

  17. RNA recognition by the DNA end-binding Ku heterodimer.

    Science.gov (United States)

    Dalby, Andrew B; Goodrich, Karen J; Pfingsten, Jennifer S; Cech, Thomas R

    2013-06-01

    Most nucleic acid-binding proteins selectively bind either DNA or RNA, but not both nucleic acids. The Saccharomyces cerevisiae Ku heterodimer is unusual in that it has two very different biologically relevant binding modes: (1) Ku is a sequence-nonspecific double-stranded DNA end-binding protein with prominent roles in nonhomologous end-joining and telomeric capping, and (2) Ku associates with a specific stem-loop of TLC1, the RNA subunit of budding yeast telomerase, and is necessary for proper nuclear localization of this ribonucleoprotein enzyme. TLC1 RNA-binding and dsDNA-binding are mutually exclusive, so they may be mediated by the same site on Ku. Although dsDNA binding by Ku is well studied, much less is known about what features of an RNA hairpin enable specific recognition by Ku. To address this question, we localized the Ku-binding site of the TLC1 hairpin with single-nucleotide resolution using phosphorothioate footprinting, used chemical modification to identify an unpredicted motif within the hairpin secondary structure, and carried out mutagenesis of the stem-loop to ascertain the critical elements within the RNA that permit Ku binding. Finally, we provide evidence that the Ku-binding site is present in additional budding yeast telomerase RNAs and discuss the possibility that RNA binding is a conserved function of the Ku heterodimer.

  18. RNA-binding protein Dnd1 inhibits microRNA access to target mRNA

    DEFF Research Database (Denmark)

    Kedde, Martijn; Strasser, Markus J; Boldajipour, Bijan

    2007-01-01

    MicroRNAs (miRNAs) are inhibitors of gene expression capable of controlling processes in normal development and cancer. In mammals, miRNAs use a seed sequence of 6-8 nucleotides (nt) to associate with 3' untranslated regions (3'UTRs) of mRNAs and inhibit their expression. Intriguingly, occasionally...... not only the miRNA-targeting site but also sequences in its vicinity are highly conserved throughout evolution. We therefore hypothesized that conserved regions in mRNAs may serve as docking platforms for modulators of miRNA activity. Here we demonstrate that the expression of dead end 1 (Dnd1......), an evolutionary conserved RNA-binding protein (RBP), counteracts the function of several miRNAs in human cells and in primordial germ cells of zebrafish by binding mRNAs and prohibiting miRNAs from associating with their target sites. These effects of Dnd1 are mediated through uridine-rich regions present...

  19. A probabilistic approach to microRNA-target binding

    Energy Technology Data Exchange (ETDEWEB)

    Ogul, Hasan, E-mail: hogul@baskent.edu.tr [Department of Computer Engineering, Baskent University, Baglica TR-06810, Ankara (Turkey); Umu, Sinan U. [Department of Chemistry, Middle East Technical University, Cankaya TR-06800, Ankara (Turkey); Bioinformatics Program, Informatics Institute, Middle East Technical University, Cankaya TR-06800, Ankara (Turkey); Tuncel, Y. Yener [Bioinformatics Program, Informatics Institute, Middle East Technical University, Cankaya TR-06800, Ankara (Turkey); Akkaya, Mahinur S. [Department of Chemistry, Middle East Technical University, Cankaya TR-06800, Ankara (Turkey)

    2011-09-16

    Highlights: {yields} A new probabilistic model is introduced for microRNA-target binding. {yields} The new model significantly outperforms RNAHybrid and miRTif. {yields} The experiments can unveil the effects of the type and directions of distinct base pairings. -- Abstract: Elucidation of microRNA activity is a crucial step in understanding gene regulation. One key problem in this effort is how to model the pairwise interactions of microRNAs with their targets. As this interaction is strongly mediated by their sequences, it is desired to set-up a probabilistic model to explain the binding preferences between a microRNA sequence and the sequence of a putative target. To this end, we introduce a new model of microRNA-target binding, which transforms an aligned duplex to a new sequence and defines the likelihood of this sequence using a Variable Length Markov Chain. It offers a complementary representation of microRNA-mRNA pairs for microRNA target prediction tools or other probabilistic frameworks of integrative gene regulation analysis. The performance of present model is evaluated by its ability to predict microRNA-target mRNA interaction given a mature microRNA sequence and a putative mRNA binding site. In regard to classification accuracy, it outperforms two recent methods based on thermodynamic stability and sequence complementarity. The experiments can also unveil the effects of base pairing types and non-seed region in duplex formation.

  20. RNA-binding region of Macrobrachium rosenbergii nodavirus capsid protein.

    Science.gov (United States)

    Goh, Zee Hong; Mohd, Nur Azmina Syakirin; Tan, Soon Guan; Bhassu, Subha; Tan, Wen Siang

    2014-09-01

    White tail disease (WTD) kills prawn larvae and causes drastic losses to the freshwater prawn (Macrobrachium rosenbergii) industry. The main causative agent of WTD is Macrobrachium rosenbergii nodavirus (MrNV). The N-terminal end of the MrNV capsid protein is very rich in positively charged amino acids and is postulated to interact with RNA molecules. N-terminal and internal deletion mutagenesis revealed that the RNA-binding region is located at positions 20-29, where 80 % of amino acids are positively charged. Substitution of all these positively charged residues with alanine abolished the RNA binding. Mutants without the RNA-binding region still assembled into virus-like particles, suggesting that this region is not a part of the capsid assembly domain. This paper is, to the best of our knowledge, the first to report the specific RNA-binding region of MrNV capsid protein.

  1. Signatures of RNA binding proteins globally coupled to effective microRNA target sites

    DEFF Research Database (Denmark)

    Jacobsen, Anders; Wen, Jiayu; Marks, Debora S

    2010-01-01

    may be modulated by other mRNA sequence elements such as binding sites for the hundreds of RNA binding proteins (RNA-BPs) expressed in any cell, and this aspect has not been systematically explored. Across a panel of published experiments, we systematically investigated to what extent sequence motifs...... proteins. This is the first systematic investigation of 3' UTR motifs that globally couple to regulation by miRNAs and may potentially antagonize or cooperate with miRNA/siRNA regulation. Our results suggest that binding sites of miRNAs and RNA-BPs should be considered in combination when interpreting......MicroRNAs (miRNAs) and small interfering RNAs (siRNAs), bound to Argonaute proteins (RISC), destabilize mRNAs through base-pairing with the mRNA. However, the gene expression changes after perturbations of these small RNAs are only partially explained by predicted miRNA/siRNA targeting. Targeting...

  2. Probing binding hot spots at protein-RNA recognition sites.

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    Barik, Amita; Nithin, Chandran; Karampudi, Naga Bhushana Rao; Mukherjee, Sunandan; Bahadur, Ranjit Prasad

    2016-01-29

    We use evolutionary conservation derived from structure alignment of polypeptide sequences along with structural and physicochemical attributes of protein-RNA interfaces to probe the binding hot spots at protein-RNA recognition sites. We find that the degree of conservation varies across the RNA binding proteins; some evolve rapidly compared to others. Additionally, irrespective of the structural class of the complexes, residues at the RNA binding sites are evolutionary better conserved than those at the solvent exposed surfaces. For recognitions involving duplex RNA, residues interacting with the major groove are better conserved than those interacting with the minor groove. We identify multi-interface residues participating simultaneously in protein-protein and protein-RNA interfaces in complexes where more than one polypeptide is involved in RNA recognition, and show that they are better conserved compared to any other RNA binding residues. We find that the residues at water preservation site are better conserved than those at hydrated or at dehydrated sites. Finally, we develop a Random Forests model using structural and physicochemical attributes for predicting binding hot spots. The model accurately predicts 80% of the instances of experimental ΔΔG values in a particular class, and provides a stepping-stone towards the engineering of protein-RNA recognition sites with desired affinity.

  3. Probing binding hot spots at protein–RNA recognition sites

    Science.gov (United States)

    Barik, Amita; Nithin, Chandran; Karampudi, Naga Bhushana Rao; Mukherjee, Sunandan; Bahadur, Ranjit Prasad

    2016-01-01

    We use evolutionary conservation derived from structure alignment of polypeptide sequences along with structural and physicochemical attributes of protein–RNA interfaces to probe the binding hot spots at protein–RNA recognition sites. We find that the degree of conservation varies across the RNA binding proteins; some evolve rapidly compared to others. Additionally, irrespective of the structural class of the complexes, residues at the RNA binding sites are evolutionary better conserved than those at the solvent exposed surfaces. For recognitions involving duplex RNA, residues interacting with the major groove are better conserved than those interacting with the minor groove. We identify multi-interface residues participating simultaneously in protein–protein and protein–RNA interfaces in complexes where more than one polypeptide is involved in RNA recognition, and show that they are better conserved compared to any other RNA binding residues. We find that the residues at water preservation site are better conserved than those at hydrated or at dehydrated sites. Finally, we develop a Random Forests model using structural and physicochemical attributes for predicting binding hot spots. The model accurately predicts 80% of the instances of experimental ΔΔG values in a particular class, and provides a stepping-stone towards the engineering of protein–RNA recognition sites with desired affinity. PMID:26365245

  4. Unravelling Mg2+-RNA binding with atomistic molecular dynamics.

    Science.gov (United States)

    Cunha, Richard A; Bussi, Giovanni

    2017-02-01

    Interaction with divalent cations is of paramount importance for RNA structural stability and function. We here report a detailed molecular dynamics study of all the possible binding sites for Mg(2+) on a RNA duplex, including both direct (inner sphere) and indirect (outer sphere) binding. In order to tackle sampling issues, we develop a modified version of bias-exchange metadynamics which allows us to simultaneously compute affinities with previously unreported statistical accuracy. Results correctly reproduce trends observed in crystallographic databases. Based on this, we simulate a carefully chosen set of models that allows us to quantify the effects of competition with monovalent cations, RNA flexibility, and RNA hybridization. Our simulations reproduce the decrease and increase of Mg(2+) affinity due to ion competition and hybridization respectively, and predict that RNA flexibility has a site dependent effect. This suggests a non trivial interplay between RNA conformational entropy and divalent cation binding.

  5. Comparative analyses of the thermodynamic RNA binding signatures of different types of RNA recognition motifs

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    Cléry, Antoine; Allain, Frédéric H-T

    2017-01-01

    Abstract RNA recognition motifs (RRMs) are structurally versatile domains important in regulation of alternative splicing. Structural mechanisms of sequence-specific recognition of single-stranded RNAs (ssRNAs) by RRMs are well understood. The thermodynamic strategies are however unclear. Therefore, we utilized microcalorimetry and semi-empirical analyses to comparatively analyze the cognate ssRNA binding thermodynamics of four different RRM domains, each with a different RNA binding mode. The different binding modes are: canonical binding to the β-sheet surface; canonical binding with involvement of N- and C-termini; binding to conserved loops; and binding to an α-helix. Our results identify enthalpy as the sole and general force driving association at physiological temperatures. Also, networks of weak interactions are a general feature regulating stability of the different RRM–ssRNA complexes. In agreement, non-polyelectrolyte effects contributed between ∼75 and 90% of the overall free energy of binding in the considered complexes. The various RNA binding modes also displayed enormous heat capacity differences, that upon dissection revealed large differential changes in hydration, conformations and dynamics upon binding RNA. Altogether, different modes employed by RRMs to bind cognate ssRNAs utilize various thermodynamics strategies during the association process. PMID:28334819

  6. Modulation of RNase E activity by alternative RNA binding sites.

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    Daeyoung Kim

    Full Text Available Endoribonuclease E (RNase E affects the composition and balance of the RNA population in Escherichia coli via degradation and processing of RNAs. In this study, we investigated the regulatory effects of an RNA binding site between amino acid residues 25 and 36 (24LYDLDIESPGHEQK37 of RNase E. Tandem mass spectrometry analysis of the N-terminal catalytic domain of RNase E (N-Rne that was UV crosslinked with a 5'-32P-end-labeled, 13-nt oligoribonucleotide (p-BR13 containing the RNase E cleavage site of RNA I revealed that two amino acid residues, Y25 and Q36, were bound to the cytosine and adenine of BR13, respectively. Based on these results, the Y25A N-Rne mutant was constructed, and was found to be hypoactive in comparison to wild-type and hyperactive Q36R mutant proteins. Mass spectrometry analysis showed that Y25A and Q36R mutations abolished the RNA binding to the uncompetitive inhibition site of RNase E. The Y25A mutation increased the RNA binding to the multimer formation interface between amino acid residues 427 and 433 (427LIEEEALK433, whereas the Q36R mutation enhanced the RNA binding to the catalytic site of the enzyme (65HGFLPL*K71. Electrophoretic mobility shift assays showed that the stable RNA-protein complex formation was positively correlated with the extent of RNA binding to the catalytic site and ribonucleolytic activity of the N-Rne proteins. These mutations exerted similar effects on the ribonucleolytic activity of the full-length RNase E in vivo. Our findings indicate that RNase E has two alternative RNA binding sites for modulating RNA binding to the catalytic site and the formation of a functional catalytic unit.

  7. RNA binding efficacy of theophylline, theobromine and caffeine.

    Science.gov (United States)

    Johnson, I Maria; Kumar, S G Bhuvan; Malathi, R

    2003-04-01

    The binding of naturally occurring methylxanthines such as theophylline, theobromine and caffeine to nucleic acids are reckoned to be pivotal as they are able to modulate the cellular activities. We explore the interaction of yeast RNA binding efficacy of the above xanthine derivatives by using UV absorption differential spectroscopy and Fourier Transform Infrared (FTIR) spectroscopy. Both the analyses show discrimination in their binding affinity to RNA. The differential UV-spectrum at P/D 3.3 reveals the greater RNA binding activity for theophylline (85 +/- 5%), whereas moderate and comparatively less binding activity for theobromine (45 +/- 5%) and caffeine (30 +/- 5%) and the binding activity was found to depend on concentration of the drugs. In FTIR analysis we observed changes in the amino group (NH) of RNA complexed by drugs, where the NH band is found to become very broad, indicating hydrogen bonding (H-bonding) with theophylline (3343.4 cm(-1)), theobromine (3379.8 cm(-1)) and caffeine (3343 cm(-1)) as compared to the free RNA (3341.6 cm(-1)). Furthermore in RNA-theophylline complex, it is observed that the carbonyl (C=O) vibration frequency (nu(C=O)) of both drug (nu(C=O)=1718, 1666 cm(-1)) as well as RNA (nu(C=O)=1699, 1658 cm(-1)) disappeared and a new vibration band appeared around 1703 cm(-1), indicating that the C=O and NH groups of drug and RNA are effectively involved in H-bonding. Whereas in RNA-theobromine and RNA-caffeine complexes, we found very little changes in C=O frequency and only broadening of the NH band of RNA due to complexation is observed in these groups. The changes in the vibrations of G-C/A-U bands and other bending frequencies are discussed. Thus the discrimination in the binding affinity of methylxanthines with RNA molecule shows that strong RNA binding drugs like theophylline can selectively be delivered to RNA targets of microbial pathogens having the mechanism of RNA catalysis.

  8. The RNA-binding landscapes of two SR proteins reveal unique functions and binding to diverse RNA classes.

    Science.gov (United States)

    Änkö, Minna-Liisa; Müller-McNicoll, Michaela; Brandl, Holger; Curk, Tomaz; Gorup, Crtomir; Henry, Ian; Ule, Jernej; Neugebauer, Karla M

    2012-01-01

    The SR proteins comprise a family of essential, structurally related RNA binding proteins. The complexity of their RNA targets and specificity of RNA recognition in vivo is not well understood. Here we use iCLIP to globally analyze and compare the RNA binding properties of two SR proteins, SRSF3 and SRSF4, in murine cells. SRSF3 and SRSF4 binding sites mapped to largely non-overlapping target genes, and in vivo consensus binding motifs were distinct. Interactions with intronless and intron-containing mRNAs as well as non-coding RNAs were detected. Surprisingly, both SR proteins bound to the 3' ends of the majority of intronless histone transcripts, implicating SRSF3 and SRSF4 in histone mRNA metabolism. In contrast, SRSF3 but not SRSF4 specifically bound transcripts encoding numerous RNA binding proteins. Remarkably, SRSF3 was shown to modulate alternative splicing of its own as well as three other transcripts encoding SR proteins. These SRSF3-mediated splicing events led to downregulation of heterologous SR proteins via nonsense-mediated decay. SRSF3 and SRSF4 display unique RNA binding properties underlying diverse cellular regulatory mechanisms, with shared as well as unique coding and non-coding targets. Importantly, CLIP analysis led to the discovery that SRSF3 cross-regulates the expression of other SR protein family members.

  9. RNA-Binding Proteins Revisited – The Emerging Arabidopsis mRNA Interactome

    KAUST Repository

    Köster, Tino

    2017-04-13

    RNA–protein interaction is an important checkpoint to tune gene expression at the RNA level. Global identification of proteins binding in vivo to mRNA has been possible through interactome capture – where proteins are fixed to target RNAs by UV crosslinking and purified through affinity capture of polyadenylated RNA. In Arabidopsis over 500 RNA-binding proteins (RBPs) enriched in UV-crosslinked samples have been identified. As in mammals and yeast, the mRNA interactomes came with a few surprises. For example, a plethora of the proteins caught on RNA had not previously been linked to RNA-mediated processes, for example proteins of intermediary metabolism. Thus, the studies provide unprecedented insights into the composition of the mRNA interactome, highlighting the complexity of RNA-mediated processes.

  10. A Nucleolar PUF RNA-binding Protein with Specificity for a Unique RNA Sequence.

    Science.gov (United States)

    Zhang, Chi; Muench, Douglas G

    2015-12-11

    PUF proteins are a conserved group of sequence specific RNA-binding proteins that bind to RNA in a modular fashion. The RNA-binding domain of PUF proteins typically consists of eight clustered Puf repeats. Plant genomes code for large families of PUF proteins that show significant variability in their predicted Puf repeat number, organization, and amino acid sequence. Here we sought to determine whether the observed variability in the RNA-binding domains of four plant PUFs results in a preference for nonclassical PUF RNA target sequences. We report the identification of a novel RNA binding sequence for a nucleolar Arabidopsis PUF protein that contains an atypical RNA-binding domain. The Arabidopsis PUM23 (APUM23) binding sequence was 10 nucleotides in length, contained a centrally located UUGA core element, and had a preferred cytosine at nucleotide position 8. These RNA sequence characteristics differ from those of other PUF proteins, because all natural PUFs studied to date bind to RNAs that contain a conserved UGU sequence at their 5' end and lack specificity for cytosine. Gel mobility shift assays validated the identity of the APUM23 binding sequence and supported the location of 3 of the 10 predicted Puf repeats in APUM23, including the cytosine-binding repeat. The preferred 10-nucleotide sequence bound by APUM23 is present within the 18S rRNA sequence, supporting the known role of APUM23 in 18S rRNA maturation. This work also reveals that APUM23, an ortholog of yeast Nop9, could provide an advanced structural backbone for Puf repeat engineering and target-specific regulation of cellular RNAs.

  11. RNA-binding protein RBM20 represses splicing to orchestrate cardiac pre-mRNA processing.

    NARCIS (Netherlands)

    Maatz, H.; Jens, M.; Liss, M.; Schafer, S.; Heinig, M.; Kirchner, M.; Adami, E.; Rintisch, C.; Dauksaite, V.; Radke, M.H.; Selbach, M.; Barton, P.J.; Cook, S.A.; Rajewsky, N.; Gotthardt, M.; Landthaler, M.; Hubner, N.

    2014-01-01

    Mutations in the gene encoding the RNA-binding protein RBM20 have been implicated in dilated cardiomyopathy (DCM), a major cause of chronic heart failure, presumably through altering cardiac RNA splicing. Here, we combined transcriptome-wide crosslinking immunoprecipitation (CLIP-seq), RNA-seq, and

  12. RNA-binding protein RBM20 represses splicing to orchestrate cardiac pre-mRNA processing.

    NARCIS (Netherlands)

    Maatz, H.; Jens, M.; Liss, M.; Schafer, S.; Heinig, M.; Kirchner, M.; Adami, E.; Rintisch, C.; Dauksaite, V.; Radke, M.H.; Selbach, M.; Barton, P.J.; Cook, S.A.; Rajewsky, N.; Gotthardt, M.; Landthaler, M.; Hubner, N.

    2014-01-01

    Mutations in the gene encoding the RNA-binding protein RBM20 have been implicated in dilated cardiomyopathy (DCM), a major cause of chronic heart failure, presumably through altering cardiac RNA splicing. Here, we combined transcriptome-wide crosslinking immunoprecipitation (CLIP-seq), RNA-seq, and

  13. The RNA-binding protein repertoire of Arabidopsis thaliana

    KAUST Repository

    Marondedze, Claudius

    2016-07-11

    RNA-binding proteins (RBPs) have essential roles in determining the fate of RNA from synthesis to decay and have been studied on a protein-by-protein basis, or computationally based on a number of well-characterised RNA-binding domains. Recently, high-throughput methods enabled the capture of mammalian RNA-binding proteomes. To gain insight into the role of Arabidopsis thaliana RBPs at the systems level, we have employed interactome capture techniques using cells from different ecotypes grown in cultures and leaves. In vivo UV-crosslinking of RNA to RBPs, oligo(dT) capture and mass spectrometry yielded 1,145 different proteins including 550 RBPs that either belong to the functional category ‘RNA-binding’, have known RNA-binding domains or have orthologs identified in mammals, C. elegans, or S. cerevisiae in addition to 595 novel candidate RBPs. We noted specific subsets of RBPs in cultured cells and leaves and a comparison of Arabidopsis, mammalian, C. elegans, and S. cerevisiae RBPs reveals a common set of proteins with a role in intermediate metabolism, as well as distinct differences suggesting that RBPs are also species and tissue specific. This study provides a foundation for studies that will advance our understanding of the biological significance of RBPs in plant developmental and stimulus specific responses.

  14. Pumilio Puf domain RNA-binding proteins in Arabidopsis.

    Science.gov (United States)

    Abbasi, Nazia; Park, Youn-Il; Choi, Sang-Bong

    2011-03-01

    Pumilio proteins are a class of RNA-binding proteins harboring Puf domains (or PUM-HD; Pumilio-Homology Domain), named after the founding members, Pumilio (from Drosophila melanogaster) and FBF (Fem-3 mRNA-Binding Factor from Caenorhabditis elegans). The domains contain multiple tandem repeats each of which recognizes one RNA base and is comprised of 35-39 amino acids. Puf domain proteins have been reported in organisms ranging from single-celled yeast to higher multicellular eukaryotes, such as humans and plants. In yeast and animals, they are involved in a variety of posttranscriptional RNA metabolism including RNA decay, RNA transport, rRNA processing and translational repression. However, their roles in plants are largely unknown. Recently, we have characterized the first member of the Puf family of RNA-binding proteins, APUM23, in Arabidopsis. Here, we discuss and summarize the diverse roles and targets of Puf proteins previously reported in other organisms and then highlight the potential regulatory roles of Puf proteins in Arabidopsis, using our recent study as an example.

  15. How do ADARs bind RNA? New protein-RNA structures illuminate substrate recognition by the RNA editing ADARs.

    Science.gov (United States)

    Thomas, Justin M; Beal, Peter A

    2017-02-20

    Deamination of adenosine in RNA to form inosine has wide ranging consequences on RNA function including amino acid substitution to give proteins not encoded in the genome. What determines which adenosines in an mRNA are subject to this modification reaction? The answer lies in an understanding of the mechanism and substrate recognition properties of adenosine deaminases that act on RNA (ADARs). Our recent publication of X-ray crystal structures of the human ADAR2 deaminase domain bound to RNA editing substrates shed considerable light on how the catalytic domains of these enzymes bind RNA and promote adenosine deamination. Here we review in detail the deaminase domain-RNA contact surfaces and present models of how full length ADARs, bearing double stranded RNA-binding domains (dsRBDs) and deaminase domains, could process naturally occurring substrate RNAs.

  16. Phloem RNA-binding proteins as potential components of the long-distance RNA transport system.

    Directory of Open Access Journals (Sweden)

    VICENTE ePALLAS

    2013-05-01

    Full Text Available RNA-binding proteins (RBPs govern a myriad of different essential processes in eukaryotic cells. Recent evidence reveals that apart from playing critical roles in RNA metabolism and RNA transport, RBPs perform a key function in plant adaption to various environmental conditions. Long distance RNA transport occurs in land plants through the phloem, a conducting tissue that integrates the wide range of signalling pathways required to regulate plant development and response to stress processes. The macromolecules in the phloem pathway vary greatly and include defence proteins, transcription factors, chaperones acting in long distance trafficking, and RNAs (mRNAs, siRNAs and miRNAs. How these RNA molecules translocate through the phloem is not well understood, but recent evidence indicates the presence of translocatable RNA-binding proteins in the phloem, which act as potential components of long distance RNA transport system. This review updates our knowledge on the characteristics and functions of RBPs present in the phloem.

  17. Importance of diffuse metal ion binding to RNA.

    Science.gov (United States)

    Tan, Zhi-Jie; Chen, Shi-Jie

    2011-01-01

    RNAs are highly charged polyanionic molecules. RNA structure and function are strongly correlated with the ionic condition of the solution. The primary focus of this article is on the role of diffusive ions in RNA folding. Due to the long-range nature of electrostatic interactions, the diffuse ions can contribute significantly to RNA structural stability and folding kinetics. We present an overview of the experimental findings as well as the theoretical developments on the diffuse ion effects in RNA folding. This review places heavy emphasis on the effect of magnesium ions. Magnesium ions play a highly efficient role in stabilizing RNA tertiary structures and promoting tertiary structural folding. The highly efficient role goes beyond the mean-field effect such as the ionic strength. In addition to the effects of specific ion binding and ion dehydration, ion-ion correlation for the diffuse ions can contribute to the efficient role of the multivalent ions such as the magnesium ions in RNA folding.

  18. RNA-binding IMPs promote cell adhesion and invadopodia formation

    DEFF Research Database (Denmark)

    Vikesaa, Jonas; Hansen, Thomas V O; Jønson, Lars

    2006-01-01

    Oncofetal RNA-binding IMPs have been implicated in mRNA localization, nuclear export, turnover and translational control. To depict the cellular actions of IMPs, we performed a loss-of-function analysis, which showed that IMPs are necessary for proper cell adhesion, cytoplasmic spreading...... and invadopodia formation. Loss of IMPs was associated with a coordinate downregulation of mRNAs encoding extracellular matrix and adhesion proteins. The transcripts were present in IMP RNP granules, implying that IMPs were directly involved in the post-transcriptional control of the transcripts. In particular......, we show that a 5.0 kb CD44 mRNA contained multiple IMP-binding sites in its 3'UTR, and following IMP depletion this species became unstable. Direct knockdown of the CD44 transcript mimicked the effect of IMPs on invadopodia, and we infer that CD44 mRNA stabilization may be involved in IMP...

  19. Trans-Regulation of RNA-Binding Protein Motifs by MicroRNA

    Directory of Open Access Journals (Sweden)

    Scott eTenenbaum

    2014-04-01

    Full Text Available The wide array of vital functions that RNA performs is dependent on its ability to dynamically fold into different structures in response to intracellular and extracellular changes. RNA-binding proteins regulate much of this activity by targeting specific RNA structures or motifs. One of these structures, the 3-way RNA junction, is characteristically found in ribosomal RNA and results from the RNA folding in cis, to produce three separate helices that meet around a central unpaired region. Here we demonstrate that 3-way junctions can also form in trans as a result of the binding of microRNAs in an unconventional manner with mRNA by splinting two non-contiguous regions together. This may be used to reinforce the base of a stem-loop motif being targeted by an RNA-binding protein. Trans interactions between non-coding RNA and mRNA may be used to control the post-transcriptional regulatory code and suggests a possible role for some of the recently described transcripts of unknown function expressed from the human genome.

  20. A human microRNA precursor binding to folic acid discovered by small RNA transcriptomic SELEX.

    Science.gov (United States)

    Terasaka, Naohiro; Futai, Kazuki; Katoh, Takayuki; Suga, Hiroaki

    2016-12-01

    RNA aptamers are structured motifs that bind to specific molecules. A growing number of RNAs bearing aptamer elements, whose functions are modulated by direct binding of metabolites, have been found in living cells. Recent studies have suggested that more small RNAs binding to metabolites likely exist and may be involved in diverse cellular processes. However, conventional methods are not necessarily suitable for the discovery of such RNA aptamer elements in small RNAs with lengths ranging from 50 to 200 nucleotides, due to the far more abundant tRNAs in this size range. Here, we describe a new in vitro selection method to uncover naturally occurring small RNAs capable of binding to a ligand of interest, referred to as small RNA transcriptomic SELEX (smaRt-SELEX). By means of this method, we identified a motif in human precursor microRNA 125a (hsa-pre-miR-125a) that interacts with folic acid. Mutation studies revealed that the terminal loop region of hsa-pre-miR-125a is important for this binding interaction. This method has potential for the discovery of new RNA aptamer elements or catalytic motifs in biological small RNA fractions. © 2016 Terasaka et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  1. Cooperativity in RNA-Protein Interactions: Global Analysis of RNA Binding Specificity

    Directory of Open Access Journals (Sweden)

    Zachary T. Campbell

    2012-05-01

    Full Text Available The control and function of RNA are governed by the specificity of RNA binding proteins. Here, we describe a method for global unbiased analysis of RNA-protein interactions that uses in vitro selection, high-throughput sequencing, and sequence-specificity landscapes. The method yields affinities for a vast array of RNAs in a single experiment, including both low- and high-affinity sites. It is reproducible and accurate. Using this approach, we analyzed members of the PUF (Pumilio and FBF family of eukaryotic mRNA regulators. Our data identify effects of a specific protein partner on PUF-RNA interactions, reveal subsets of target sites not previously detected, and demonstrate that designer PUF proteins can precisely alter specificity. The approach described here is, in principle, broadly applicable for analysis of any molecule that binds RNA, including proteins, nucleic acids, and small molecules.

  2. Hacking RNA: Hakai promotes tumorigenesis by switching on the RNA-binding function of PSF

    Science.gov (United States)

    Figueroa, Angélica; Fujita, Yasuyuki; Gorospe, Myriam

    2009-01-01

    Hakai, an E3 ubiquitin ligase for the E-cadherin complex, plays a crucial role in lowering cell-cell contacts in epithelial cells, a hallmark feature of tumor progression. Recently, Hakai was also found to interact with PSF (PTB-associated splicing factor). While PSF can function as a DNA-binding protein with a tumor suppressive function, its association with Hakai promotes PSF’s RNA-binding ability and post-transcriptional influence on target mRNAs. Hakai overexpression enhanced the binding of PSF to mRNAs encoding cancer-related proteins, while knockdown of Hakai reduced the RNA-binding ability of PSF. Furthermore, the knockdown of PSF suppressed Hakai-induced cell proliferation. Thus, Hakai can affect the oncogenic phenotype both by altering E-cadherin-based intercellular adhesions and by increasing PSF’s ability to bind RNAs that promote cancer-related gene expression. PMID:19855157

  3. Hacking RNA: Hakai promotes tumorigenesis by enhancing the RNA-binding function of PSF.

    Science.gov (United States)

    Figueroa, Angélica; Fujita, Yasuyuki; Gorospe, Myriam

    2009-11-15

    Hakai, an E3 ubiquitin ligase for the E-cadherin complex, plays a crucial role in lowering cell-cell contacts in epithelial cells, a hallmark feature of tumor progression. Recently, Hakai was also found to interact with PSF (PTB-associated splicing factor). While PSF can function as a DNA-binding protein with a tumor suppressive function, its association with Hakai promotes PSF's RNA-binding ability and post-transcriptional influence on target mRNAs. Hakai overexpression enhanced the binding of PSF to mRNAs encoding cancer-related proteins, while knockdown of Hakai reduced the RNA-binding ability of PSF. Furthermore, the knockdown of PSF suppressed Hakai-induced cell proliferation. Thus, Hakai can affect the oncogenic phenotype both by altering E-cadherin-based intercellular adhesions and by increasing PSF's ability to bind RNAs that promote cancer-related gene expression.

  4. RNA-protein binding kinetics in an automated microfluidic reactor.

    Science.gov (United States)

    Ridgeway, William K; Seitaridou, Effrosyni; Phillips, Rob; Williamson, James R

    2009-11-01

    Microfluidic chips can automate biochemical assays on the nanoliter scale, which is of considerable utility for RNA-protein binding reactions that would otherwise require large quantities of proteins. Unfortunately, complex reactions involving multiple reactants cannot be prepared in current microfluidic mixer designs, nor is investigation of long-time scale reactions possible. Here, a microfluidic 'Riboreactor' has been designed and constructed to facilitate the study of kinetics of RNA-protein complex formation over long time scales. With computer automation, the reactor can prepare binding reactions from any combination of eight reagents, and is optimized to monitor long reaction times. By integrating a two-photon microscope into the microfluidic platform, 5-nl reactions can be observed for longer than 1000 s with single-molecule sensitivity and negligible photobleaching. Using the Riboreactor, RNA-protein binding reactions with a fragment of the bacterial 30S ribosome were prepared in a fully automated fashion and binding rates were consistent with rates obtained from conventional assays. The microfluidic chip successfully combines automation, low sample consumption, ultra-sensitive fluorescence detection and a high degree of reproducibility. The chip should be able to probe complex reaction networks describing the assembly of large multicomponent RNPs such as the ribosome.

  5. RNA targeting by small molecules: Binding of protoberberine, benzophenanthridine and aristolochia alkaloids to various RNA structures

    Indian Academy of Sciences (India)

    Gopinatha Suresh Kumar

    2012-07-01

    Studies on RNA targeting by small molecules to specifically control certain cellular functions is an area of remarkable current interest. For this purpose, a basic understanding of the molecular aspects of the interaction of small molecules with various RNA structures is essential. Alkaloids are a group of natural products with potential therapeutic utility, and very recently, their interaction with many RNA structures have been reported. Especially noteworthy are the protoberberines and aristolochia alkaloids distributed widely in many botanical families. Many of the alkaloids of these group exhibit excellent binding affinity to many RNA structures that may be exploited to develop RNA targeted therapeutics. This review attempts to present the current status on the understanding of the interaction of these alkaloids with various RNA structures, mainly highlighting the biophysical aspects.

  6. Poly(A) RNA-binding proteins and polyadenosine RNA: new members and novel functions.

    Science.gov (United States)

    Wigington, Callie P; Williams, Kathryn R; Meers, Michael P; Bassell, Gary J; Corbett, Anita H

    2014-01-01

    Poly(A) RNA-binding proteins (Pabs) bind with high affinity and specificity to polyadenosine RNA. Textbook models show a nuclear Pab, PABPN1, and a cytoplasmic Pab, PABPC, where the nuclear PABPN1 modulates poly(A) tail length and the cytoplasmic PABPC stabilizes poly(A) RNA in the cytoplasm and also enhances translation. While these conventional roles are critically important, the Pab family has expanded recently both in number and in function. A number of novel roles have emerged for both PAPBPN1 and PABPC that contribute to the fine-tuning of gene expression. Furthermore, as the characterization of the nucleic acid binding properties of RNA-binding proteins advances, additional proteins that show high affinity and specificity for polyadenosine RNA are being discovered. With this expansion of the Pab family comes a concomitant increase in the potential for Pabs to modulate gene expression. Further complication comes from an expansion of the potential binding sites for Pab proteins as revealed by an analysis of templated polyadenosine stretches present within the transcriptome. Thus, Pabs could influence mRNA fate and function not only by binding to the nontemplated poly(A) tail but also to internal stretches of adenosine. Understanding the diverse functions of Pab proteins is not only critical to understand how gene expression is regulated but also to understand the molecular basis for tissue-specific diseases that occur when Pab proteins are altered. Here we describe both conventional and recently emerged functions for PABPN1 and PABPC and then introduce and discuss three new Pab family members, ZC3H14, hnRNP-Q1, and LARP4.

  7. RNA-binding proteins in microsatellite expansion disorders: mediators of RNA toxicity.

    Science.gov (United States)

    Echeverria, Gloria V; Cooper, Thomas A

    2012-06-26

    Although protein-mediated toxicity in neurological disease has been extensively characterized, RNA-mediated toxicity is an emerging mechanism of pathogenesis. In microsatellite expansion disorders, expansion of repeated sequences in noncoding regions gives rise to RNA that produces a toxic gain of function, while expansions in coding regions can disrupt protein function as well as produce toxic RNA. The toxic RNA typically aggregates into nuclear foci and contributes to disease pathogenesis. In many cases, toxicity of the RNA is caused by the disrupted functions of RNA-binding proteins. We will discuss evidence for RNA-mediated toxicity in microsatellite expansion disorders. Different microsatellite expansion disorders are linked with alterations in the same as well as disease-specific RNA-binding proteins. Recent studies have shown that microsatellite expansions can encode multiple repeat-containing toxic RNAs through bidirectional transcription and protein species through repeat-associated non-ATG translation. We will discuss approaches that have characterized the toxic contributions of these various factors.

  8. Evolutionary Conservation and Diversification of Puf RNA Binding Proteins and Their mRNA Targets.

    Science.gov (United States)

    Hogan, Gregory J; Brown, Patrick O; Herschlag, Daniel

    2015-01-01

    Reprogramming of a gene's expression pattern by acquisition and loss of sequences recognized by specific regulatory RNA binding proteins may be a major mechanism in the evolution of biological regulatory programs. We identified that RNA targets of Puf3 orthologs have been conserved over 100-500 million years of evolution in five eukaryotic lineages. Focusing on Puf proteins and their targets across 80 fungi, we constructed a parsimonious model for their evolutionary history. This model entails extensive and coordinated changes in the Puf targets as well as changes in the number of Puf genes and alterations of RNA binding specificity including that: 1) Binding of Puf3 to more than 200 RNAs whose protein products are predominantly involved in the production and organization of mitochondrial complexes predates the origin of budding yeasts and filamentous fungi and was maintained for 500 million years, throughout the evolution of budding yeast. 2) In filamentous fungi, remarkably, more than 150 of the ancestral Puf3 targets were gained by Puf4, with one lineage maintaining both Puf3 and Puf4 as regulators and a sister lineage losing Puf3 as a regulator of these RNAs. The decrease in gene expression of these mRNAs upon deletion of Puf4 in filamentous fungi (N. crassa) in contrast to the increase upon Puf3 deletion in budding yeast (S. cerevisiae) suggests that the output of the RNA regulatory network is different with Puf4 in filamentous fungi than with Puf3 in budding yeast. 3) The coregulated Puf4 target set in filamentous fungi expanded to include mitochondrial genes involved in the tricarboxylic acid (TCA) cycle and other nuclear-encoded RNAs with mitochondrial function not bound by Puf3 in budding yeast, observations that provide additional evidence for substantial rewiring of post-transcriptional regulation. 4) Puf3 also expanded and diversified its targets in filamentous fungi, gaining interactions with the mRNAs encoding the mitochondrial electron transport

  9. Evolutionary Conservation and Diversification of Puf RNA Binding Proteins and Their mRNA Targets.

    Directory of Open Access Journals (Sweden)

    Gregory J Hogan

    Full Text Available Reprogramming of a gene's expression pattern by acquisition and loss of sequences recognized by specific regulatory RNA binding proteins may be a major mechanism in the evolution of biological regulatory programs. We identified that RNA targets of Puf3 orthologs have been conserved over 100-500 million years of evolution in five eukaryotic lineages. Focusing on Puf proteins and their targets across 80 fungi, we constructed a parsimonious model for their evolutionary history. This model entails extensive and coordinated changes in the Puf targets as well as changes in the number of Puf genes and alterations of RNA binding specificity including that: 1 Binding of Puf3 to more than 200 RNAs whose protein products are predominantly involved in the production and organization of mitochondrial complexes predates the origin of budding yeasts and filamentous fungi and was maintained for 500 million years, throughout the evolution of budding yeast. 2 In filamentous fungi, remarkably, more than 150 of the ancestral Puf3 targets were gained by Puf4, with one lineage maintaining both Puf3 and Puf4 as regulators and a sister lineage losing Puf3 as a regulator of these RNAs. The decrease in gene expression of these mRNAs upon deletion of Puf4 in filamentous fungi (N. crassa in contrast to the increase upon Puf3 deletion in budding yeast (S. cerevisiae suggests that the output of the RNA regulatory network is different with Puf4 in filamentous fungi than with Puf3 in budding yeast. 3 The coregulated Puf4 target set in filamentous fungi expanded to include mitochondrial genes involved in the tricarboxylic acid (TCA cycle and other nuclear-encoded RNAs with mitochondrial function not bound by Puf3 in budding yeast, observations that provide additional evidence for substantial rewiring of post-transcriptional regulation. 4 Puf3 also expanded and diversified its targets in filamentous fungi, gaining interactions with the mRNAs encoding the mitochondrial electron

  10. RNA Detection in Live Bacterial Cells Using Fluorescent Protein Complementation Triggered by Interaction of Two RNA Aptamers with Two RNA-Binding Peptides

    Directory of Open Access Journals (Sweden)

    Charles R. Cantor

    2011-03-01

    Full Text Available Many genetic and infectious diseases can be targeted at the RNA level as RNA is more accessible than DNA. We seek to develop new approaches for detection and tracking RNA in live cells, which is necessary for RNA-based diagnostics and therapy. We recently described a method for RNA visualization in live bacterial cells based on fluorescent protein complementation [1-3]. The RNA is tagged with an RNA aptamer that binds an RNA-binding protein with high affinity. This RNA-binding protein is expressed as two split fragments fused to the fragments of a split fluorescent protein. In the presence of RNA the fragments of the RNA-binding protein bind the aptamer and bring together the fragments of the fluorescent protein, which results in its re-assembly and fluorescence development [1-3]. Here we describe a new version of the RNA labeling method where fluorescent protein complementation is triggered by paired interactions of two different closely-positioned RNA aptamers with two different RNA-binding viral peptides. The new method, which has been developed in bacteria as a model system, uses a smaller ribonucleoprotein complementation complex, as compared with the method using split RNA-binding protein, and it can potentially be applied to a broad variety of RNA targets in both prokaryotic and eukaryotic cells. We also describe experiments exploring background fluorescence in these RNA detection systems and conditions that improve the signal-to-background ratio.

  11. RNA-binding protein Lin28 in cancer and immunity.

    Science.gov (United States)

    Jiang, Shuai; Baltimore, David

    2016-05-28

    The highly conserved RNA-binding protein, Lin28, is involved in many biological processes, including development, reprogramming, pluripotency, and metabolism. Importantly, Lin28 functions as an oncogene, promoting tumor progression and metastasis in various human cancers. Lin28 can regulate gene expression either by directly binding to mRNAs or by blocking microRNA biogenesis, and the underlying mechanisms include Let-7-dependent and Let-7-independent modes of action. Recent evidence shows that Lin28 also plays a fundamental role in immunity. The roles of Lin28 in disease are complex and require characterization of its physiological functions in cancer and immunological contexts. Here we review emerging information on the role of Lin28 in cancer and immunity and the molecular mechanisms it uses. We discuss our present knowledge of the system and highlight remaining mysteries related to the functions of this small RNA-binding protein. This knowledge may lead to Lin28 becoming a diagnostic marker for cancer or immune-related diseases and a possible therapeutic target.

  12. Metabolic Enzymes Enjoying New Partnerships as RNA-Binding Proteins.

    Science.gov (United States)

    Castello, Alfredo; Hentze, Matthias W; Preiss, Thomas

    2015-12-01

    In the past century, few areas of biology advanced as much as our understanding of the pathways of intermediary metabolism. Initially considered unimportant in terms of gene regulation, crucial cellular fate changes, cell differentiation, or malignant transformation are now known to involve 'metabolic remodeling' with profound changes in the expression of many metabolic enzyme genes. This review focuses on the recent identification of RNA-binding activity of numerous metabolic enzymes. We discuss possible roles of this unexpected second activity in feedback gene regulation ('moonlighting') and/or in the control of enzymatic function. We also consider how metabolism-driven post-translational modifications could regulate enzyme-RNA interactions. Thus, RNA emerges as a new partner of metabolic enzymes with far-reaching possible consequences to be unraveled in the future.

  13. Plant coilin: structural characteristics and RNA-binding properties.

    Directory of Open Access Journals (Sweden)

    Valentine Makarov

    Full Text Available Cajal bodies (CBs are dynamic subnuclear compartments involved in the biogenesis of ribonucleoproteins. Coilin is a major structural scaffolding protein necessary for CB formation, composition and activity. The predicted secondary structure of Arabidopsis thaliana coilin (Atcoilin suggests that the protein is composed of three main domains. Analysis of the physical properties of deletion mutants indicates that Atcoilin might consist of an N-terminal globular domain, a central highly disordered domain and a C-terminal domain containing a presumable Tudor-like structure adjacent to a disordered C terminus. Despite the low homology in amino acid sequences, a similar type of domain organization is likely shared by human and animal coilin proteins and coilin-like proteins of various plant species. Atcoilin is able to bind RNA effectively and in a non-specific manner. This activity is provided by three RNA-binding sites: two sets of basic amino acids in the N-terminal domain and one set in the central domain. Interaction with RNA induces the multimerization of the Atcoilin molecule, a consequence of the structural alterations in the N-terminal domain. The interaction with RNA and subsequent multimerization may facilitate coilin's function as a scaffolding protein. A model of the N-terminal domain is also proposed.

  14. In vitro RNA-binding assay for studying trans-factors for RNA editing in chloroplasts.

    Science.gov (United States)

    Shikanai, Toshiharu; Okuda, Kenji

    2011-01-01

    In plant organelles, specific C residues are modified to U by RNA editing. Short RNA sequences surrounding the target site (i.e., cis-elements) are recognized by trans-factors, which were recently shown to be pentatricopeptide repeat (PPR) proteins. PPR proteins consist of tandem arrays of a highly degenerate unit of 35 (pentatrico) amino acids, and PPR motifs are believed to recognize specific RNA sequences. In Arabidopsis thaliana, more than 450 sites are edited in mitochondria and plastids, and a similar number of PPR proteins are encoded in the nuclear genome. To study how the tandem array of a PPR motif facilitates the recognition of RNA sequences, an efficient biochemical strategy is an in vitro binding assay of recombinant PPR proteins with target RNA. This analysis is especially powerful with a combination of in vivo analyses based on the phenotypes of mutants and transgenic plants. In this chapter, we describe methods for the expression of recombinant PPR proteins in Escherichia coli, preparation of probe RNAs, and RNA gel shift assays. These methods can also be utilized for other RNA-binding proteins.

  15. Visualizing double-stranded RNA distribution and dynamics in living cells by dsRNA binding-dependent fluorescence complementation

    Energy Technology Data Exchange (ETDEWEB)

    Cheng, Xiaofei [Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, London, Ontario N5V 4T3 (Canada); College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou, Zhejiang 310036 (China); Deng, Ping; Cui, Hongguang [Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, London, Ontario N5V 4T3 (Canada); Wang, Aiming, E-mail: aiming.wang@agr.gc.ca [Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, London, Ontario N5V 4T3 (Canada)

    2015-11-15

    Double-stranded RNA (dsRNA) is an important type of RNA that plays essential roles in diverse cellular processes in eukaryotic organisms and a hallmark in infections by positive-sense RNA viruses. Currently, no in vivo technology has been developed for visualizing dsRNA in living cells. Here, we report a dsRNA binding-dependent fluorescence complementation (dRBFC) assay that can be used to efficiently monitor dsRNA distribution and dynamics in vivo. The system consists of two dsRNA-binding proteins, which are fused to the N- and C-terminal halves of the yellow fluorescent protein (YFP). Binding of the two fusion proteins to a common dsRNA brings the split YFP halves in close proximity, leading to the reconstitution of the fluorescence-competent structure and restoration of fluorescence. Using this technique, we were able to visualize the distribution and trafficking of the replicative RNA intermediates of positive-sense RNA viruses in living cells. - Highlights: • A live-cell imaging system was developed for visualizing dsRNA in vivo. • It uses dsRNA binding proteins fused with two halves of a fluorescent protein. • Binding to a common dsRNA enables the reporter to become fluorescent. • The system can efficiently monitor viral RNA replication in living cells.

  16. CLIPZ: a database and analysis environment for experimentally determined binding sites of RNA-binding proteins.

    Science.gov (United States)

    Khorshid, Mohsen; Rodak, Christoph; Zavolan, Mihaela

    2011-01-01

    The stability, localization and translation rate of mRNAs are regulated by a multitude of RNA-binding proteins (RBPs) that find their targets directly or with the help of guide RNAs. Among the experimental methods for mapping RBP binding sites, cross-linking and immunoprecipitation (CLIP) coupled with deep sequencing provides transcriptome-wide coverage as well as high resolution. However, partly due to their vast volume, the data that were so far generated in CLIP experiments have not been put in a form that enables fast and interactive exploration of binding sites. To address this need, we have developed the CLIPZ database and analysis environment. Binding site data for RBPs such as Argonaute 1-4, Insulin-like growth factor II mRNA-binding protein 1-3, TNRC6 proteins A-C, Pumilio 2, Quaking and Polypyrimidine tract binding protein can be visualized at the level of the genome and of individual transcripts. Individual users can upload their own sequence data sets while being able to limit the access to these data to specific users, and analyses of the public and private data sets can be performed interactively. CLIPZ, available at http://www.clipz.unibas.ch, aims to provide an open access repository of information for post-transcriptional regulatory elements.

  17. Regulation of RNA binding proteins in trypanosomatid protozoan parasites

    Institute of Scientific and Technical Information of China (English)

    María Albertina Romaniuk; Gabriela Cervini; Alejandro Cassola

    2016-01-01

    Posttranscriptional mechanisms have a critical role in the overall outcome of gene expression. These mechanisms are especially relevant in protozoa from the genus Trypanosoma, which is composed by death threatening parasites affecting people in Sub-saharan Africa or in the Americas. In these parasites the classic view of regulation of transcription initiation to modulate the products of a given gene cannot be applied. This is due to the presence of transcription start sites that give rise to long polycistronic units that need to be processed costranscriptionally by trans-splicing and polyadenylation to give mature monocistronic mRNAs. Posttranscriptional mechanisms such as mRNA degradation and translational repression are responsible for the final synthesis of the required protein products. In this context, RNA-binding proteins(RBPs) in trypanosomes have a relevant role as modulators of mRNA abundance and translational repression by associating to the 3’ untranslated regions in mRNA. Many different RBPs have been proposed to modulate cohorts of mRNAs in trypanosomes. However, the current understanding of their functions lacks a dynamic view on the different steps at which these RBPs are regulated. Here, we discuss different evidences to propose regulatory events for different RBPs in these parasites. These events vary from regulated developmental expression, to biogenesis of cytoplasmic ribonucleoprotein complexes in the nucleus, and condensation of RBPs and mRNA into large cytoplasmic granules. Finally, we discuss how newly identified posttranslational modifications of RBPs and mRNA metabolism-related proteins could have an enormous impact on the modulation of m RNA abundance. To understand these modifications is especially relevant in these parasites due to the fact that the enzymes involved could be interesting targets for drug therapy.

  18. Regulation of RNA binding proteins in trypanosomatid protozoan parasites.

    Science.gov (United States)

    Romaniuk, María Albertina; Cervini, Gabriela; Cassola, Alejandro

    2016-02-26

    Posttranscriptional mechanisms have a critical role in the overall outcome of gene expression. These mechanisms are especially relevant in protozoa from the genus Trypanosoma, which is composed by death threatening parasites affecting people in Sub-saharan Africa or in the Americas. In these parasites the classic view of regulation of transcription initiation to modulate the products of a given gene cannot be applied. This is due to the presence of transcription start sites that give rise to long polycistronic units that need to be processed costranscriptionally by trans-splicing and polyadenylation to give mature monocistronic mRNAs. Posttranscriptional mechanisms such as mRNA degradation and translational repression are responsible for the final synthesis of the required protein products. In this context, RNA-binding proteins (RBPs) in trypanosomes have a relevant role as modulators of mRNA abundance and translational repression by associating to the 3' untranslated regions in mRNA. Many different RBPs have been proposed to modulate cohorts of mRNAs in trypanosomes. However, the current understanding of their functions lacks a dynamic view on the different steps at which these RBPs are regulated. Here, we discuss different evidences to propose regulatory events for different RBPs in these parasites. These events vary from regulated developmental expression, to biogenesis of cytoplasmic ribonucleoprotein complexes in the nucleus, and condensation of RBPs and mRNA into large cytoplasmic granules. Finally, we discuss how newly identified posttranslational modifications of RBPs and mRNA metabolism-related proteins could have an enormous impact on the modulation of mRNA abundance. To understand these modifications is especially relevant in these parasites due to the fact that the enzymes involved could be interesting targets for drug therapy.

  19. Annealing to sequences within the primer binding site loop promotes an HIV-1 RNA conformation favoring RNA dimerization and packaging

    OpenAIRE

    Seif, Elias; Niu, Meijuan; Kleiman, Lawrence

    2013-01-01

    Experiments are presented which suggest that the binding of the primer tRNA to the primer binding site of the HIV-1 5′ UTR is involved in the dimerization of the genome, as part of the packaging process.

  20. Structural basis underlying CAC RNA recognition by the RRM domain of dimeric RNA-binding protein RBPMS.

    Science.gov (United States)

    Teplova, Marianna; Farazi, Thalia A; Tuschl, Thomas; Patel, Dinshaw J

    2016-01-01

    RNA-binding protein with multiple splicing (designated RBPMS) is a higher vertebrate mRNA-binding protein containing a single RNA recognition motif (RRM). RBPMS has been shown to be involved in mRNA transport, localization and stability, with key roles in axon guidance, smooth muscle plasticity, as well as regulation of cancer cell proliferation and migration. We report on structure-function studies of the RRM domain of RBPMS bound to a CAC-containing single-stranded RNA. These results provide insights into potential topologies of complexes formed by the RBPMS RRM domain and the tandem CAC repeat binding sites as detected by photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation. These studies establish that the RRM domain of RBPMS forms a symmetrical dimer in the free state, with each monomer binding sequence-specifically to all three nucleotides of a CAC segment in the RNA bound state. Structure-guided mutations within the dimerization and RNA-binding interfaces of RBPMS RRM on RNA complex formation resulted in both disruption of dimerization and a decrease in RNA-binding affinity as observed by size exclusion chromatography and isothermal titration calorimetry. As anticipated from biochemical binding studies, over-expression of dimerization or RNA-binding mutants of Flag-HA-tagged RBPMS were no longer able to track with stress granules in HEK293 cells, thereby documenting the deleterious effects of such mutations in vivo.

  1. The sweet side of RNA regulation: glyceraldehyde-3-phosphate dehydrogenase as a noncanonical RNA-binding protein.

    Science.gov (United States)

    White, Michael R; Garcin, Elsa D

    2016-01-01

    The glycolytic protein, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), has a vast array of extraglycolytic cellular functions, including interactions with nucleic acids. GAPDH has been implicated in the translocation of transfer RNA (tRNA), the regulation of cellular messenger RNA (mRNA) stability and translation, as well as the regulation of replication and gene expression of many single-stranded RNA viruses. A growing body of evidence supports GAPDH-RNA interactions serving as part of a larger coordination between intermediary metabolism and RNA biogenesis. Despite the established role of GAPDH in nucleic acid regulation, it is still unclear how and where GAPDH binds to its RNA targets, highlighted by the absence of any conserved RNA-binding sequences. This review will summarize our current understanding of GAPDH-mediated regulation of RNA function. WIREs RNA 2016, 7:53-70. doi: 10.1002/wrna.1315 For further resources related to this article, please visit the WIREs website.

  2. RNA-binding protein RBM20 represses splicing to orchestrate cardiac pre-mRNA processing

    Science.gov (United States)

    Maatz, Henrike; Jens, Marvin; Liss, Martin; Schafer, Sebastian; Heinig, Matthias; Kirchner, Marieluise; Adami, Eleonora; Rintisch, Carola; Dauksaite, Vita; Radke, Michael H.; Selbach, Matthias; Barton, Paul J.R.; Cook, Stuart A.; Rajewsky, Nikolaus; Gotthardt, Michael; Landthaler, Markus; Hubner, Norbert

    2014-01-01

    Mutations in the gene encoding the RNA-binding protein RBM20 have been implicated in dilated cardiomyopathy (DCM), a major cause of chronic heart failure, presumably through altering cardiac RNA splicing. Here, we combined transcriptome-wide crosslinking immunoprecipitation (CLIP-seq), RNA-seq, and quantitative proteomics in cell culture and rat and human hearts to examine how RBM20 regulates alternative splicing in the heart. Our analyses revealed the presence of a distinct RBM20 RNA-recognition element that is predominantly found within intronic binding sites and linked to repression of exon splicing with RBM20 binding near 3′ and 5′ splice sites. Proteomic analysis determined that RBM20 interacts with both U1 and U2 small nuclear ribonucleic particles (snRNPs) and suggested that RBM20-dependent splicing repression occurs through spliceosome stalling at complex A. Direct RBM20 targets included several genes previously shown to be involved in DCM as well as genes not typically associated with this disease. In failing human hearts, reduced expression of RBM20 affected alternative splicing of several direct targets, indicating that differences in RBM20 expression may affect cardiac function. Together, these findings identify RBM20-regulated targets and provide insight into the pathogenesis of human heart failure. PMID:24960161

  3. RNA-binding proteins in plants: the tip of an iceberg?

    Science.gov (United States)

    Fedoroff, Nina V.; Federoff, N. V. (Principal Investigator)

    2002-01-01

    RNA-binding proteins, which are involved in the synthesis, processing, transport, translation, and degradation of RNA, are emerging as important, often multifunctional, cellular regulatory proteins. Although relatively few RNA-binding proteins have been studied in plants, they are being identified with increasing frequency, both genetically and biochemically. RNA-binding proteins that regulate chloroplast mRNA stability and translation in response to light and that have been elegantly analyzed in Clamydomonas reinhardtii have counterparts with similar functions in higher plants. Several recent reports describe mutations in genes encoding RNA-binding proteins that affect plant development and hormone signaling.

  4. Characterization of RNA-Protein Interactions: Lessons from Two RNA-Binding Proteins, SRSF1 and SRSF2.

    Science.gov (United States)

    Skrdlant, Lindsey; Lin, Ren-Jang

    2016-01-01

    SR proteins are a class of RNA-binding proteins whose RNA-binding ability is required for both constitutive and alternative splicing. While members of the SR protein family were once thought to have redundant functions, in-depth biochemical analysis of their RNA-binding abilities has revealed distinct binding profiles for each SR protein, that often lead to either synergistic or antagonistic functions. SR protein family members SRSF1 and SRSF2 are two of the most highly studied RNA-binding proteins. Here we examine the various methods used to differentiate SRSF1 and SRSF2 RNA-binding ability. We discuss the benefits and type of information that can be determined using each method.

  5. A Compendium of RNA-Binding Proteins that Regulate MicroRNA Biogenesis.

    Science.gov (United States)

    Treiber, Thomas; Treiber, Nora; Plessmann, Uwe; Harlander, Simone; Daiß, Julia-Lisa; Eichner, Norbert; Lehmann, Gerhard; Schall, Kevin; Urlaub, Henning; Meister, Gunter

    2017-04-20

    During microRNA (miRNA) biogenesis, two endonucleolytic reactions convert stem-loop-structured precursors into mature miRNAs. These processing steps can be posttranscriptionally regulated by RNA-binding proteins (RBPs). Here, we have used a proteomics-based pull-down approach to map and characterize the interactome of a multitude of pre-miRNAs. We identify ∼180 RBPs that interact specifically with distinct pre-miRNAs. For functional validation, we combined RNAi and CRISPR/Cas-mediated knockout experiments to analyze RBP-dependent changes in miRNA levels. Indeed, a large number of the investigated candidates, including splicing factors and other mRNA processing proteins, have effects on miRNA processing. As an example, we show that TRIM71/LIN41 is a potent regulator of miR-29a processing and its inactivation directly affects miR-29a targets. We provide an extended database of RBPs that interact with pre-miRNAs in extracts of different cell types, highlighting a widespread layer of co- and posttranscriptional regulation of miRNA biogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. RNA-binding properties and RNA chaperone activity of human peroxiredoxin 1

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Ji-Hee; Lee, Jeong-Mi; Lee, Hae Na; Kim, Eun-Kyung; Ha, Bin [Lee Gil Ya Cancer and Diabetes Institute, Gachon University (Korea, Republic of); Ahn, Sung-Min, E-mail: smahn@gachon.ac.kr [Lee Gil Ya Cancer and Diabetes Institute, Gachon University (Korea, Republic of); Department of Translational Medicine, Gachon University Gil Hospital, Incheon (Korea, Republic of); Jang, Ho Hee, E-mail: hhjang@gachon.ac.kr [Lee Gil Ya Cancer and Diabetes Institute, Gachon University (Korea, Republic of); Lee, Sang Yeol [Division of Applied Life Sciences (Brain Korea 21 program), Gyeongsang National University, Jinju 660-701 (Korea, Republic of)

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer hPrx1 has RNA-binding properties. Black-Right-Pointing-Pointer hPrx1 exhibits helix-destabilizing activity. Black-Right-Pointing-Pointer Cold stress increases hPrx1 level in the nuclear fraction. Black-Right-Pointing-Pointer hPrx1 enhances the viability of cells exposed to cold stress. -- Abstract: Human peroxiredoxin 1 (hPrx1), a member of the peroxiredoxin family, detoxifies peroxide substrates and has been implicated in numerous biological processes, including cell growth, proliferation, differentiation, apoptosis, and redox signaling. To date, Prx1 has not been implicated in RNA metabolism. Here, we investigated the ability of hPrx1 to bind RNA and act as an RNA chaperone. In vitro, hPrx1 bound to RNA and DNA, and unwound nucleic acid duplexes. hPrx1 also acted as a transcription anti-terminator in an assay using an Escherichia coli strain containing a stem-loop structure upstream of the chloramphenicol resistance gene. The overall cellular level of hPrx1 expression was not increased at low temperatures, but the nuclear level of hPrx1 was increased. In addition, hPrx1 overexpression enhanced the survival of cells exposed to cold stress, whereas hPrx1 knockdown significantly reduced cell survival under the same conditions. These findings suggest that hPrx1 may perform biological functions as a RNA-binding protein, which are distinctive from known functions of hPrx1 as a reactive oxygen species scavenger.

  7. Targeting of cytosolic mRNA to mitochondria: naked RNA can bind to the mitochondrial surface.

    Science.gov (United States)

    Michaud, Morgane; Maréchal-Drouard, Laurence; Duchêne, Anne-Marie

    2014-05-01

    Mitochondria contain hundreds of proteins but only a few are encoded by the mitochondrial genome. The other proteins are nuclear-encoded and imported into mitochondria. These proteins can be translated on free cytosolic polysomes, then targeted and imported into mitochondria. Nonetheless, numerous cytosolic mRNAs encoding mitochondrial proteins are detected at the surface of mitochondria in yeast, plants and animals. The localization of mRNAs to the vicinity of mitochondria would be a way for mitochondrial protein sorting. The mechanisms responsible for mRNA targeting to mitochondria are not clearly identified. Sequences within the mRNA molecules (cis-elements), as well as a few trans-acting factors, have been shown to be essential for targeting of some mRNAs. In order to identify receptors involved in mRNA docking to the mitochondrial surface, we have developed an in vitro mRNA binding assay with isolated plant mitochondria. We show that naked mRNAs are able to bind to isolated mitochondria, and our results strongly suggest that mRNA docking to the plant mitochondrial outer membrane requires at least one component of TOM complex.

  8. A bioinformatic survey of RNA-binding proteins in Plasmodium.

    Science.gov (United States)

    Reddy, B P Niranjan; Shrestha, Sony; Hart, Kevin J; Liang, Xiaoying; Kemirembe, Karen; Cui, Liwang; Lindner, Scott E

    2015-11-02

    The malaria parasites in the genus Plasmodium have a very complicated life cycle involving an invertebrate vector and a vertebrate host. RNA-binding proteins (RBPs) are critical factors involved in every aspect of the development of these parasites. However, very few RBPs have been functionally characterized to date in the human parasite Plasmodium falciparum. Using different bioinformatic methods and tools we searched P. falciparum genome to list and annotate RBPs. A representative 3D models for each of the RBD domain identified in P. falciparum was created using I-TESSAR and SWISS-MODEL. Microarray and RNAseq data analysis pertaining PfRBPs was performed using MeV software. Finally, Cytoscape was used to create protein-protein interaction network for CITH-Dozi and Caf1-CCR4-Not complexes. We report the identification of 189 putative RBP genes belonging to 13 different families in Plasmodium, which comprise 3.5% of all annotated genes. Almost 90% (169/189) of these genes belong to six prominent RBP classes, namely RNA recognition motifs, DEAD/H-box RNA helicases, K homology, Zinc finger, Puf and Alba gene families. Interestingly, almost all of the identified RNA-binding helicases and KH genes have cognate homologs in model species, suggesting their evolutionary conservation. Exploration of the existing P. falciparum blood-stage transcriptomes revealed that most RBPs have peak mRNA expression levels early during the intraerythrocytic development cycle, which taper off in later stages. Nearly 27% of RBPs have elevated expression in gametocytes, while 47 and 24% have elevated mRNA expression in ookinete and asexual stages. Comparative interactome analyses using human and Plasmodium protein-protein interaction datasets suggest extensive conservation of the PfCITH/PfDOZI and PfCaf1-CCR4-NOT complexes. The Plasmodium parasites possess a large number of putative RBPs belonging to most of RBP families identified so far, suggesting the presence of extensive post

  9. Split End Family RNA Binding Proteins: Novel Tumor Suppressors Coupling Transcriptional Regulation with RNA Processing

    Directory of Open Access Journals (Sweden)

    Hairui Su

    2015-01-01

    Full Text Available Split End (SPEN family proteins have three members: SPEN, RBM15, and RBM15B. SPEN family proteins contain three conserved RNA recognition motifs on the N-terminal region and an SPOC domain on the C-terminal region. RBM15 is fused to MKL1 in chromosome translocation t (1;22, which causes childhood acute megakaryoblastic leukemia (AMKL. Haploinsufficiency of RBM15 in AMKL indicates that RBM15 is a tumor suppressor. Both SPEN and RBM15 are mutated in a variety of cancer types, implying that they are tumor suppressors. SPEN and RBM15are required for the development of multiple organs including hematopoiesis partly via regulating the NOTCH signaling pathway, as well as the WNT signaling pathway in species ranging from Drosophila to mammals. Besides transcriptional regulation, RBM15 regulates RNA export and RNA splicing. In this review, we summarized data in the literature on how the members in SPEN family regulate gene expression at transcription and RNA processing steps. The crosstalk between epigenetic regulation and RNA metabolism is increasingly appreciated in understanding tumorigenesis. Studying the SPEN family of RNA binding proteins will create new perspectives for cancer therapy.

  10. BindUP: a web server for non-homology-based prediction of DNA and RNA binding proteins.

    Science.gov (United States)

    Paz, Inbal; Kligun, Efrat; Bengad, Barak; Mandel-Gutfreund, Yael

    2016-07-08

    Gene expression is a multi-step process involving many layers of regulation. The main regulators of the pathway are DNA and RNA binding proteins. While over the years, a large number of DNA and RNA binding proteins have been identified and extensively studied, it is still expected that many other proteins, some with yet another known function, are awaiting to be discovered. Here we present a new web server, BindUP, freely accessible through the website http://bindup.technion.ac.il/, for predicting DNA and RNA binding proteins using a non-homology-based approach. Our method is based on the electrostatic features of the protein surface and other general properties of the protein. BindUP predicts nucleic acid binding function given the proteins three-dimensional structure or a structural model. Additionally, BindUP provides information on the largest electrostatic surface patches, visualized on the server. The server was tested on several datasets of DNA and RNA binding proteins, including proteins which do not possess DNA or RNA binding domains and have no similarity to known nucleic acid binding proteins, achieving very high accuracy. BindUP is applicable in either single or batch modes and can be applied for testing hundreds of proteins simultaneously in a highly efficient manner.

  11. RNA-Binding Proteins in Trichomonas vaginalis: Atypical Multifunctional Proteins

    Directory of Open Access Journals (Sweden)

    Elisa E. Figueroa-Angulo

    2015-11-01

    Full Text Available 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.

  12. RNA-Binding Proteins in Trichomonas vaginalis: Atypical Multifunctional Proteins.

    Science.gov (United States)

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

    2015-11-26

    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.

  13. Identification of the RNA recognition element of the RBPMS family of RNA-binding proteins and their transcriptome-wide mRNA targets.

    Science.gov (United States)

    Farazi, Thalia A; Leonhardt, Carl S; Mukherjee, Neelanjan; Mihailovic, Aleksandra; Li, Song; Max, Klaas E A; Meyer, Cindy; Yamaji, Masashi; Cekan, Pavol; Jacobs, Nicholas C; Gerstberger, Stefanie; Bognanni, Claudia; Larsson, Erik; Ohler, Uwe; Tuschl, Thomas

    2014-07-01

    Recent studies implicated the RNA-binding protein with multiple splicing (RBPMS) family of proteins in oocyte, retinal ganglion cell, heart, and gastrointestinal smooth muscle development. These RNA-binding proteins contain a single RNA recognition motif (RRM), and their targets and molecular function have not yet been identified. We defined transcriptome-wide RNA targets using photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) in HEK293 cells, revealing exonic mature and intronic pre-mRNA binding sites, in agreement with the nuclear and cytoplasmic localization of the proteins. Computational and biochemical approaches defined the RNA recognition element (RRE) as a tandem CAC trinucleotide motif separated by a variable spacer region. Similar to other mRNA-binding proteins, RBPMS family of proteins relocalized to cytoplasmic stress granules under oxidative stress conditions suggestive of a support function for mRNA localization in large and/or multinucleated cells where it is preferentially expressed.

  14. Aggregation of ALS-linked FUS mutant sequesters RNA binding proteins and impairs RNA granules formation

    Energy Technology Data Exchange (ETDEWEB)

    Takanashi, Keisuke; Yamaguchi, Atsushi, E-mail: atsyama@restaff.chiba-u.jp

    2014-09-26

    Highlights: • Aggregation of ALS-linked FUS mutant sequesters ALS-associated RNA-binding proteins (FUS wt, hnRNP A1, and hnRNP A2). • Aggregation of ALS-linked FUS mutant sequesters SMN1 in the detergent-insoluble fraction. • Aggregation of ALS-linked FUS mutant reduced the number of speckles in the nucleus. • Overproduced ALS-linked FUS mutant reduced the number of processing-bodies (PBs). - Abstract: Protein aggregate/inclusion is one of hallmarks for neurodegenerative disorders including amyotrophic lateral sclerosis (ALS). FUS/TLS, one of causative genes for familial ALS, encodes a multifunctional DNA/RNA binding protein predominantly localized in the nucleus. C-terminal mutations in FUS/TLS cause the retention and the inclusion of FUS/TLS mutants in the cytoplasm. In the present study, we examined the effects of ALS-linked FUS mutants on ALS-associated RNA binding proteins and RNA granules. FUS C-terminal mutants were diffusely mislocalized in the cytoplasm as small granules in transiently transfected SH-SY5Y cells, whereas large aggregates were spontaneously formed in ∼10% of those cells. hnRNP A1, hnRNP A2, and SMN1 as well as FUS wild type were assembled into stress granules under stress conditions, and these were also recruited to FUS mutant-derived spontaneous aggregates in the cytoplasm. These aggregates stalled poly(A) mRNAs and sequestered SMN1 in the detergent insoluble fraction, which also reduced the number of nuclear oligo(dT)-positive foci (speckles) in FISH (fluorescence in situ hybridization) assay. In addition, the number of P-bodies was decreased in cells harboring cytoplasmic granules of FUS P525L. These findings raise the possibility that ALS-linked C-terminal FUS mutants could sequester a variety of RNA binding proteins and mRNAs in the cytoplasmic aggregates, which could disrupt various aspects of RNA equilibrium and biogenesis.

  15. Modeling RNA-ligand interactions: the Rev-binding element RNA-aminoglycoside complex.

    Science.gov (United States)

    Leclerc, F; Cedergren, R

    1998-01-15

    An approach to the modeling of ligand-RNA complexes has been developed by combining three-dimensional structure-activity relationship (3D-SAR) computations with a docking protocol. The ability of 3D-SAR to predict bound conformations of flexible ligands was first assessed by attempting to reconstruct the known, bound conformations of phenyloxazolines complexed with human rhinovirus 14 (HRV14) RNA. Subsequently, the same 3D-SAR analysis was applied to the identification of bound conformations of aminoglycosides which associate with the Rev-binding element (RBE) RNA. Bound conformations were identified by parsing ligand conformational data sets with pharmacophores determined by the 3D-SAR analysis. These "bioactive" structures were docked to the receptor RNA, and optimization of the complex was undertaken by extensive searching of ligand conformational space coupled with molecular dynamics computations. The similarity between the bound conformations of the ligand from the 3D-SAR analysis and those found in the docking protocol suggests that this methodology is valid for the prediction of bound ligand conformations and the modeling of the structure of the ligand-RNA complexes.

  16. Dihedral angle preferences of DNA and RNA binding amino acid residues in proteins.

    Science.gov (United States)

    Ponnuraj, Karthe; Saravanan, Konda Mani

    2017-04-01

    A protein can interact with DNA or RNA molecules to perform various cellular processes. Identifying or analyzing DNA/RNA binding site amino acid residues is important to understand molecular recognition process. It is quite possible to accurately model DNA/RNA binding amino acid residues in experimental protein-DNA/RNA complex by using the electron density map whereas, locating/modeling the binding site amino acid residues in the predicted three dimensional structures of DNA/RNA binding proteins is still a difficult task. Considering the above facts, in the present work, we have carried out a comprehensive analysis of dihedral angle preferences of DNA and RNA binding site amino acid residues by using a classical Ramachandran map. We have computed backbone dihedral angles of non-DNA/RNA binding residues and used as control dataset to make a comparative study. The dihedral angle preference of DNA and RNA binding site residues of twenty amino acid type is presented. Our analysis clearly revealed that the dihedral angles (φ, ψ) of DNA/RNA binding amino acid residues prefer to occupy (-89° to -60°, -59° to -30°) bins. The results presented in this paper will help to model/locate DNA/RNA binding amino acid residues with better accuracy. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Structural delineation of stem-loop RNA binding by human TAF15 protein.

    Science.gov (United States)

    Kashyap, Maruthi; Ganguly, Akshay Kumar; Bhavesh, Neel Sarovar

    2015-11-27

    Human TATA binding protein associated factor 2 N (TAF15) and Fused in sarcoma (FUS) are nucleic acid binding proteins belonging to the conserved FET family of proteins. They are involved in diverse processes such as pre-mRNA splicing, mRNA transport, and DNA binding. The absence of information regarding the structural mechanism employed by the FET family in recognizing and discriminating their cognate and non-cognate RNA targets has hampered the attainment of consensus on modes of protein-RNA binding for this family. Our study provides a molecular basis of this RNA recognition using a combination of solution-state NMR spectroscopy, calorimetry, docking and molecular dynamics simulation. Analysis of TAF15-RRM solution structure and its binding with stem-loop RNA has yielded conclusive evidence of a non-canonical mode of RNA recognition. Rather than classical stacking interactions that occur across nitrogen bases and aromatic amino acids on ribonucleoprotein sites, moderate-affinity hydrogen bonding network between the nitrogen bases in the stem-loop RNA and a concave face on the RRM surface primarily mediate TAF15-RRM RNA interaction. We have compared the binding affinities across a set of single-stranded RNA oligonucleotides to conclusively establish that RNA binding is dependent upon structural elements in the RNA rather than sequence.

  18. RNA-binding protein CPEB1 remodels host and viral RNA landscapes.

    Science.gov (United States)

    Batra, Ranjan; Stark, Thomas J; Clark, Elizabeth; Belzile, Jean-Philippe; Wheeler, Emily C; Yee, Brian A; Huang, Hui; Gelboin-Burkhart, Chelsea; Huelga, Stephanie C; Aigner, Stefan; Roberts, Brett T; Bos, Tomas J; Sathe, Shashank; Donohue, John Paul; Rigo, Frank; Ares, Manuel; Spector, Deborah H; Yeo, Gene W

    2016-12-01

    Host and virus interactions occurring at the post-transcriptional level are critical for infection but remain poorly understood. Here, we performed comprehensive transcriptome-wide analyses revealing that human cytomegalovirus (HCMV) infection results in widespread alternative splicing (AS), shortening of 3' untranslated regions (3' UTRs) and lengthening of poly(A)-tails in host gene transcripts. We found that the host RNA-binding protein CPEB1 was highly induced after infection, and ectopic expression of CPEB1 in noninfected cells recapitulated infection-related post-transcriptional changes. CPEB1 was also required for poly(A)-tail lengthening of viral RNAs important for productive infection. Strikingly, depletion of CPEB1 reversed infection-related cytopathology and post-transcriptional changes, and decreased productive HCMV titers. Host RNA processing was also altered in herpes simplex virus-2 (HSV-2)-infected cells, thereby indicating that this phenomenon might be a common occurrence during herpesvirus infections. We anticipate that our work may serve as a starting point for therapeutic targeting of host RNA-binding proteins in herpesvirus infections.

  19. Novel RNA- and FMRP-binding protein TRF2-S regulates axonal mRNA transport and presynaptic plasticity.

    Science.gov (United States)

    Zhang, Peisu; Abdelmohsen, Kotb; Liu, Yong; Tominaga-Yamanaka, Kumiko; Yoon, Je-Hyun; Ioannis, Grammatikakis; Martindale, Jennifer L; Zhang, Yongqing; Becker, Kevin G; Yang, In Hong; Gorospe, Myriam; Mattson, Mark P

    2015-11-20

    Despite considerable evidence that RNA-binding proteins (RBPs) regulate mRNA transport and local translation in dendrites, roles for axonal RBPs are poorly understood. Here we demonstrate that a non-telomeric isoform of telomere repeat-binding factor 2 (TRF2-S) is a novel RBP that regulates axonal plasticity. TRF2-S interacts directly with target mRNAs to facilitate their axonal delivery. The process is antagonized by fragile X mental retardation protein (FMRP). Distinct from the current RNA-binding model of FMRP, we show that FMRP occupies the GAR domain of TRF2-S protein to block the assembly of TRF2-S-mRNA complexes. Overexpressing TRF2-S and silencing FMRP promotes mRNA entry to axons and enhances axonal outgrowth and neurotransmitter release from presynaptic terminals. Our findings suggest a pivotal role for TRF2-S in an axonal mRNA localization pathway that enhances axon outgrowth and neurotransmitter release.

  20. Roles of RNA-Binding Proteins in DNA Damage Response

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    Mihoko Kai

    2016-02-01

    Full Text Available Living cells experience DNA damage as a result of replication errors and oxidative metabolism, exposure to environmental agents (e.g., ultraviolet light, ionizing radiation (IR, and radiation therapies and chemotherapies for cancer treatments. Accumulation of DNA damage can lead to multiple diseases such as neurodegenerative disorders, cancers, immune deficiencies, infertility, and also aging. Cells have evolved elaborate mechanisms to deal with DNA damage. Networks of DNA damage response (DDR pathways are coordinated to detect and repair DNA damage, regulate cell cycle and transcription, and determine the cell fate. Upstream factors of DNA damage checkpoints and repair, “sensor” proteins, detect DNA damage and send the signals to downstream factors in order to maintain genomic integrity. Unexpectedly, we have discovered that an RNA-processing factor is involved in DNA repair processes. We have identified a gene that contributes to glioblastoma multiforme (GBM’s treatment resistance and recurrence. This gene, RBM14, is known to function in transcription and RNA splicing. RBM14 is also required for maintaining the stem-like state of GBM spheres, and it controls the DNA-PK-dependent non-homologous end-joining (NHEJ pathway by interacting with KU80. RBM14 is a RNA-binding protein (RBP with low complexity domains, called intrinsically disordered proteins (IDPs, and it also physically interacts with PARP1. Furthermore, RBM14 is recruited to DNA double-strand breaks (DSBs in a poly(ADP-ribose (PAR-dependent manner (unpublished data. DNA-dependent PARP1 (poly-(ADP ribose polymerase 1 makes key contributions in the DNA damage response (DDR network. RBM14 therefore plays an important role in a PARP-dependent DSB repair process. Most recently, it was shown that the other RBPs with intrinsically disordered domains are recruited to DNA damage sites in a PAR-dependent manner, and that these RBPs form liquid compartments (also known as

  1. Crystal structure and RNA-binding properties of an Hfq homolog from the deep-branching Aquificae: conservation of the lateral RNA-binding mode.

    Science.gov (United States)

    Stanek, Kimberly A; Patterson-West, Jennifer; Randolph, Peter S; Mura, Cameron

    2017-04-01

    The host factor Hfq, as the bacterial branch of the Sm family, is an RNA-binding protein involved in the post-transcriptional regulation of mRNA expression and turnover. Hfq facilitates pairing between small regulatory RNAs (sRNAs) and their corresponding mRNA targets by binding both RNAs and bringing them into close proximity. Hfq homologs self-assemble into homo-hexameric rings with at least two distinct surfaces that bind RNA. Recently, another binding site, dubbed the `lateral rim', has been implicated in sRNA·mRNA annealing; the RNA-binding properties of this site appear to be rather subtle, and its degree of evolutionary conservation is unknown. An Hfq homolog has been identified in the phylogenetically deep-branching thermophile Aquifex aeolicus (Aae), but little is known about the structure and function of Hfq from basal bacterial lineages such as the Aquificae. Therefore, Aae Hfq was cloned, overexpressed, purified, crystallized and biochemically characterized. Structures of Aae Hfq were determined in space groups P1 and P6, both to 1.5 Å resolution, and nanomolar-scale binding affinities for uridine- and adenosine-rich RNAs were discovered. Co-crystallization with U6 RNA reveals that the outer rim of the Aae Hfq hexamer features a well defined binding pocket that is selective for uracil. This Aae Hfq structure, combined with biochemical and biophysical characterization of the homolog, reveals deep evolutionary conservation of the lateral RNA-binding mode, and lays a foundation for further studies of Hfq-associated RNA biology in ancient bacterial phyla.

  2. Recombinant human MDM2 oncoprotein shows sequence composition selectivity for binding to both RNA and DNA.

    Science.gov (United States)

    Challen, Christine; Anderson, John J; Chrzanowska-Lightowlers, Zofia M A; Lightowlers, Robert N; Lunec, John

    2012-03-01

    MDM2 is a 90 kDa nucleo-phosphoprotein that binds p53 and other proteins contributing to its oncogenic properties. Its structure includes an amino proximal p53 binding site, a central acidic domain and a carboxy region which incorporates Zinc and Ring Finger domains suggestive of nucleic acid binding or transcription factor function. It has previously been reported that a bacculovirus expressed MDM2 protein binds RNA in a sequence-specific manner through the Ring Finger domain, however, its ability to bind DNA has yet to be examined. We report here that a bacterially expressed human MDM2 protein binds both DNA as well as the previously defined RNA consensus sequence. DNA binding appears selective and involves the carboxy-terminal domain of the molecule. RNA binding is inhibited by an MDM2 specific antibody, which recognises an epitope within the carboxy region of the protein. Selection cloning and sequence analysis of MDM2 DNA binding sequences, unlike RNA binding sequences, revealed no obvious DNA binding consensus sequence, but preferential binding to oligopurine:pyrimidine-rich stretches. Our results suggest that the observed preferential DNA binding may occur through the Zinc Finger or in a charge-charge interaction through the Ring Finger, thereby implying potentially different mechanisms for DNA and RNA MDM2 binding.

  3. RNAcontext: a new method for learning the sequence and structure binding preferences of RNA-binding proteins.

    Directory of Open Access Journals (Sweden)

    Hilal Kazan

    Full Text Available Metazoan genomes encode hundreds of RNA-binding proteins (RBPs. These proteins regulate post-transcriptional gene expression and have critical roles in numerous cellular processes including mRNA splicing, export, stability and translation. Despite their ubiquity and importance, the binding preferences for most RBPs are not well characterized. In vitro and in vivo studies, using affinity selection-based approaches, have successfully identified RNA sequence associated with specific RBPs; however, it is difficult to infer RBP sequence and structural preferences without specifically designed motif finding methods. In this study, we introduce a new motif-finding method, RNAcontext, designed to elucidate RBP-specific sequence and structural preferences with greater accuracy than existing approaches. We evaluated RNAcontext on recently published in vitro and in vivo RNA affinity selected data and demonstrate that RNAcontext identifies known binding preferences for several control proteins including HuR, PTB, and Vts1p and predicts new RNA structure preferences for SF2/ASF, RBM4, FUSIP1 and SLM2. The predicted preferences for SF2/ASF are consistent with its recently reported in vivo binding sites. RNAcontext is an accurate and efficient motif finding method ideally suited for using large-scale RNA-binding affinity datasets to determine the relative binding preferences of RBPs for a wide range of RNA sequences and structures.

  4. NEAT1 scaffolds RNA-binding proteins and the Microprocessor to globally enhance pri-miRNA processing.

    Science.gov (United States)

    Jiang, Li; Shao, Changwei; Wu, Qi-Jia; Chen, Geng; Zhou, Jie; Yang, Bo; Li, Hairi; Gou, Lan-Tao; Zhang, Yi; Wang, Yangming; Yeo, Gene W; Zhou, Yu; Fu, Xiang-Dong

    2017-08-28

    MicroRNA (miRNA) biogenesis is known to be modulated by a variety of RNA-binding proteins (RBPs), but in most cases, individual RBPs appear to influence the processing of a small subset of target miRNAs. Here, we report that the RNA-binding NONO-PSF heterodimer binds a large number of expressed pri-miRNAs in HeLa cells to globally enhance pri-miRNA processing by the Drosha-DGCR8 Microprocessor. NONO and PSF are key components of paraspeckles organized by the long noncoding RNA (lncRNA) NEAT1. We further demonstrate that NEAT1 also has a profound effect on global pri-miRNA processing. Mechanistic dissection reveals that NEAT1 broadly interacts with the NONO-PSF heterodimer as well as many other RBPs and that multiple RNA segments in NEAT1, including a 'pseudo pri-miRNA' near its 3' end, help attract the Microprocessor. These findings suggest a 'bird nest' model in which an lncRNA orchestrates efficient processing of potentially an entire class of small noncoding RNAs in the nucleus.

  5. Divergence of Pumilio/fem-3 mRNA binding factor (PUF) protein specificity through variations in an RNA-binding pocket.

    Science.gov (United States)

    Qiu, Chen; Kershner, Aaron; Wang, Yeming; Holley, Cynthia P; Wilinski, Daniel; Keles, Sunduz; Kimble, Judith; Wickens, Marvin; Hall, Traci M Tanaka

    2012-02-24

    mRNA control networks depend on recognition of specific RNA sequences. Pumilio-fem-3 mRNA binding factor (PUF) RNA-binding proteins achieve that specificity through variations on a conserved scaffold. Saccharomyces cerevisiae Puf3p achieves specificity through an additional binding pocket for a cytosine base upstream of the core RNA recognition site. Here we demonstrate that this chemically simple adaptation is prevalent and contributes to the diversity of RNA specificities among PUF proteins. Bioinformatics analysis shows that mRNAs associated with Caenorhabditis elegans fem-3 mRNA binding factor (FBF)-2 in vivo contain an upstream cytosine required for biological regulation. Crystal structures of FBF-2 and C. elegans PUF-6 reveal binding pockets structurally similar to that of Puf3p, whereas sequence alignments predict a pocket in PUF-11. For Puf3p, FBF-2, PUF-6, and PUF-11, the upstream pockets and a cytosine are required for maximal binding to RNA, but the quantitative impact on binding affinity varies. Furthermore, the position of the upstream cytosine relative to the core PUF recognition site can differ, which in the case of FBF-2 originally masked the identification of this consensus sequence feature. Importantly, other PUF proteins lack the pocket and so do not discriminate upstream bases. A structure-based alignment reveals that these proteins lack key residues that would contact the cytosine, and in some instances, they also present amino acid side chains that interfere with binding. Loss of the pocket requires only substitution of one serine, as appears to have occurred during the evolution of certain fungal species.

  6. Backbone resonance assignments of the micro-RNA precursor binding region of human TRBP.

    Science.gov (United States)

    Benoit, Matthieu P M H; Plevin, Michael J

    2013-10-01

    TAR-RNA binding protein (TRBP) is a multidomain human protein involved in micro-RNA (miRNA) biogenesis. TRBP is a component of both the Dicer complex, which processes precursor miRNAs, and the RNA-induced silencing complex-loading complex. In addition, TRBP is implicated in the human immunodeficiency virus replication cycle and interferon-protein kinase R activity. TRBP contains 3 double-stranded RNA binding domains the first two of which have been shown to interact with miRNA precursors. Here we present the backbone resonance assignments and secondary structure of residues 19-228 of human TRBP2.

  7. Hexanucleotide repeats in ALS/FTD form length-dependent RNA foci, sequester RNA binding proteins, and are neurotoxic.

    Science.gov (United States)

    Lee, Youn-Bok; Chen, Han-Jou; Peres, João N; Gomez-Deza, Jorge; Attig, Jan; Stalekar, Maja; Troakes, Claire; Nishimura, Agnes L; Scotter, Emma L; Vance, Caroline; Adachi, Yoshitsugu; Sardone, Valentina; Miller, Jack W; Smith, Bradley N; Gallo, Jean-Marc; Ule, Jernej; Hirth, Frank; Rogelj, Boris; Houart, Corinne; Shaw, Christopher E

    2013-12-12

    The GGGGCC (G4C2) intronic repeat expansion within C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Intranuclear neuronal RNA foci have been observed in ALS and FTD tissues, suggesting that G4C2 RNA may be toxic. Here, we demonstrate that the expression of 38× and 72× G4C2 repeats form intranuclear RNA foci that initiate apoptotic cell death in neuronal cell lines and zebrafish embryos. The foci colocalize with a subset of RNA binding proteins, including SF2, SC35, and hnRNP-H in transfected cells. Only hnRNP-H binds directly to G4C2 repeats following RNA immunoprecipitation, and only hnRNP-H colocalizes with 70% of G4C2 RNA foci detected in C9ORF72 mutant ALS and FTD brain tissues. We show that expanded G4C2 repeats are potently neurotoxic and bind hnRNP-H and other RNA binding proteins. We propose that RNA toxicity and protein sequestration may disrupt RNA processing and contribute to neurodegeneration.

  8. Combinatorial Synthesis, Screening, and Binding Studies of Highly Functionalized Polyamino-amido Oligomers for Binding to Folded RNA

    Directory of Open Access Journals (Sweden)

    Jonathan K. Pokorski

    2012-01-01

    Full Text Available Folded RNA molecules have recently emerged as critical regulatory elements in biological pathways, serving not just as carriers of genetic information but also as key components in enzymatic assemblies. In particular, the transactivation response element (TAR of the HIV genome regulates transcriptional elongation by interacting specifically with the Tat protein, initiating the recruitment of the elongation complex. Preventing this interaction from occurring in vivo halts HIV replication, thus making RNA-binding molecules an intriguing pharmaceutical target. Using α-amino acids as starting materials, we have designed and synthesized a new class of polyamino-amido oligomers, called PAAs, specifically for binding to folded RNA structures. The PAA monomers were readily incorporated into a 125-member combinatorial library of PAA trimers. In order to rapidly assess RNA binding, a quantum dot-based fluorescent screen was developed to visualize RNA binding on-resin. The binding affinities of hits were quantified using a terbium footprinting assay, allowing us to identify a ligand (SFF with low micromolar affinity (kd=14 μM for TAR RNA. The work presented herein represents the development of a flexible scaffold that can be easily synthesized, screened, and subsequently modified to provide ligands specific for binding to folded RNAs.

  9. DO-RIP-seq to quantify RNA binding sites transcriptome-wide.

    Science.gov (United States)

    Nicholson, Cindo O; Friedersdorf, Matthew B; Bisogno, Laura S; Keene, Jack D

    2016-11-10

    Post-transcriptional processes orchestrate gene expression through dynamic protein-RNA interactions. These interactions occur at specific sites determined by RNA sequence, secondary structure, or nucleotide modifications. Methods have been developed either to quantify binding of whole transcripts or to identify the binding sites, but there is none proven to quantify binding at both the whole transcript and binding site levels. Here we describe digestion optimized RNA immunoprecipitation with deep sequencing (DO-RIP-seq) as a method that quantitates at the whole transcript target (RIP-Seq-Like or RSL) level and at the binding site level (BSL) using continuous metrics. DO-RIP-seq methodology was developed using the RBP HuR/ELAVL1 as a test case (Nicholson et al., 2016). DO-RIP-seq employs treatment of cell lysates with a nuclease under optimized conditions to yield partially digested RNA fragments bound by RNA binding proteins, followed by immunoprecipitations that capture the digested RNA-protein complexes and assess non-specific or background interactions. Analyses of sequenced cDNA libraries made from the bound RNA fragments yielded two types of enrichment scores; one for RSL binding events and the other for BSL events (Nicholson et al., 2016). These analyses plus the extensive read coverage of DO-RIP-seq allows seamless integration of binding site and whole transcript information. Therefore, DO-RIP-seq is useful for quantifying RBP binding events that are regulated during dynamic biological processes.

  10. MeRNA: a Database of Metal Ion Binding Sites in RNAStructures

    Energy Technology Data Exchange (ETDEWEB)

    Stefan, Liliana R.; Zhang, Rui; Levitan, Aaron G.; Hendrix, DonnaF.; Brenner, Steven E.; Holbrook, Stephen R.

    2005-10-05

    Metal ions are essential for the folding of RNA into stable tertiary structures and for the catalytic activity of some RNA enzymes. To aid in the study of the roles of metal ions in RNA structural biology, we have created MeRNA (Metals in RNA), a comprehensive compilation of all metal binding sites identified in RNA three-dimensional structures available from the Protein Data Bank (PDB) and Nucleic Acid Database (NDB). Currently, our database contains information relating to binding of 9764 metal ions corresponding to 23 distinct elements; in 256 RNA structures. The metal ion locations were confirmed and ligands characterized using original literature references. MeRNA includes eight manually identified metal-ion binding motifs, which are described in the literature. MeRNA is searchable by PDB identifier, metal ion, method of structure determination, resolution and R-values for X-ray structure, and distance from metal to any RNA atom or to water. New structures with their respective binding motifs will be added to the database as they become available. The MeRNA database will further our understanding of the roles of metal ions in RNA folding and catalysis and have applications in structural and functional analysis, RNA design and engineering.

  11. Protein universe containing a PUA RNA-binding domain.

    Science.gov (United States)

    Cerrudo, Carolina S; Ghiringhelli, Pablo D; Gomez, Daniel E

    2014-01-01

    Here, we review current knowledge about pseudouridine synthase and archaeosine transglycosylase (PUA)-domain-containing proteins to illustrate progress in this field. A methodological analysis of the literature about the topic was carried out, together with a 'qualitative comparative analysis' to give a more comprehensive review. Bioinformatics methods for whole-protein or protein-domain identification are commonly based on pairwise protein sequence comparisons; we added comparison of structures to detect the whole universe of proteins containing the PUA domain. We present an update of proteins having this domain, focusing on the specific proteins present in Homo sapiens (dyskerin, MCT1, Nip7, eIF2D and Nsun6), and explore the existence of these in other species. We also analyze the phylogenetic distribution of the PUA domain in different species and proteins. Finally, we performed a structural comparison of the PUA domain through data mining of structural databases, determining a conserved structural motif, despite the differences in the sequence, even among eukaryotes, archaea and bacteria. All data discussed in this review, both bibliographic and analytical, corroborate the functional importance of the PUA domain in RNA-binding proteins.

  12. Functional Equivalence of Retroviral MA Domains in Facilitating Psi RNA Binding Specificity by Gag

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    Tiffiny Rye-McCurdy

    2016-09-01

    Full Text Available Retroviruses specifically package full-length, dimeric genomic RNA (gRNA even in the presence of a vast excess of cellular RNA. The “psi” (Ψ element within the 5′-untranslated region (5′UTR of gRNA is critical for packaging through interaction with the nucleocapsid (NC domain of Gag. However, in vitro Gag binding affinity for Ψ versus non-Ψ RNAs is not significantly different. Previous salt-titration binding assays revealed that human immunodeficiency virus type 1 (HIV-1 Gag bound to Ψ RNA with high specificity and relatively few charge interactions, whereas binding to non-Ψ RNA was less specific and involved more electrostatic interactions. The NC domain was critical for specific Ψ binding, but surprisingly, a Gag mutant lacking the matrix (MA domain was less effective at discriminating Ψ from non-Ψ RNA. We now find that Rous sarcoma virus (RSV Gag also effectively discriminates RSV Ψ from non-Ψ RNA in a MA-dependent manner. Interestingly, Gag chimeras, wherein the HIV-1 and RSV MA domains were swapped, maintained high binding specificity to cognate Ψ RNAs. Using Ψ RNA mutant constructs, determinants responsible for promoting high Gag binding specificity were identified in both systems. Taken together, these studies reveal the functional equivalence of HIV-1 and RSV MA domains in facilitating Ψ RNA selectivity by Gag, as well as Ψ elements that promote this selectivity.

  13. Shared RNA-binding sites for interacting members of the Drosophila ELAV family of neuronal proteins

    OpenAIRE

    Borgeson, Claudia D.; Samson, Marie-Laure

    2005-01-01

    The product of the Drosophila embryonic lethal abnormal visual system is a conserved protein (ELAV) necessary for normal neuronal differentiation and maintenance. It possesses three RNA-binding domains and is involved in the regulation of RNA metabolism. The long elav 3′-untranslated region (3′-UTR) is necessary for autoregulation. We used RNA-binding assays and in vitro selection to identify the ELAV best binding site in the elav 3′-UTR. This site resembles ELAV-binding sites identified prev...

  14. Crystal structure of the single-stranded RNA binding protein HutP from Geobacillus thermodenitrificans.

    Science.gov (United States)

    Thiruselvam, Viswanathan; Sivaraman, Padavattan; Kumarevel, Thirumananseri; Ponnuswamy, Mondikalipudur Nanjappagounder

    2014-04-18

    RNA binding proteins control gene expression by the attenuation/antitermination mechanism. HutP is an RNA binding antitermination protein. It regulates the expression of hut operon when it binds with RNA by modulating the secondary structure of single-stranded hut mRNA. HutP necessitates the presence of l-histidine and divalent metal ion to bind with RNA. Herein, we report the crystal structures of ternary complex (HutP-l-histidine-Mg(2+)) and EDTA (0.5 M) treated ternary complex (HutP-l-histidine-Mg(2+)), solved at 1.9 Å and 2.5 Å resolutions, respectively, from Geobacillus thermodenitrificans. The addition of 0.5 M EDTA does not affect the overall metal-ion mediated ternary complex structure and however, the metal ions at the non-specific binding sites are chelated, as evidenced from the results of structural features.

  15. Rapid NMR screening of RNA secondary structure and binding

    Energy Technology Data Exchange (ETDEWEB)

    Helmling, Christina; Keyhani, Sara; Sochor, Florian; Fürtig, Boris; Hengesbach, Martin; Schwalbe, Harald, E-mail: schwalbe@nmr.uni-frankfurt.de [Johann Wolfgang Goethe-Universität, Institut für Organische Chemie und Chemische Biologie, Center for Biomolecular Magnetic Resonance (BMRZ) (Germany)

    2015-09-15

    Determination of RNA secondary structures by NMR spectroscopy is a useful tool e.g. to elucidate RNA folding space or functional aspects of regulatory RNA elements. However, current approaches of RNA synthesis and preparation are usually time-consuming and do not provide analysis with single nucleotide precision when applied for a large number of different RNA sequences. Here, we significantly improve the yield and 3′ end homogeneity of RNA preparation by in vitro transcription. Further, by establishing a native purification procedure with increased throughput, we provide a shortcut to study several RNA constructs simultaneously. We show that this approach yields μmol quantities of RNA with purities comparable to PAGE purification, while avoiding denaturation of the RNA.

  16. Annealing to sequences within the primer binding site loop promotes an HIV-1 RNA conformation favoring RNA dimerization and packaging.

    Science.gov (United States)

    Seif, Elias; Niu, Meijuan; Kleiman, Lawrence

    2013-10-01

    The 5' untranslated region (5' UTR) of HIV-1 genomic RNA (gRNA) includes structural elements that regulate reverse transcription, transcription, translation, tRNA(Lys3) annealing to the gRNA, and gRNA dimerization and packaging into viruses. It has been reported that gRNA dimerization and packaging are regulated by changes in the conformation of the 5'-UTR RNA. In this study, we show that annealing of tRNA(Lys3) or a DNA oligomer complementary to sequences within the primer binding site (PBS) loop of the 5' UTR enhances its dimerization in vitro. Structural analysis of the 5'-UTR RNA using selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) shows that the annealing promotes a conformational change of the 5' UTR that has been previously reported to favor gRNA dimerization and packaging into virus. The model predicted by SHAPE analysis is supported by antisense experiments designed to test which annealed sequences will promote or inhibit gRNA dimerization. Based on reports showing that the gRNA dimerization favors its incorporation into viruses, we tested the ability of a mutant gRNA unable to anneal to tRNA(Lys3) to be incorporated into virions. We found a ∼60% decrease in mutant gRNA packaging compared with wild-type gRNA. Together, these data further support a model for viral assembly in which the initial annealing of tRNA(Lys3) to gRNA is cytoplasmic, which in turn aids in the promotion of gRNA dimerization and its incorporation into virions.

  17. Combinatorial Control of mRNA Fates by RNA-Binding Proteins and Non-Coding RNAs

    Directory of Open Access Journals (Sweden)

    Valentina Iadevaia

    2015-09-01

    Full Text Available Post-transcriptional control of gene expression is mediated by RNA-binding proteins (RBPs and small non-coding RNAs (e.g., microRNAs that bind to distinct elements in their mRNA targets. Here, we review recent examples describing the synergistic and/or antagonistic effects mediated by RBPs and miRNAs to determine the localisation, stability and translation of mRNAs in mammalian cells. From these studies, it is becoming increasingly apparent that dynamic rearrangements of RNA-protein complexes could have profound implications in human cancer, in synaptic plasticity, and in cellular differentiation.

  18. Targeted siRNA Delivery and mRNA Knockdown Mediated by Bispecific Digoxigenin-binding Antibodies

    Directory of Open Access Journals (Sweden)

    Britta Schneider

    2012-01-01

    Full Text Available Bispecific antibodies (bsAbs that bind to cell surface antigens and to digoxigenin (Dig were used for targeted small interfering RNA (siRNA delivery. They are derivatives of immunoglobulins G (IgGs that bind tumor antigens, such as Her2, IGF1-R, CD22, and LeY, with stabilized Dig-binding variable domains fused to the C-terminal ends of the heavy chains. siRNA that was digoxigeninylated at its 3′end was bound in a 2:1 ratio to the bsAbs. These bsAb–siRNA complexes delivered siRNAs specifically to cells that express the corresponding antigen as demonstrated by flow cytometry and confocal microscopy. The complexes internalized into endosomes and Dig-siRNAs separated from bsAbs, but Dig-siRNA was not released into the cytoplasm; bsAb-targeting alone was thus not sufficient for effective mRNA knockdown. This limitation was overcome by formulating the Dig-siRNA into nanoparticles consisting of dynamic polyconjugates (DPCs or into lipid-based nanoparticles (LNPs. The resulting complexes enabled bsAb-targeted siRNA-specific messenger RNA (mRNA knockdown with IC50 siRNA values in the low nanomolar range for a variety of bsAbs, siRNAs, and target cells. Furthermore, pilot studies in mice bearing tumor xenografts indicated mRNA knockdown in endothelial cells following systemic co-administration of bsAbs and siRNA formulated in LNPs that were targeted to the tumor vasculature.

  19. SONAR Discovers RNA-Binding Proteins from Analysis of Large-Scale Protein-Protein Interactomes.

    Science.gov (United States)

    Brannan, Kristopher W; Jin, Wenhao; Huelga, Stephanie C; Banks, Charles A S; Gilmore, Joshua M; Florens, Laurence; Washburn, Michael P; Van Nostrand, Eric L; Pratt, Gabriel A; Schwinn, Marie K; Daniels, Danette L; Yeo, Gene W

    2016-10-20

    RNA metabolism is controlled by an expanding, yet incomplete, catalog of RNA-binding proteins (RBPs), many of which lack characterized RNA binding domains. Approaches to expand the RBP repertoire to discover non-canonical RBPs are currently needed. Here, HaloTag fusion pull down of 12 nuclear and cytoplasmic RBPs followed by quantitative mass spectrometry (MS) demonstrates that proteins interacting with multiple RBPs in an RNA-dependent manner are enriched for RBPs. This motivated SONAR, a computational approach that predicts RNA binding activity by analyzing large-scale affinity precipitation-MS protein-protein interactomes. Without relying on sequence or structure information, SONAR identifies 1,923 human, 489 fly, and 745 yeast RBPs, including over 100 human candidate RBPs that contain zinc finger domains. Enhanced CLIP confirms RNA binding activity and identifies transcriptome-wide RNA binding sites for SONAR-predicted RBPs, revealing unexpected RNA binding activity for disease-relevant proteins and DNA binding proteins.

  20. Systemic RNA Interference Deficiency-1 (SID-1) Extracellular Domain Selectively Binds Long Double-stranded RNA and Is Required for RNA Transport by SID-1.

    Science.gov (United States)

    Li, Weiqiang; Koutmou, Kristin S; Leahy, Daniel J; Li, Min

    2015-07-31

    During systemic RNA interference (RNAi) in Caenorhabditis elegans, RNA spreads across different cells and tissues in a process that requires the systemic RNA interference deficient-1 (sid-1) gene, which encodes an integral membrane protein. SID-1 acts cell-autonomously and is required for cellular import of interfering RNAs. Heterologous expression of SID-1 in Drosophila Schneider 2 cells enables passive uptake of dsRNA and subsequent soaking RNAi. Previous studies have suggested that SID-1 may serve as an RNA channel, but its precise molecular role remains unclear. To test the hypothesis that SID-1 mediates a direct biochemical recognition of RNA molecule and subsequent permeation, we expressed the extracellular domain (ECD) of SID-1 and purified it to near homogeneity. Recombinant purified SID-1 ECD selectively binds dsRNA but not dsDNA in a length-dependent and sequence-independent manner. Genetic missense mutations in SID-1 ECD causal for deficient systemic RNAi resulted in significant reduction in its affinity for dsRNA. Furthermore, full-length proteins with these mutations decrease SID-1-mediated RNA transport efficiency, providing evidence that dsRNA binding to SID-1 ECD is related to RNA transport. To examine the functional similarity of mammalian homologs of SID-1 (SIDT1 and SIDT2), we expressed and purified mouse SIDT1 and SIDT2 ECDs. We show that they bind long dsRNA in vitro, supportive of dsRNA recognition. In summary, our study illustrates the functional importance of SID-1 ECD as a dsRNA binding domain that contributes to RNA transport.

  1. A novel RNA binding protein that interacts with NMDA R1 mRNA: regulation by ethanol.

    Science.gov (United States)

    Anji, Antje; Kumari, Meena

    2006-05-01

    Excitatory NMDA receptors are an important target of ethanol. Chronic ethanol exposure, in vivo and in vitro, increases polypeptide levels of NR1 subunit, the key subunit of functional NMDA receptors. In vitro, chronic ethanol treatment increases the half-life of NR1 mRNA and this observation is dependent on new protein synthesis. The present study was undertaken to locate cis-acting region(s) within the NR1 3'-untranslated region (UTR) and identify NR1 3'-UTR binding trans-acting proteins expressed in mouse fetal cortical neurons. Utilizing RNA gel shift assays we identified a 156-nt cis-acting region that binds to polysomal trans-acting proteins. This binding was highly specific as inclusion of cyclophilin RNA or tRNA did not interfere with cis-trans interactions. Importantly, the 3'-UTR binding activity was significantly up-regulated in the presence of ethanol. UV cross-link analysis detected three NR1 3'-UTR binding proteins and their molecular mass calculated by Northwestern analysis was approximately 88, 60 and 47 kDa, respectively. Northwestern analysis showed a significant up-regulation of the 88-kDa protein after chronic ethanol treatment. The 88-kDa protein was purified and identified by tandem mass spectrometry as the beta subunit of alpha glucosidase II (GIIbeta). That GIIbeta is indeed a trans-acting protein and binds specifically to 3'-UTR of NR1 mRNA was confirmed by RNA gel mobility supershift assays and immuno RT-PCR. Western blotting data established a significant increase of GIIbeta polypeptide in chronic ethanol-exposed fetal cortical neurons. We hypothesize that the identified cis-acting region and the associated RNA-binding proteins are important regulators of NR1 subunit gene expression.

  2. The expanding universe of ribonucleoproteins: of novel RNA-binding proteins and unconventional interactions.

    Science.gov (United States)

    Beckmann, Benedikt M; Castello, Alfredo; Medenbach, Jan

    2016-06-01

    Post-transcriptional regulation of gene expression plays a critical role in almost all cellular processes. Regulation occurs mostly by RNA-binding proteins (RBPs) that recognise RNA elements and form ribonucleoproteins (RNPs) to control RNA metabolism from synthesis to decay. Recently, the repertoire of RBPs was significantly expanded owing to methodological advances such as RNA interactome capture. The newly identified RNA binders are involved in diverse biological processes and belong to a broad spectrum of protein families, many of them exhibiting enzymatic activities. This suggests the existence of an extensive crosstalk between RNA biology and other, in principle unrelated, cell functions such as intermediary metabolism. Unexpectedly, hundreds of new RBPs do not contain identifiable RNA-binding domains (RBDs), raising the question of how they interact with RNA. Despite the many functions that have been attributed to RNA, our understanding of RNPs is still mostly governed by a rather protein-centric view, leading to the idea that proteins have evolved to bind to and regulate RNA and not vice versa. However, RNPs formed by an RNA-driven interaction mechanism (RNA-determined RNPs) are abundant and offer an alternative explanation for the surprising lack of classical RBDs in many RNA-interacting proteins. Moreover, RNAs can act as scaffolds to orchestrate and organise protein networks and directly control their activity, suggesting that nucleic acids might play an important regulatory role in many cellular processes, including metabolism.

  3. The use of calorimetry in the biophysical characterization of small molecule alkaloids binding to RNA structures.

    Science.gov (United States)

    Kumar, Gopinatha Suresh; Basu, Anirban

    2016-05-01

    RNA has now emerged as a potential target for therapeutic intervention. RNA targeted drug design requires detailed thermodynamic characterization that provides new insights into the interactions and this together with structural data, may be used in rational drug design. The use of calorimetry to characterize small molecule-RNA interactions has emerged as a reliable and sensitive tool after the recent advancements in biocalorimetry. This review summarizes the recent advancements in thermodynamic characterization of small molecules, particularly some natural alkaloids binding to various RNA structures. Thermodynamic characterization provides information that can supplement structural data leading to more effective drug development protocols. This review provides a concise report on the use of isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC) techniques in characterizing small molecules, mostly alkaloids-RNA interactions with particular reference to binding of tRNA, single stranded RNA, double stranded RNA, poly(A), triplex RNA. It is now apparent that a combination of structural and thermodynamic data is essential for rational design of specific RNA targeted drugs. Recent advancements in biocalorimetry instrumentation have led to detailed understanding of the thermodynamics of small molecules binding to various RNA structures paving the path for the development of many new natural and synthetic molecules as specific binders to various RNA structures. RNA targeted drug design, that remained unexplored, will immensely benefit from the calorimetric studies leading to the development of effective drugs for many diseases. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Adding energy minimization strategy to peptide-design algorithm enables better search for RNA-binding peptides: Redesigned λ N peptide binds boxB RNA.

    Science.gov (United States)

    Xiao, Xingqing; Hung, Michelle E; Leonard, Joshua N; Hall, Carol K

    2016-10-15

    Our previously developed peptide-design algorithm was improved by adding an energy minimization strategy which allows the amino acid sidechains to move in a broad configuration space during sequence evolution. In this work, the new algorithm was used to generate a library of 21-mer peptides which could substitute for λ N peptide in binding to boxB RNA. Six potential peptides were obtained from the algorithm, all of which exhibited good binding capability with boxB RNA. Atomistic molecular dynamics simulations were then conducted to examine the ability of the λ N peptide and three best evolved peptides, viz. Pept01, Pept26, and Pept28, to bind to boxB RNA. Simulation results demonstrated that our evolved peptides are better at binding to boxB RNA than the λ N peptide. Sequence searches using the old (without energy minimization strategy) and new (with energy minimization strategy) algorithms confirm that the new algorithm is more effective at finding good RNA-binding peptides than the old algorithm. © 2016 Wiley Periodicals, Inc.

  5. Annexin A2 binds RNA and reduces the frameshifting efficiency of infectious bronchitis virus.

    Directory of Open Access Journals (Sweden)

    Hoyun Kwak

    Full Text Available Annexin A2 (ANXA2 is a protein implicated in diverse cellular functions, including exocytosis, DNA synthesis and cell proliferation. It was recently proposed to be involved in RNA metabolism because it was shown to associate with some cellular mRNA. Here, we identified ANXA2 as a RNA binding protein (RBP that binds IBV (Infectious Bronchitis Virus pseudoknot RNA. We first confirmed the binding of ANXA2 to IBV pseudoknot RNA by ultraviolet crosslinking and showed its binding to RNA pseudoknot with ANXA2 protein in vitro and in the cells. Since the RNA pseudoknot located in the frameshifting region of IBV was used as bait for cellular RBPs, we tested whether ANXA2 could regulate the frameshfting of IBV pseudoknot RNA by dual luciferase assay. Overexpression of ANXA2 significantly reduced the frameshifting efficiency from IBV pseudoknot RNA and knockdown of the protein strikingly increased the frameshifting efficiency. The results suggest that ANXA2 is a cellular RBP that can modulate the frameshifting efficiency of viral RNA, enabling it to act as an anti-viral cellular protein, and hinting at roles in RNA metabolism for other cellular mRNAs.

  6. Cellular RNA binding proteins NS1-BP and hnRNP K regulate influenza A virus RNA splicing.

    Science.gov (United States)

    Tsai, Pei-Ling; Chiou, Ni-Ting; Kuss, Sharon; García-Sastre, Adolfo; Lynch, Kristen W; Fontoura, Beatriz M A

    2013-01-01

    Influenza A virus is a major human pathogen with a genome comprised of eight single-strand, negative-sense, RNA segments. Two viral RNA segments, NS1 and M, undergo alternative splicing and yield several proteins including NS1, NS2, M1 and M2 proteins. However, the mechanisms or players involved in splicing of these viral RNA segments have not been fully studied. Here, by investigating the interacting partners and function of the cellular protein NS1-binding protein (NS1-BP), we revealed novel players in the splicing of the M1 segment. Using a proteomics approach, we identified a complex of RNA binding proteins containing NS1-BP and heterogeneous nuclear ribonucleoproteins (hnRNPs), among which are hnRNPs involved in host pre-mRNA splicing. We found that low levels of NS1-BP specifically impaired proper alternative splicing of the viral M1 mRNA segment to yield the M2 mRNA without affecting splicing of mRNA3, M4, or the NS mRNA segments. Further biochemical analysis by formaldehyde and UV cross-linking demonstrated that NS1-BP did not interact directly with viral M1 mRNA but its interacting partners, hnRNPs A1, K, L, and M, directly bound M1 mRNA. Among these hnRNPs, we identified hnRNP K as a major mediator of M1 mRNA splicing. The M1 mRNA segment generates the matrix protein M1 and the M2 ion channel, which are essential proteins involved in viral trafficking, release into the cytoplasm, and budding. Thus, reduction of NS1-BP and/or hnRNP K levels altered M2/M1 mRNA and protein ratios, decreasing M2 levels and inhibiting virus replication. Thus, NS1-BP-hnRNPK complex is a key mediator of influenza A virus gene expression.

  7. Predicting sequence and structural specificities of RNA binding regions recognized by splicing factor SRSF1

    Directory of Open Access Journals (Sweden)

    Wang Xin

    2011-12-01

    Full Text Available Abstract Background RNA-binding proteins (RBPs play diverse roles in eukaryotic RNA processing. Despite their pervasive functions in coding and noncoding RNA biogenesis and regulation, elucidating the sequence specificities that define protein-RNA interactions remains a major challenge. Recently, CLIP-seq (Cross-linking immunoprecipitation followed by high-throughput sequencing has been successfully implemented to study the transcriptome-wide binding patterns of SRSF1, PTBP1, NOVA and fox2 proteins. These studies either adopted traditional methods like Multiple EM for Motif Elicitation (MEME to discover the sequence consensus of RBP's binding sites or used Z-score statistics to search for the overrepresented nucleotides of a certain size. We argue that most of these methods are not well-suited for RNA motif identification, as they are unable to incorporate the RNA structural context of protein-RNA interactions, which may affect to binding specificity. Here, we describe a novel model-based approach--RNAMotifModeler to identify the consensus of protein-RNA binding regions by integrating sequence features and RNA secondary structures. Results As an example, we implemented RNAMotifModeler on SRSF1 (SF2/ASF CLIP-seq data. The sequence-structural consensus we identified is a purine-rich octamer 'AGAAGAAG' in a highly single-stranded RNA context. The unpaired probabilities, the probabilities of not forming pairs, are significantly higher than negative controls and the flanking sequence surrounding the binding site, indicating that SRSF1 proteins tend to bind on single-stranded RNA. Further statistical evaluations revealed that the second and fifth bases of SRSF1octamer motif have much stronger sequence specificities, but weaker single-strandedness, while the third, fourth, sixth and seventh bases are far more likely to be single-stranded, but have more degenerate sequence specificities. Therefore, we hypothesize that nucleotide specificity and

  8. Trends in the Binding of Cell Penetrating Peptides to siRNA: A Molecular Docking Study

    Science.gov (United States)

    Gunathunge, B. G. C. M.; Wimalasiri, P. N.; Karunaratne, D. N.

    2017-01-01

    The use of gene therapeutics, including short interfering RNA (siRNA), is limited by the lack of efficient delivery systems. An appealing approach to deliver gene therapeutics involves noncovalent complexation with cell penetrating peptides (CPPs) which are able to penetrate the cell membranes of mammals. Although a number of CPPs have been discovered, our understanding of their complexation and translocation of siRNA is as yet insufficient. Here, we report on computational studies comparing the binding affinities of CPPs with siRNA, considering a variety of CPPs. Specifically, seventeen CPPs from three different categories, cationic, amphipathic, and hydrophobic CPPs, were studied. Molecular mechanics were used to minimize structures, while molecular docking calculations were used to predict the orientation and favorability of sequentially binding multiple peptides to siRNA. Binding scores from docking calculations were highest for amphipathic peptides over cationic and hydrophobic peptides. Results indicate that initial complexation of peptides will likely occur along the major groove of the siRNA, driven by electrostatic interactions. Subsequent binding of CPPs is likely to occur in the minor groove and later on bind randomly, to siRNA or previously bound CPPs, through hydrophobic interactions. However, hydrophobic CPPs do not show this binding pattern. Ultimately binding yields a positively charged nanoparticle capable of noninvasive cellular import of therapeutic molecules.

  9. MBSTAR: multiple instance learning for predicting specific functional binding sites in microRNA targets

    Science.gov (United States)

    Bandyopadhyay, Sanghamitra; Ghosh, Dip; Mitra, Ramkrishna; Zhao, Zhongming

    2015-01-01

    MicroRNA (miRNA) regulates gene expression by binding to specific sites in the 3'untranslated regions of its target genes. Machine learning based miRNA target prediction algorithms first extract a set of features from potential binding sites (PBSs) in the mRNA and then train a classifier to distinguish targets from non-targets. However, they do not consider whether the PBSs are functional or not, and consequently result in high false positive rates. This substantially affects the follow up functional validation by experiments. We present a novel machine learning based approach, MBSTAR (Multiple instance learning of Binding Sites of miRNA TARgets), for accurate prediction of true or functional miRNA binding sites. Multiple instance learning framework is adopted to handle the lack of information about the actual binding sites in the target mRNAs. Biologically validated 9531 interacting and 973 non-interacting miRNA-mRNA pairs are identified from Tarbase 6.0 and confirmed with PAR-CLIP dataset. It is found that MBSTAR achieves the highest number of binding sites overlapping with PAR-CLIP with maximum F-Score of 0.337. Compared to the other methods, MBSTAR also predicts target mRNAs with highest accuracy. The tool and genome wide predictions are available at http://www.isical.ac.in/~bioinfo_miu/MBStar30.htm.

  10. From face to interface recognition: a differential geometric approach to distinguish DNA from RNA binding surfaces.

    Science.gov (United States)

    Shazman, Shula; Elber, Gershon; Mandel-Gutfreund, Yael

    2011-09-01

    Protein nucleic acid interactions play a critical role in all steps of the gene expression pathway. Nucleic acid (NA) binding proteins interact with their partners, DNA or RNA, via distinct regions on their surface that are characterized by an ensemble of chemical, physical and geometrical properties. In this study, we introduce a novel methodology based on differential geometry, commonly used in face recognition, to characterize and predict NA binding surfaces on proteins. Applying the method on experimentally solved three-dimensional structures of proteins we successfully classify double-stranded DNA (dsDNA) from single-stranded RNA (ssRNA) binding proteins, with 83% accuracy. We show that the method is insensitive to conformational changes that occur upon binding and can be applicable for de novo protein-function prediction. Remarkably, when concentrating on the zinc finger motif, we distinguish successfully between RNA and DNA binding interfaces possessing the same binding motif even within the same protein, as demonstrated for the RNA polymerase transcription-factor, TFIIIA. In conclusion, we present a novel methodology to characterize protein surfaces, which can accurately tell apart dsDNA from an ssRNA binding interfaces. The strength of our method in recognizing fine-tuned differences on NA binding interfaces make it applicable for many other molecular recognition problems, with potential implications for drug design.

  11. A Caenorhabditis elegans PUF protein family with distinct RNA binding specificity.

    Science.gov (United States)

    Stumpf, Craig R; Kimble, Judith; Wickens, Marvin

    2008-08-01

    PUF proteins comprise a highly conserved family of sequence-specific RNA binding proteins that regulate target mRNAs via binding directly to their 3'UTRs. The Caenorhabditis elegans genome encodes several PUF proteins, which cluster into four groups based on sequence similarity; all share amino acids that interact with the RNA in the cocrystal of human Pumilio with RNA. Members of the FBF and the PUF-8/9 groups bind different but related RNA sequences. We focus here on the binding specificity of representatives of a third cluster, comprising PUF-5, -6, and -7. We performed in vivo selection experiments using the yeast three-hybrid system to identify RNA sequences that bind PUF-5 and PUF-6, and we confirmed binding to optimal sites in vitro. The consensus sequences derived from the screens are similar for PUF-5 and PUF-6 but differ from those of the FBF or PUF-8/-9 groups. Similarly, neither PUF-5 nor PUF-6 bind the recognition sites preferred by the other clusters. Mutagenesis studies confirmed the unique RNA specificity of PUF-5/-6. Using the PUF-5 consensus derived from our experiments, we searched a database of C. elegans 3'UTRs to identify potential targets of PUF-5, several of which indeed bind PUF-5. Therefore the consensus has predictive value and provides a route to finding genuine targets of these proteins.

  12. A strategy to enhance the binding affinity of fluorophore-aptamer pairs for RNA tagging with neomycin conjugation.

    Science.gov (United States)

    Jeon, Jongho; Lee, Kyung Hyun; Rao, Jianghong

    2012-10-14

    Fluorogenic sulforhodamine-neomycin conjugates have been designed and synthesized for RNA tagging. Conjugates were fluorescently activated by binding to RNA aptamers and exhibited greater than 250-400 fold enhancement in binding affinity relative to corresponding unconjugated fluorophores.

  13. EBV noncoding RNA EBER2 interacts with host RNA-binding proteins to regulate viral gene expression.

    Science.gov (United States)

    Lee, Nara; Yario, Therese A; Gao, Jessica S; Steitz, Joan A

    2016-03-22

    Epstein-Barr virus (EBV) produces a highly abundant noncoding RNA called EBV-encoded RNA 2 (EBER2) that interacts indirectly with the host transcription factor paired box protein 5 (PAX5) to regulate viral latent membrane protein 1/2 (LMP1/2) gene expression as well as EBV lytic replication. To identify intermediary proteins, we isolated EBER2-PAX5-containing complexes and analyzed the protein components by mass spectrometry. The top candidates include three host proteins splicing factor proline and glutamine rich (SFPQ), non-POU domain-containing octamer-binding protein (NONO), and RNA binding motif protein 14 (RBM14), all reported to be components of nuclear bodies called paraspeckles. In vivo RNA-protein crosslinking indicates that SFPQ and RBM14 contact EBER2 directly. Binding studies using recombinant proteins demonstrate that SFPQ and NONO associate with PAX5, potentially bridging its interaction with EBER2. Similar to EBER2 or PAX5 depletion, knockdown of any of the three host RNA-binding proteins results in the up-regulation of viral LMP2A mRNA levels, supporting a physiologically relevant interaction of these newly identified factors with EBER2 and PAX5. Identification of these EBER2-interacting proteins enables the search for cellular noncoding RNAs that regulate host gene expression in a manner similar to EBER2.

  14. Selection and characterization of small molecules that bind the HIV-1 frameshift site RNA.

    Science.gov (United States)

    Marcheschi, Ryan J; Mouzakis, Kathryn D; Butcher, Samuel E

    2009-10-16

    HIV-1 requires a -1 translational frameshift to properly synthesize the viral enzymes required for replication. The frameshift mechanism is dependent upon two RNA elements, a seven-nucleotide slippery sequence (UUUUUUA) and a downstream RNA structure. Frameshifting occurs with a frequency of approximately 5%, and increasing or decreasing this frequency may result in a decrease in viral replication. Here, we report the results of a high-throughput screen designed to find small molecules that bind to the HIV-1 frameshift site RNA. Out of 34,500 compounds screened, 202 were identified as positive hits. We show that one of these compounds, doxorubicin, binds the HIV-1 RNA with low micromolar affinity (K(d) = 2.8 microM). This binding was confirmed and localized to the RNA using NMR. Further analysis revealed that this compound increased the RNA stability by approximately 5 degrees C and decreased translational frameshifting by 28% (+/-14%), as measured in vitro.

  15. Structures of the Bacterial Ribosome in Classical and Hybrid States of tRNA Binding

    Energy Technology Data Exchange (ETDEWEB)

    Dunkle, Jack A.; Wang, Leyi; Feldman, Michael B.; Pulk, Arto; Chen, Vincent B.; Kapral, Gary J.; Noeske, Jonas; Richardson, Jane S.; Blanchard, Scott C.; Cate, Jamie H. Doudna (Cornell); (UCB); (Duke)

    2011-09-06

    During protein synthesis, the ribosome controls the movement of tRNA and mRNA by means of large-scale structural rearrangements. We describe structures of the intact bacterial ribosome from Escherichia coli that reveal how the ribosome binds tRNA in two functionally distinct states, determined to a resolution of {approx}3.2 angstroms by means of x-ray crystallography. One state positions tRNA in the peptidyl-tRNA binding site. The second, a fully rotated state, is stabilized by ribosome recycling factor and binds tRNA in a highly bent conformation in a hybrid peptidyl/exit site. The structures help to explain how the ratchet-like motion of the two ribosomal subunits contributes to the mechanisms of translocation, termination, and ribosome recycling.

  16. U2 toggles iteratively between the stem IIa and stem IIc conformations to promote pre-mRNA splicing.

    Science.gov (United States)

    Hilliker, Angela K; Mefford, Melissa A; Staley, Jonathan P

    2007-04-01

    To ligate exons in pre-messenger RNA (pre-mRNA) splicing, the spliceosome must reposition the substrate after cleaving the 5' splice site. Because spliceosomal small nuclear RNAs (snRNAs) bind the substrate, snRNA structures may rearrange to reposition the substrate. However, such rearrangements have remained undefined. Although U2 stem IIc inhibits binding of U2 snRNP to pre-mRNA during assembly, we found that weakening U2 stem IIc suppressed a mutation in prp16, a DExD/H box ATPase that promotes splicing after 5' splice site cleavage. The prp16 mutation was also suppressed by mutations flanking stem IIc, suggesting that Prp16p facilitates a switch from stem IIc to the mutually exclusive U2 stem IIa, which activates binding of U2 to pre-mRNA during assembly. Providing evidence that stem IIa switches back to stem IIc before exon ligation, disrupting stem IIa suppressed 3' splice site mutations, and disrupting stem IIc impaired exon ligation. Disrupting stem IIc also exacerbated the 5' splice site cleavage defects of certain substrate mutations, suggesting a parallel role for stem IIc at both catalytic stages. We propose that U2, much like the ribosome, toggles between two conformations--a closed stem IIc conformation that promotes catalysis and an open stem IIa conformation that promotes substrate binding and release.

  17. Binding by TRBP-dsRBD2 Does Not Induce Bending of Double-Stranded RNA.

    Science.gov (United States)

    Acevedo, Roderico; Evans, Declan; Penrod, Katheryn A; Showalter, Scott A

    2016-06-21

    Protein-nucleic acid interactions are central to a variety of biological processes, many of which involve large-scale conformational changes that lead to bending of the nucleic acid helix. Here, we focus on the nonsequence-specific protein TRBP, whose double-stranded RNA-binding domains (dsRBDs) interact with the A-form geometry of double-stranded RNA (dsRNA). Crystal structures of dsRBD-dsRNA interactions suggest that the dsRNA helix must bend in such a way that its major groove expands to conform to the dsRBD's binding surface. We show through isothermal titration calorimetry experiments that dsRBD2 of TRBP binds dsRNA with a temperature-independent observed binding affinity (KD ∼500 nM). Furthermore, a near-zero observed heat capacity change (ΔCp = 70 ± 40 cal·mol(-1)·K(-1)) suggests that large-scale conformational changes do not occur upon binding. This result is bolstered by molecular-dynamics simulations in which dsRBD-dsRNA interactions generate only modest bending of the RNA along its helical axis. Overall, these results suggest that this particular dsRBD-dsRNA interaction produces little to no change in the A-form geometry of dsRNA in solution. These results further support our previous hypothesis, based on extensive gel-shift assays, that TRBP preferentially binds to sites of nearly ideal A-form structure while being excluded from sites of local deformation in the RNA helical structure. The implications of this mechanism for efficient micro-RNA processing will be discussed.

  18. RNAs nonspecifically inhibit RNA polymerase II by preventing binding to the DNA template.

    Science.gov (United States)

    Pai, Dave A; Kaplan, Craig D; Kweon, Hye Kyong; Murakami, Kenji; Andrews, Philip C; Engelke, David R

    2014-05-01

    Many RNAs are known to act as regulators of transcription in eukaryotes, including certain small RNAs that directly inhibit RNA polymerases both in prokaryotes and eukaryotes. We have examined the potential for a variety of RNAs to directly inhibit transcription by yeast RNA polymerase II (Pol II) and find that unstructured RNAs are potent inhibitors of purified yeast Pol II. Inhibition by RNA is achieved by blocking binding of the DNA template and requires binding of the RNA to Pol II prior to open complex formation. RNA is not able to displace a DNA template that is already stably bound to Pol II, nor can RNA inhibit elongating Pol II. Unstructured RNAs are more potent inhibitors than highly structured RNAs and can also block specific transcription initiation in the presence of basal transcription factors. Crosslinking studies with ultraviolet light show that unstructured RNA is most closely associated with the two large subunits of Pol II that comprise the template binding cleft, but the RNA has contacts in a basic residue channel behind the back wall of the active site. These results are distinct from previous observations of specific inhibition by small, structured RNAs in that they demonstrate a sensitivity of the holoenzyme to inhibition by unstructured RNA products that bind to a surface outside the DNA cleft. These results are discussed in terms of the need to prevent inhibition by RNAs, either though sequestration of nascent RNA or preemptive interaction of Pol II with the DNA template.

  19. A viral suppressor of RNA silencing inhibits ARGONAUTE 1 function by precluding target RNA binding to pre-assembled RISC.

    Science.gov (United States)

    Kenesi, Erzsébet; Carbonell, Alberto; Lózsa, Rita; Vértessy, Beáta; Lakatos, Lóránt

    2017-07-27

    In most eukaryotes, RNA silencing is an adaptive immune system regulating key biological processes including antiviral defense. To evade this response, viruses of plants, worms and insects have evolved viral suppressors of RNA silencing proteins (VSRs). Various VSRs, such as P1 from Sweet potato mild mottle virus (SPMMV), inhibit the activity of RNA-induced silencing complexes (RISCs) including an ARGONAUTE (AGO) protein loaded with a small RNA. However, the specific mechanisms explaining this class of inhibition are unknown. Here, we show that SPMMV P1 interacts with AGO1 and AGO2 from Arabidopsis thaliana, but solely interferes with AGO1 function. Moreover, a mutational analysis of a newly identified zinc finger domain in P1 revealed that this domain could represent an effector domain as it is required for P1 suppressor activity but not for AGO1 binding. Finally, a comparative analysis of the target RNA binding capacity of AGO1 in the presence of wild-type or suppressor-defective P1 forms revealed that P1 blocks target RNA binding to AGO1. Our results describe the negative regulation of RISC, the small RNA containing molecular machine. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  20. Unzipping and binding of small interfering RNA with single walled Carbon Nanotube: a platform for small interfering RNA delivery

    CERN Document Server

    Santosh, Mogurampelly; Bhattacharyya, Dhananjay; Sood, A K; Maiti, Prabal K; 10.1063/1.3682780

    2012-01-01

    In an effort to design efficient platform for siRNA delivery, we combine all atom classical and quantum simulations to study the binding of small interfering RNA (siRNA) by pristine single wall carbon nanotube (SWCNT). Our results show that siRNA strongly binds to SWCNT surface via unzipping its base-pairs and the propensity of unzipping increases with the increase in the diameter of the SWCNTs. The unzipping and subsequent wrapping events are initiated and driven by van der Waals interactions between the aromatic rings of siRNA nucleobases and the SWCNT surface. However, MD simulations of double strand DNA (dsDNA) of the same sequence show that the dsDNA undergoes much less unzipping and wrapping on the SWCNT in the simulation time scale of 70 ns. This interesting difference is due to smaller interaction energy of thymidine of dsDNA with the SWCNT compared to that of uridine of siRNA, as calculated by dispersion corrected density functional theory (DFT) methods. After the optimal binding of siRNA to SWCNT, t...

  1. DNA-Damage Response RNA-Binding Proteins (DDRBPs): Perspectives from a New Class of Proteins and Their RNA Targets.

    Science.gov (United States)

    Dutertre, Martin; Vagner, Stéphan

    2016-09-29

    Upon DNA damage, cells trigger an early DNA-damage response (DDR) involving DNA repair and cell cycle checkpoints, and late responses involving gene expression regulation that determine cell fate. Screens for genes involved in the DDR have found many RNA-binding proteins (RBPs), while screens for novel RBPs have identified DDR proteins. An increasing number of RBPs are involved in early and/or late DDR. We propose to call this new class of actors of the DDR, which contain an RNA-binding activity, DNA-damage response RNA-binding proteins (DDRBPs). We then discuss how DDRBPs contribute not only to gene expression regulation in the late DDR but also to early DDR signaling, DNA repair, and chromatin modifications at DNA-damage sites through interactions with both long and short noncoding RNAs.

  2. A versatile assay for RNA-binding proteins in living cells.

    Science.gov (United States)

    Strein, Claudia; Alleaume, Anne-Marie; Rothbauer, Ulrich; Hentze, Matthias W; Castello, Alfredo

    2014-05-01

    RNA-binding proteins (RBPs) control RNA fate from synthesis to decay. Since their cellular expression levels frequently do not reflect their in vivo activity, methods are needed to assess the steady state RNA-binding activity of RBPs as well as their responses to stimuli. While electrophoresis mobility shift assays (EMSA) have been used for such determinations, their results serve at best as proxies for the RBP activities in living cells. Here, we describe a quantitative dual fluorescence method to analyze protein-mRNA interactions in vivo. Known or candidate RBPs are fused to fluorescent proteins (eGFP, YFP), expressed in cells, cross-linked in vivo to RNA by ultraviolet light irradiation, and immunoprecipitated, after lysis, with a single chain antibody fragment directed against eGFP (GFP-binding protein, GBP). Polyadenylated RNA-binding activity of fusion proteins is assessed by hybridization with an oligo(DT) probe coupled with a red fluorophore. Since UV light is directly applied to living cells, the assay can be used to monitor dynamic changes in RNA-binding activities in response to biological or pharmacological stimuli. Notably, immunoprecipitation and hybridization can also be performed with commercially available GBP-coupled 96-well plates (GFP-multiTrap), allowing highly parallel RNA-binding measurements in a single experiment. Therefore, this method creates the possibility to conduct in vivo high-throughput RNA-binding assays. We believe that this fast and simple radioactivity-free method will find many useful applications in RNA biology.

  3. Diverse role of three tyrosines in binding of the RNA 5' cap to the human nuclear cap binding complex.

    Science.gov (United States)

    Worch, Remigiusz; Jankowska-Anyszka, Marzena; Niedzwiecka, Anna; Stepinski, Janusz; Mazza, Catherine; Darzynkiewicz, Edward; Cusack, Stephen; Stolarski, Ryszard

    2009-01-16

    The heterodimeric nuclear cap-binding complex (CBC) specifically recognizes the monomethylguanosine 5' cap structure of the eukaryotic RNA polymerase II transcripts such as mRNA and U snRNA. The binding is essential for nuclear maturation of mRNA, for nuclear export of U snRNA in metazoans, and for nonsense-mediated decay of mRNA and the pioneer round of translation. We analysed the recognition of the cap by native human CBC and mutants in which each tyrosine that stacks with the 7-methylguanosine moiety was replaced by phenylalanine or alanine and both tyrosines were replaced by phenylalanines. The equilibrium association constants (K(as)) for two selected cap analogues, P(1)-7-methylguanosine-5' P(3)-guanosine-5' triphosphate and 7-methylguanosine triphosphate, were determined by two independent methods, fluorescence titration and surface plasmon resonance. We could distinguish two tyrosines, Y43 and Y20, in stabilization of the cap inside the CBC-binding pocket. In particular, lack of Y20 in CBC leads to a greater affinity of the mono- than the dinucleotide cap analogue, in contrast to the wild-type protein. A crucial role of cation-pi stacking in the mechanism of the specific cap recognition by CBC was postulated from the comparison of the experimentally derived Gibbs free binding energy (DeltaG degrees) with the stacking energy (DeltaE) of the 7-methylguanosine/Y binary and ternary complexes calculated by the Møller-Plesset second-order perturbation method. The resulting kinetic model of the association between the capped RNA and CBC, based on the experimental data and quantum calculations, is discussed with respect to the "CBC-to-eukaryotic initiation factor 4E handoff" of mRNA.

  4. Inhibition of RNA polymerase by captan at both DNA and substrate binding sites.

    Science.gov (United States)

    Luo, G; Lewis, R A

    1992-12-01

    RNA synthesis carried out in vitro by Escherichia coli RNA polymerase was inhibited irreversibly by captan when T7 DNA was used as template. An earlier report and this one show that captan blocks the DNA binding site on the enzyme. Herein, it is also revealed that captan acts at the nucleoside triphosphate (NTP) binding site, and kinetic relationships of the action of captan at the two sites are detailed. The inhibition by captan via the DNA binding site of the enzyme was confirmed by kinetic studies and it was further shown that [14C]captan bound to the beta' subunit of RNA polymerase. This subunit contains the DNA binding site. Competitive-like inhibition by captan versus UTP led to the conclusion that captan also blocked the NTP binding site. In support of this conclusion, [14C]captan was observed to bind to the beta subunit which contains the NTP binding site. Whereas, preincubation of RNA polymerase with both DNA and NTPs prevented captan inhibition, preincubation with either DNA or NTPs alone was insufficient to protect the enzyme from the action of captan. Furthermore, the interaction of [14C]captan with the beta and beta' subunits was not prevented by a similar preincubation. Captan also bound, to a lesser extent, to the alpha and sigma subunits. Therefore, captan binding appears to involve interaction with RNA polymerase at sites in addition to those for DNA and NTP; however, this action does not inhibit the polymerase activity.

  5. Inhibition of RNA Polymerase II Transcription in Human Cells by Synthetic DNA-Binding Ligands

    Science.gov (United States)

    Dickinson, Liliane A.; Gulizia, Richard J.; Trauger, John W.; Baird, Eldon E.; Mosier, Donald E.; Gottesfeld, Joel M.; Dervan, Peter B.

    1998-10-01

    Sequence-specific DNA-binding small molecules that can permeate human cells potentially could regulate transcription of specific genes. Multiple cellular DNA-binding transcription factors are required by HIV type 1 for RNA synthesis. Two pyrrole--imidazole polyamides were designed to bind DNA sequences immediately adjacent to binding sites for the transcription factors Ets-1, lymphoid-enhancer binding factor 1, and TATA-box binding protein. These synthetic ligands specifically inhibit DNA-binding of each transcription factor and HIV type 1 transcription in cell-free assays. When used in combination, the polyamides inhibit virus replication by >99% in isolated human peripheral blood lymphocytes, with no detectable cell toxicity. The ability of small molecules to target predetermined DNA sequences located with RNA polymerase II promoters suggests a general approach for regulation of gene expression, as well as a mechanism for the inhibition of viral replication.

  6. The VP3 factor from viruses of Birnaviridae family suppresses RNA silencing by binding both long and small RNA duplexes.

    Science.gov (United States)

    Valli, Adrian; Busnadiego, Idoia; Maliogka, Varvara; Ferrero, Diego; Castón, José R; Rodríguez, José Francisco; García, Juan Antonio

    2012-01-01

    RNA silencing is directly involved in antiviral defense in a wide variety of eukaryotic organisms, including plants, fungi, invertebrates, and presumably vertebrate animals. The study of RNA silencing-mediated antiviral defences in vertebrates is hampered by the overlap with other antiviral mechanisms; thus, heterologous systems are often used to study the interplay between RNA silencing and vertebrate-infecting viruses. In this report we show that the VP3 protein of the avian birnavirus Infectious bursal disease virus (IBDV) displays, in addition to its capacity to bind long double-stranded RNA, the ability to interact with double-stranded small RNA molecules. We also demonstrate that IBDV VP3 prevents the silencing mediated degradation of a reporter mRNA, and that this silencing suppression activity depends on its RNA binding ability. Furthermore, we find that the anti-silencing activity of IBDV VP3 is shared with the homologous proteins expressed by both insect- and fish-infecting birnaviruses. Finally, we show that IBDV VP3 can functionally replace the well-characterized HCPro silencing suppressor of Plum pox virus, a potyvirus that is unable to infect plants in the absence of an active silencing suppressor. Altogether, our results support the idea that VP3 protects the viral genome from host sentinels, including those of the RNA silencing machinery.

  7. The VP3 factor from viruses of Birnaviridae family suppresses RNA silencing by binding both long and small RNA duplexes.

    Directory of Open Access Journals (Sweden)

    Adrian Valli

    Full Text Available RNA silencing is directly involved in antiviral defense in a wide variety of eukaryotic organisms, including plants, fungi, invertebrates, and presumably vertebrate animals. The study of RNA silencing-mediated antiviral defences in vertebrates is hampered by the overlap with other antiviral mechanisms; thus, heterologous systems are often used to study the interplay between RNA silencing and vertebrate-infecting viruses. In this report we show that the VP3 protein of the avian birnavirus Infectious bursal disease virus (IBDV displays, in addition to its capacity to bind long double-stranded RNA, the ability to interact with double-stranded small RNA molecules. We also demonstrate that IBDV VP3 prevents the silencing mediated degradation of a reporter mRNA, and that this silencing suppression activity depends on its RNA binding ability. Furthermore, we find that the anti-silencing activity of IBDV VP3 is shared with the homologous proteins expressed by both insect- and fish-infecting birnaviruses. Finally, we show that IBDV VP3 can functionally replace the well-characterized HCPro silencing suppressor of Plum pox virus, a potyvirus that is unable to infect plants in the absence of an active silencing suppressor. Altogether, our results support the idea that VP3 protects the viral genome from host sentinels, including those of the RNA silencing machinery.

  8. THUMP--a predicted RNA-binding domain shared by 4-thiouridine, pseudouridine synthases and RNA methylases.

    Science.gov (United States)

    Aravind, L; Koonin, E V

    2001-04-01

    Sequence profile searches were used to identify an ancient domain in ThiI-like thiouridine synthases, conserved RNA methylases, archaeal pseudouridine synthases and several uncharacterized proteins. We predict that this domain is an RNA-binding domain that adopts an alpha/beta fold similar to that found in the C-terminal domain of translation initiation factor 3 and ribosomal protein S8.

  9. Identification of lncRNA MEG3 Binding Protein Using MS2-Tagged RNA Affinity Purification and Mass Spectrometry.

    Science.gov (United States)

    Liu, Shanshan; Zhu, Juanjuan; Jiang, Taifeng; Zhong, Yiran; Tie, Yi; Wu, Yongge; Zheng, Xiaofei; Jin, Yinghua; Fu, Hanjiang

    2015-08-01

    Long noncoding RNAs (lncRNAs) are nonprotein coding transcripts longer than 200 nucleotides. Recently in mammals, thousands of long noncoding RNAs have been identified and studied as key molecular players in different biological processes with protein complexes. As a long noncoding RNA, maternally expressed gene 3 (MEG3) plays an important role in many cellular processes. However, the mechanism underlying MEG3 regulatory effects remains enigmatic. By using the specific interaction between MS2 coat protein and MS2 RNA hairpin, we developed a method (MS2-tagged RNA affinity purification and mass spectrometry (MTRAP-MS)) to identify proteins that interact with MEG3. Mass spectrometry and gene ontology (GO) analysis showed that MEG3 binding proteins possess nucleotide binding properties and take part in transport, translation, and other biological processes. In addition, interleukin enhancer binding factor 3 (ILF3) and poly(A) binding protein, cytoplasmic 3 (PABPC3) were validated for their interaction with MEG3. These findings indicate that the newly developed method can effectively enrich lncRNA binding proteins and provides a strong basis for studying MEG3 functions.

  10. STarMir Tools for Prediction of microRNA binding sites

    Science.gov (United States)

    Kanoria, Shaveta; Rennie, William; Liu, Chaochun; Carmack, C. Steven; Lu, Jun; Ding, Ye

    2017-01-01

    MicroRNAs (miRNAs) are a class of endogenous short non-coding RNAs that regulate gene expression by targeting messenger RNAs (mRNAs), which results in translational repression and/or mRNA degradation. As regulatory molecules, miRNAs are involved in many mammalian biological processes and also in the manifestation of certain human diseases. As miRNAs play central role in the regulation of gene expression, understanding miRNA-binding patterns is essential to gain an insight of miRNA mediated gene regulation and also holds promise for therapeutic applications. Computational prediction of miRNA binding sites on target mRNAs facilitates experimental investigation of miRNA functions. This chapter provides protocols for using the STarMir web server for improved predictions of miRNA binding sites on a target mRNA. As an application module of the Sfold RNA package, the current version of STarMir is an implementation of logistic prediction models developed with high throughput miRNA binding data from crosslinking immuno-precipitation (CLIP) studies. The models incorporated comprehensive thermodynamic, structural and sequence features, and were found to make improved predictions of both seed and seedless sites, in comparison to the established algorithms [1]. Their broad applicability was indicated by their good performance in cross-species validation. STarMir is freely available at http://sfold.wadsworth.org/starmir.html PMID:27665594

  11. Using RNase sequence specificity to refine the identification of RNA-protein binding regions

    OpenAIRE

    Wang Xinguo; Li Lang; Shen Changyu; Wang Guohua; Wang Xin; Mooney Sean D; Edenberg Howard J; Sanford Jeremy R; Liu Yunlong

    2008-01-01

    Abstract Massively parallel pyrosequencing is a high-throughput technology that can sequence hundreds of thousands of DNA/RNA fragments in a single experiment. Combining it with immunoprecipitation-based biochemical assays, such as cross-linking immunoprecipitation (CLIP), provides a genome-wide method to detect the sites at which proteins bind DNA or RNA. In a CLIP-pyrosequencing experiment, the resolutions of the detected protein binding regions are partially determined by the length of the...

  12. Conformational-Dependent and Independent RNA Binding to the Fragile X Mental Retardation Protein

    Directory of Open Access Journals (Sweden)

    Xin Yan

    2011-01-01

    Full Text Available The interaction between the fragile X mental retardation protein (FMRP and BC1 RNA has been the subject of controversy. We probed the parameters of RNA binding to FMRP in several ways. Nondenaturing agarose gel analysis showed that BC1 RNA transcripts produced by in vitro transcription contain a population of conformers, which can be modulated by preannealing. Accordingly, FMRP differentially binds to the annealed and unannealed conformer populations. Using partial RNase digestion, we demonstrate that annealed BC1 RNA contains a unique conformer that FMRP likely binds. We further demonstrate that this interaction is 100-fold weaker than that the binding of eEF-1A mRNA and FMRP, and that preannealing is not a general requirement for FMRP's interaction with RNA. In addition, binding does not require the N-terminal 204 amino acids of FMRP, methylated arginine residues and can be recapitulated by both fragile X paralogs. Altogether, our data continue to support a model in which BC1 RNA functions independently of FMRP.

  13. UPF201 Archaeal Specific Family Members Reveal Structural Similarity to RNA-Binding Proteins but Low Likelyhood for RNA-Binding Function

    Energy Technology Data Exchange (ETDEWEB)

    Rao, K.; Burley, S; Swaminathan, S

    2008-01-01

    We have determined X-ray crystal structures of four members of an archaeal specific family of proteins of unknown function (UPF0201; Pfam classification: DUF54) to advance our understanding of the genetic repertoire of archaea. Despite low pairwise amino acid sequence identities (10-40%) and the absence of conserved sequence motifs, the three-dimensional structures of these proteins are remarkably similar to one another. Their common polypeptide chain fold, encompassing a five-stranded antiparallel {beta}-sheet and five {alpha}-helices, proved to be quite unexpectedly similar to that of the RRM-type RNA-binding domain of the ribosomal L5 protein, which is responsible for binding the 5S- rRNA. Structure-based sequence alignments enabled construction of a phylogenetic tree relating UPF0201 family members to L5 ribosomal proteins and other structurally similar RNA binding proteins, thereby expanding our understanding of the evolutionary purview of the RRM superfamily. Analyses of the surfaces of these newly determined UPF0201 structures suggest that they probably do not function as RNA binding proteins, and that this domain specific family of proteins has acquired a novel function in archaebacteria, which awaits experimental elucidation.

  14. UPF201 Archaeal Specific Family Members Reveals Structural Similarity to RNA-Binding Proteins but Low Likelihood for RNA-Binding Function

    Energy Technology Data Exchange (ETDEWEB)

    Rao, K.N.; Swaminathan, S.; Burley, S. K.

    2008-12-11

    We have determined X-ray crystal structures of four members of an archaeal specific family of proteins of unknown function (UPF0201; Pfam classification: DUF54) to advance our understanding of the genetic repertoire of archaea. Despite low pairwise amino acid sequence identities (10-40%) and the absence of conserved sequence motifs, the three-dimensional structures of these proteins are remarkably similar to one another. Their common polypeptide chain fold, encompassing a five-stranded antiparallel {beta}-sheet and five {alpha}-helices, proved to be quite unexpectedly similar to that of the RRM-type RNA-binding domain of the ribosomal L5 protein, which is responsible for binding the 5S- rRNA. Structure-based sequence alignments enabled construction of a phylogenetic tree relating UPF0201 family members to L5 ribosomal proteins and other structurally similar RNA binding proteins, thereby expanding our understanding of the evolutionary purview of the RRM superfamily. Analyses of the surfaces of these newly determined UPF0201 structures suggest that they probably do not function as RNA binding proteins, and that this domain specific family of proteins has acquired a novel function in archaebacteria, which awaits experimental elucidation.

  15. UPF201 archaeal specific family members reveal structural similarity to RNA-binding proteins but low likelihood for RNA-binding function.

    Directory of Open Access Journals (Sweden)

    Krishnamurthy N Rao

    Full Text Available We have determined X-ray crystal structures of four members of an archaeal specific family of proteins of unknown function (UPF0201; Pfam classification: DUF54 to advance our understanding of the genetic repertoire of archaea. Despite low pairwise amino acid sequence identities (10-40% and the absence of conserved sequence motifs, the three-dimensional structures of these proteins are remarkably similar to one another. Their common polypeptide chain fold, encompassing a five-stranded antiparallel beta-sheet and five alpha-helices, proved to be quite unexpectedly similar to that of the RRM-type RNA-binding domain of the ribosomal L5 protein, which is responsible for binding the 5S- rRNA. Structure-based sequence alignments enabled construction of a phylogenetic tree relating UPF0201 family members to L5 ribosomal proteins and other structurally similar RNA binding proteins, thereby expanding our understanding of the evolutionary purview of the RRM superfamily. Analyses of the surfaces of these newly determined UPF0201 structures suggest that they probably do not function as RNA binding proteins, and that this domain specific family of proteins has acquired a novel function in archaebacteria, which awaits experimental elucidation.

  16. H19 RNA binds four molecules of insulin-like growth factor II mRNA-binding protein

    DEFF Research Database (Denmark)

    Runge, Steffen; Nielsen, Finn Cilius; Nielsen, Jacob;

    2000-01-01

    attachment sites are clustered within a 700-nucleotide segment encoded by exons 4 and 5. This 3'-terminal segment targets H19 RNA to lamellipodia and perinuclear regions in dispersed fibroblasts where IMP is also localized. The results suggest that IMP participates in H19 RNA localization and provides a link...

  17. Structural Basis for Telomerase Catalytic Subunit TERT Binding to RNA Template and Telomeric DNA

    Energy Technology Data Exchange (ETDEWEB)

    Mitchell, M.; Gillis, A; Futahashi, M; Fujiwara, H; Skordalakes, E

    2010-01-01

    Telomerase is a specialized DNA polymerase that extends the 3{prime} ends of eukaryotic linear chromosomes, a process required for genomic stability and cell viability. Here we present the crystal structure of the active Tribolium castaneum telomerase catalytic subunit, TERT, bound to an RNA-DNA hairpin designed to resemble the putative RNA-templating region and telomeric DNA. The RNA-DNA hybrid adopts a helical structure, docked in the interior cavity of the TERT ring. Contacts between the RNA template and motifs 2 and B{prime} position the solvent-accessible RNA bases close to the enzyme active site for nucleotide binding and selectivity. Nucleic acid binding induces rigid TERT conformational changes to form a tight catalytic complex. Overall, TERT-RNA template and TERT-telomeric DNA associations are remarkably similar to those observed for retroviral reverse transcriptases, suggesting common mechanistic aspects of DNA replication between the two families of enzymes.

  18. Interacting protein partners of Arabidopsis RNA binding protein AtRBP45b

    Science.gov (United States)

    RNA binding proteins (RBPs) are important players in post-transcriptional gene regulation and shown to play an important role in normal development and in response to environmental perturbations. Arabidopsis RBP, AtRBP45b with triple RNA recognition motifs (RRMs) have are closely related to the yeas...

  19. Mapping Escherichia coli elongation factor Tu residues involved in binding of aminoacyl-tRNA

    DEFF Research Database (Denmark)

    Wiborg, Ove; Andersen, C; Knudsen, Charlotte Rohde

    1996-01-01

    -tRNA, which suggested an important role of Lys-89 and Asn-90 in tRNA binding. Furthermore, our results indicate helix B to be an important target site for nucleotide exchange factor EF-Ts. Also the mutants His-66 to Ala and His-118 to either Ala or Glu were characterized in an in vitro translation assay...

  20. SCA31 Flies Perform in a Balancing Act between RAN Translation and RNA-Binding Proteins.

    Science.gov (United States)

    Jackson, George R

    2017-04-05

    In this issue of Neuron, Ishiguro et al. (2017) explore the toxicity of RAN translation in spinocerebellar ataxia 31. Using a Drosophila model, the authors demonstrate that TDP-43 and other RNA-binding proteins act as chaperones to regulate the formation of toxic RNA aggregates.

  1. Binding of DNA-binding alkaloids berberine and palmatine to tRNA and comparison to ethidium: Spectroscopic and molecular modeling studies

    Science.gov (United States)

    Islam, Md. Maidul; Pandya, Prateek; Chowdhury, Sebanti Roy; Kumar, Surat; Kumar, Gopinatha Suresh

    2008-11-01

    The interaction of two natural protoberberine plant alkaloids berberine and palmatine with tRNA phe was studied using various biophysical techniques and molecular modeling and the data were compared with the binding of the classical DNA intercalator, ethidium. Circular dichroic studies revealed that the tRNA conformation was moderately perturbed on binding of the alkaloids. The cooperative binding of both the alkaloids and ethidium to tRNA was revealed from absorbance and fluorescence studies. Fluorescence quenching studies advanced a conclusion that while berberine and palmatine are partially intercalated, ethidium is fully intercalated on the tRNA molecule. The binding of the alkaloids as well as ethidium stabilized the tRNA melting, and the binding constant evaluated from the averaged optical melting temperature data was in agreement with fluorescence spectral-binding data. Differential scanning calorimetry revealed that the tRNA melting showed three close transitions that were affected on binding of these small molecules. Molecular docking calculations performed showed the preferred regions of binding of these small molecules on the tRNA. Taken together, the results suggest that the binding of the alkaloids berberine and palmatine on the tRNA structure appears to be mostly by partial intercalation while ethidium intercalates fully on the tRNA. These results further advance our knowledge on the molecular aspects on the interaction of these alkaloids to tRNA.

  2. Branched peptide boronic acids (BPBAs): a novel mode of binding towards RNA.

    Science.gov (United States)

    Zhang, Wenyu; Bryson, David I; Crumpton, Jason B; Wynn, Jessica; Santos, Webster L

    2013-03-25

    We report branched peptide boronic acids (BPBAs) that bind to RRE IIB from an on-bead high-throughput screening of a 3.3.4-library (46 656 compounds). We demonstrate that boronic acids are tunable moieties that afford a novel binding mode towards RNA.

  3. RNA-binding protein hnRNPLL as a critical regulator of lymphocyte homeostasis and differentiation.

    Science.gov (United States)

    Chang, Xing

    2016-05-01

    RNA-binding proteins orchestrate posttranscriptional regulation of gene expression, such as messenger RNA (mRNA) splicing, RNA stability regulation, and translation regulation. Heterogeneous nuclear RNA-binding proteins (hnRNPs) refer to a collection of unrelated RNA-binding proteins predominantly located in the nucleus (Han et al. Biochem J 2010, 430:379-392). Although canonical functions of hnRNPs are to promote pre-mRNA splicing, they are involved in all the processes of RNA metabolism through recognizing specific cis-elements on RNA (Dreyfuss et al. Annu Rev Biochem 1993, 62:289-321; Huelga et al. Cell Rep 2012, 1:167-178; Krecic and Swanson. Curr Opin Cell Biol 1999, 11:363-371). Heterogeneous nuclear RNA-binding protein L like (hnRNPLL) is a tissue-specific hnRNP, which was identified as a regulator of CD45RA to CD45RO switching during memory T-cell development (Oberdoerffer et al. Science 2008, 321:686-691; Topp et al. RNA 2008, 14:2038-2049; Wu et al. Immunity 2008, 29:863-875). Since then, hnRNPLL has emerged as a critical regulator of lymphocyte homeostasis and terminal differentiation, controlling alternative splicing or expression of critical genes for the lymphocytes development (Wu et al. Immunity 2008, 29:863-875; Chang et al. Proc Natl Acad Sci USA 2015, 112:E1888-E1897). This review will summarize recent advances in understanding the functions of hnRNPLL, focusing on its biochemical functions and physiological roles in lymphocyte differentiation and homeostasis. WIREs RNA 2016, 7:295-302. doi: 10.1002/wrna.1335 For further resources related to this article, please visit the WIREs website.

  4. Transcription factor binding sites are highly enriched within microRNA precursor sequences

    Directory of Open Access Journals (Sweden)

    Piriyapongsa Jittima

    2011-12-01

    Full Text Available Abstract Background Transcription factors are thought to regulate the transcription of microRNA genes in a manner similar to that of protein-coding genes; that is, by binding to conventional transcription factor binding site DNA sequences located in or near promoter regions that lie upstream of the microRNA genes. However, in the course of analyzing the genomics of human microRNA genes, we noticed that annotated transcription factor binding sites commonly lie within 70- to 110-nt long microRNA small hairpin precursor sequences. Results We report that about 45% of all human small hairpin microRNA (pre-miR sequences contain at least one predicted transcription factor binding site motif that is conserved across human, mouse and rat, and this rises to over 75% if one excludes primate-specific pre-miRs. The association is robust and has extremely strong statistical significance; it affects both intergenic and intronic pre-miRs and both isolated and clustered microRNA genes. We also confirmed and extended this finding using a separate analysis that examined all human pre-miR sequences regardless of conservation across species. Conclusions The transcription factor binding sites localized within small hairpin microRNA precursor sequences may possibly regulate their transcription. Transcription factors may also possibly bind directly to nascent primary microRNA gene transcripts or small hairpin microRNA precursors and regulate their processing. Reviewers This article was reviewed by Guillaume Bourque (nominated by Jerzy Jurka, Dmitri Pervouchine (nominated by Mikhail Gelfand, and Yuriy Gusev.

  5. Structure of the second RRM domain of Nrd1, a fission yeast MAPK target RNA binding protein, and implication for its RNA recognition and regulation

    Energy Technology Data Exchange (ETDEWEB)

    Kobayashi, Ayaho; Kanaba, Teppei [Graduate School of Science and Engineering, Tokyo Metropolitan University, Minamiosawa 1-1, Hachioji 192-0397 (Japan); Satoh, Ryosuke [Institute of Microbial Chemistry, 3-14-23 Kamiosaki, Shinagawa-ku 141-0021, Tokyo (Japan); Fujiwara, Toshinobu [Institute of Microbial Chemistry, 3-14-23 Kamiosaki, Shinagawa-ku 141-0021, Tokyo (Japan); Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku,Nagoya 467-8603 (Japan); Ito, Yutaka [Graduate School of Science and Engineering, Tokyo Metropolitan University, Minamiosawa 1-1, Hachioji 192-0397 (Japan); Sugiura, Reiko [Laboratory of Molecular Pharmacogenomics, School of Pharmaceutical Sciences, Kinki University, 3-4-1 Kowakae, Higashi-Osaka 577-8502 (Japan); Mishima, Masaki, E-mail: mishima-masaki@tmu.ac.jp [Graduate School of Science and Engineering, Tokyo Metropolitan University, Minamiosawa 1-1, Hachioji 192-0397 (Japan)

    2013-07-19

    Highlights: •Solution structure of the second RRM of Nrd1 was determined. •RNA binding site of the second RRM was estimated. •Regulatory mechanism of RNA binding by phosphorylation is discussed. -- Abstract: Negative regulator of differentiation 1 (Nrd1) is known as a negative regulator of sexual differentiation in fission yeast. Recently, it has been revealed that Nrd1 also regulates cytokinesis, in which physical separation of the cell is achieved by a contractile ring comprising many proteins including actin and myosin. Cdc4, a myosin II light chain, is known to be required for cytokinesis. Nrd1 binds and stabilizes Cdc4 mRNA, and thereby suppressing the cytokinesis defects of the cdc4 mutants. Interestingly, Pmk1 MAPK phosphorylates Nrd1, resulting in markedly reduced RNA binding activity. Furthermore, Nrd1 localizes to stress granules in response to various stresses, and Pmk1 phosphorylation enhances the localization. Nrd1 consists of four RRM domains, although the mechanism by which Pmk1 regulates the RNA binding activity of Nrd1 is unknown. In an effort to delineate the relationship between Nrd1 structure and function, we prepared each RNA binding domain of Nrd1 and examined RNA binding to chemically synthesized oligo RNA using NMR. The structure of the second RRM domain of Nrd1 was determined and the RNA binding site on the second RRM domain was mapped by NMR. A plausible mechanism pertaining to the regulation of RNA binding activity by phosphorylation is also discussed.

  6. Is tRNA binding or tRNA mimicry mandatory for translation factors?

    Science.gov (United States)

    Kristensen, Ole; Laurberg, Martin; Liljas, Anders; Selmer, Maria

    2002-02-01

    tRNA is the adaptor in the translation process. The ribosome has three sites for tRNA, the A-, P-, and E-sites. The tRNAs bridge between the ribosomal subunits with the decoding site and the mRNA on the small or 30S subunit and the peptidyl transfer site on the large or 50S subunit. The possibility that translation release factors could mimic tRNA has been discussed for a long time, since their function is very similar to that of tRNA. They identify stop codons of the mRNA presented in the decoding site and hydrolyse the nascent peptide from the peptidyl tRNA in the peptidyl transfer site. The structures of eubacterial release factors are not yet known, and the first example of tRNA mimicry was discovered when elongation factor G (EF-G) was found to have a closely similar shape to a complex of elongation factor Tu (EF-Tu) with aminoacyl-tRNA. An even closer imitation of the tRNA shape is seen in ribosome recycling factor (RRF). The number of proteins mimicking tRNA is rapidly increasing. This primarily concerns translation factors. It is now evident that in some sense they are either tRNA mimics, GTPases or possibly both.

  7. 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

    IP-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. 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...

  8. CUGBP1 and MBNL1 preferentially bind to 3 ' UTRs and facilitate mRNA decay

    DEFF Research Database (Denmark)

    Masuda, A.; Andersen, H. S.; Doktor, T. K.

    2012-01-01

    CUGBP1 and MBNL1 are developmentally regulated RNA-binding proteins that are causally associated with myotonic dystrophy type 1. We globally determined the in vivo RNA-binding sites of CUGBP1 and MBNL1. Interestingly, CUGBP1 and MBNL1 are both preferentially bound to 39 UTRs. Analysis of CUGBP1...... that CUGBP1 and MBNL1 regulate alternative splicing. Screening by exon array and validation by RT-PCR revealed position dependence of CUGBP1- and MBNL1-binding sites on the resulting alternative splicing pattern. This study suggests that regulation of CUGBP1 and MBNL1 is essential for accurate control...

  9. CLIP Identifies Nova-Regulated RNA Networks in the Brain

    Science.gov (United States)

    Ule, Jernej; Jensen, Kirk B.; Ruggiu, Matteo; Mele, Aldo; Ule, Aljaž; Darnell, Robert B.

    2003-11-01

    Nova proteins are neuron-specific antigens targeted in paraneoplastic opsoclonus myoclonus ataxia (POMA), an autoimmune neurologic disease characterized by abnormal motor inhibition. Nova proteins regulate neuronal pre-messenger RNA splicing by directly binding to RNA. To identify Nova RNA targets, we developed a method to purify protein-RNA complexes from mouse brain with the use of ultraviolet cross-linking and immunoprecipitation (CLIP). Thirty-four transcripts were identified multiple times by Nova CLIP. Three-quarters of these encode proteins that function at the neuronal synapse, and one-third are involved in neuronal inhibition. Splicing targets confirmed in Nova-/- mice include c-Jun N-terminal kinase 2, neogenin, and gephyrin; the latter encodes a protein that clusters inhibitory γ-aminobutyric acid and glycine receptors, two previously identified Nova splicing targets. Thus, CLIP reveals that Nova coordinately regulates a biologically coherent set of RNAs encoding multiple components of the inhibitory synapse, an observation that may relate to the cause of abnormal motor inhibition in POMA.

  10. Evolution of the B-Block Binding Subunit of TFIIIC That Binds to the Internal Promoter for RNA Polymerase III

    Directory of Open Access Journals (Sweden)

    Sachiko Matsutani

    2014-01-01

    Full Text Available Eukaryotic RNA polymerase III transcribes tRNA genes, and this requires the transcription factor TFIIIC. Promoters are within genes, with which the B-block binding subunit of TFIIIC associates to initiate transcription. The binding subunits are more than 1000 amino acids in length in various eukaryotic species. There are four regions with conserved sequence similarities in the subunits. The helix-turn-helix motif is included in one of these regions and has been characterized as the B-block_TFIIIC family in the Pfam database. In the NCBI and EMBL translated protein databases, there are archaeal proteins (approximately 100 amino acids in length referred to as B-block binding subunits. Most of them contain a B-block_TFIIIC motif. DELTA-BLAST searches using these archaeal proteins as queries showed significant multiple blast hits for many eukaryotic B-block binding subunits on the same proteins. This result suggests that eukaryotic B-block binding subunits were constituted by repeating a small unit of B-block_TFIIIC over a long evolutionary period. Bacterial proteins have also been annotated as B-block binding subunits in the databases. Here, some of them were confirmed to have significant similarities to B-block_TFIIIC. These results may imply that part of the RNAP III transcription machinery existed in the common ancestry of prokaryotes and eukaryotes.

  11. The CRM domain: an RNA binding module derived from an ancient ribosome-associated protein.

    Science.gov (United States)

    Barkan, Alice; Klipcan, Larik; Ostersetzer, Oren; Kawamura, Tetsuya; Asakura, Yukari; Watkins, Kenneth P

    2007-01-01

    The CRS1-YhbY domain (also called the CRM domain) is represented as a stand-alone protein in Archaea and Bacteria, and in a family of single- and multidomain proteins in plants. The function of this domain is unknown, but structural data and the presence of the domain in several proteins known to interact with RNA have led to the proposal that it binds RNA. Here we describe a phylogenetic analysis of the domain, its incorporation into diverse proteins in plants, and biochemical properties of a prokaryotic and eukaryotic representative of the domain family. We show that a bacterial member of the family, Escherichia coli YhbY, is associated with pre-50S ribosomal subunits, suggesting that YhbY functions in ribosome assembly. GFP fused to a single-domain CRM protein from maize localizes to the nucleolus, suggesting that an analogous activity may have been retained in plants. We show further that an isolated maize CRM domain has RNA binding activity in vitro, and that a small motif shared with KH RNA binding domains, a conserved "GxxG" loop, contributes to its RNA binding activity. These and other results suggest that the CRM domain evolved in the context of ribosome function prior to the divergence of Archaea and Bacteria, that this function has been maintained in extant prokaryotes, and that the domain was recruited to serve as an RNA binding module during the evolution of plant genomes.

  12. Having it both ways: transcription factors that bind DNA and RNA.

    Science.gov (United States)

    Cassiday, Laura A; Maher, L James

    2002-10-01

    Multifunctional proteins challenge the conventional 'one protein-one function' paradigm. Here we note apparent multifunctional proteins with nucleic acid partners, tabulating eight examples. We then focus on eight additional cases of transcription factors that bind double-stranded DNA with sequence specificity, but that also appear to lead alternative lives as RNA-binding proteins. Exemplified by the prototypic Xenopus TFIIIA protein, and more recently by mammalian p53, this list of transcription factors includes WT-1, TRA-1, bicoid, the bacterial sigma(70) subunit, STAT1 and TLS/FUS. The existence of transcription factors that bind both DNA and RNA provides an interesting puzzle. Little is known concerning the biological roles of these alternative protein-nucleic acid interactions, and even less is known concerning the structural basis for dual nucleic acid specificity. We discuss how these natural examples have motivated us to identify artificial RNA sequences that competitively inhibit a DNA-binding transcription factor not known to have a natural RNA partner. The identification of such RNAs raises the possibility that RNA binding by DNA-binding proteins is more common than currently appreciated.

  13. False positive RNA binding activities after Ni-affinity purification from Escherichia coli.

    Science.gov (United States)

    Milojevic, Tetyana; Sonnleitner, Elisabeth; Romeo, Alessandra; Djinović-Carugo, Kristina; Bläsi, Udo

    2013-06-01

    A His-tag is often added by means of recombinant DNA technology to a heterologous protein of interest, which is then over-produced in Escherchia coli and purified by one-step immobilized metal-affinity chromatography (IMAC). Owing to the presence of 24 histidines at the C-termini of the hexameric E. coli RNA chaperone Hfq, the protein co-purifies with His-tagged proteins of interest. As Hfq can bind to distinct RNA substrates with high affinity, its presence can obscure studies performed with (putative) RNA binding activities purified by IMAC. Here, we present results for a seemingly positive RNA-binding activity, exemplifying that false-positive results can be avoided if the protein of interest is either subjected to further purification step(s) or produced in an E. coli hfq- strain.

  14. Lin28a uses distinct mechanisms of binding to RNA and affects miRNA levels positively and negatively.

    Science.gov (United States)

    Nowak, Jakub Stanislaw; Hobor, Fruzsina; Downie Ruiz Velasco, Angela; Choudhury, Nila Roy; Heikel, Gregory; Kerr, Alastair; Ramos, Andres; Michlewski, Gracjan

    2017-03-01

    Lin28a inhibits the biogenesis of let-7 miRNAs by triggering the polyuridylation and degradation of their precursors by terminal uridylyltransferases TUT4/7 and 3'-5' exoribonuclease Dis3l2, respectively. Previously, we showed that Lin28a also controls the production of neuro-specific miRNA-9 via a polyuridylation-independent mechanism. Here we reveal that the sequences and structural characteristics of pre-let-7 and pre-miRNA-9 are eliciting two distinct modes of binding to Lin28a. We present evidence that Dis3l2 controls miRNA-9 production. Finally, we show that the constitutive expression of untagged Lin28a during neuronal differentiation in vitro positively and negatively affects numerous other miRNAs. Our findings shed light on the role of Lin28a in differentiating cells and on the ways in which one RNA-binding protein can perform multiple roles in the regulation of RNA processing. © 2017 Nowak et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  15. Control of signaling in a MAP-kinase pathway by an RNA-binding protein.

    Directory of Open Access Journals (Sweden)

    Susanne Prinz

    Full Text Available Signaling-protein mRNAs tend to have long untranslated regions (UTRs containing binding sites for RNA-binding proteins regulating gene expression. Here we show that a PUF-family RNA-binding protein, Mpt5, represses the yeast MAP-kinase pathway controlling differentiation to the filamentous form. Mpt5 represses the protein levels of two pathway components, the Ste7 MAP-kinase kinase and the Tec1 transcriptional activator, and negatively regulates the kinase activity of the Kss1 MAP kinase. Moreover, Mpt5 specifically inhibits the output of the pathway in the absence of stimuli, and thereby prevents inappropriate cell differentiation. The results provide an example of what may be a genome-scale level of regulation at the interface of signaling networks and protein-RNA binding networks.

  16. Dynamic mechanisms for pre-miRNA binding and export by Exportin-5.

    Science.gov (United States)

    Wang, Xia; Xu, Xue; Ma, Zhi; Huo, Yingqiu; Xiao, Zhengtao; Li, Yan; Wang, Yonghua

    2011-08-01

    The biogenesis and function of mature microRNAs (miRNAs) is dependent on the nuclear export of miRNA precursors (pre-miRNA) by Exportin-5 (Exp5). To characterize the molecular mechanisms of how pre-miRNA is recognized and transported by Exp5, we have performed 21 molecular dynamic (MD) simulations of RNA-bound Exp5 (Exp5-RanGTP-premiRNA, Exp5-RanGDP-premiRNA, Exp5-premiRNA), RNA-unbound Exp5 (Exp5-RanGTP, Exp5-RanGDP, apo-Exp5), and pre-miRNA. Our simulations with standard MD, steered molecular dynamics (SMD), and energy analysis have shown that (1) Free Exp5 undergoes extensive opening motion, and in this way facilitates the RanGTP binding. (2) RanGTP efficiently regulates the association/dissociation of pre-miRNA to its complex by inducing conformational changes in the HEAT-repeat helix stacking of Exp5. (3) The GTP hydrolysis prevents Ran from rebinding to Exp5 by regulating the hydrophobic interfaces and salt bridges between Ran and Exp5. (4) The transition from the A'-form to the A-form of the pre-miRNA modulates the structural complementarities between the protein and the pre-miRNA, thus promoting efficient assembly of the complex. (5) The base-flipping process (from the closed to the fully flipped state) of the 2-nt 3' overhang is a prerequisite for the pre-miRNA recognition by Exp5, which occurs in a sequence-independent manner as evidenced by the fact that different 2-nt 3' overhangs bind to Exp5 in essentially the same way. And finally, a plausible mechanism of the pre-miRNA export cycle has been proposed explaining how the protein-protein and protein-RNA interactions are coordinated in physiological conditions.

  17. Three-dimensional model of a selective theophylline-binding RNA molecule

    Energy Technology Data Exchange (ETDEWEB)

    Tung, Chang-Shung; Oprea, T.I.; Hummer, G.; Garcia, A.E.

    1995-07-01

    We propose a three-dimensional (3D) model for an RNA molecule that selectively binds theophylline but not caffeine. This RNA, which was found using SELEX [Jenison, R.D., et al., Science (1994) 263:1425] is 10,000 times more specific for theophylline (Kd=320 nM) than for caffeine (Kd=3.5 mM), although the two ligands are identical except for a methyl group substituted at N7 (present only in caffeine). The binding affinity for ten xanthine-based ligands was used to derive a Comparative Molecular Field Analysis (CoMFA) model (R{sup 2} = 0.93 for 3 components, with cross-validated R{sup 2} of 0.73), using the SYBYL and GOLPE programs. A pharmacophoric map was generated to locate steric and electrostatic interactions between theophylline and the RNA binding site. This information was used to identify putative functional groups of the binding pocket and to generate distance constraints. Based on a model for the secondary structure (Jenison et al., idem), the 3D structure of this RNA was then generated using the following method: each helical region of the RNA molecule was treated as a rigid body; single-stranded loops with specific end-to-end distances were generated. The structures of RNA-xanthine complexes were studied using a modified Monte Carlo algorithm. The detailed structure of an RNA-ligand complex model, as well as possible explanations for the theophylline selectivity will be discussed.

  18. High-throughput prediction of RNA, DNA and protein binding regions mediated by intrinsic disorder.

    Science.gov (United States)

    Peng, Zhenling; Kurgan, Lukasz

    2015-10-15

    Intrinsically disordered proteins and regions (IDPs and IDRs) lack stable 3D structure under physiological conditions in-vitro, are common in eukaryotes, and facilitate interactions with RNA, DNA and proteins. Current methods for prediction of IDPs and IDRs do not provide insights into their functions, except for a handful of methods that address predictions of protein-binding regions. We report first-of-its-kind computational method DisoRDPbind for high-throughput prediction of RNA, DNA and protein binding residues located in IDRs from protein sequences. DisoRDPbind is implemented using a runtime-efficient multi-layered design that utilizes information extracted from physiochemical properties of amino acids, sequence complexity, putative secondary structure and disorder and sequence alignment. Empirical tests demonstrate that it provides accurate predictions that are competitive with other predictors of disorder-mediated protein binding regions and complementary to the methods that predict RNA- and DNA-binding residues annotated based on crystal structures. Application in Homo sapiens, Mus musculus, Caenorhabditis elegans and Drosophila melanogaster proteomes reveals that RNA- and DNA-binding proteins predicted by DisoRDPbind complement and overlap with the corresponding known binding proteins collected from several sources. Also, the number of the putative protein-binding regions predicted with DisoRDPbind correlates with the promiscuity of proteins in the corresponding protein-protein interaction networks. Webserver: http://biomine.ece.ualberta.ca/DisoRDPbind/.

  19. Sequence-specific binding of a hormonally regulated mRNA binding protein to cytidine-rich sequences in the lutropin receptor open reading frame.

    Science.gov (United States)

    Kash, J C; Menon, K M

    1999-12-21

    In previous studies, a lutropin receptor mRNA binding protein implicated in the hormonal regulation of lutropin receptor mRNA stability was identified. This protein, termed LRBP-1, was shown by RNA gel electrophoretic mobility shift assay to specifically interact with lutropin receptor RNA sequences. The present studies have examined the specificity of lutropin receptor mRNA recognition by LRBP-1 and mapped the contact site by RNA footprinting and by site-directed mutagenesis. LRBP-1 was partially purified by cation-exchange chromatography, and the mRNA binding properties of the partially purified LRBP-1 were examined by RNA gel electrophoretic mobility shift assay and hydroxyl-radical RNA footprinting. These data showed that the LRBP-1 binding site is located between nucleotides 203 and 220 of the receptor open reading frame, and consists of the bipartite polypyrimidine sequence 5'-UCUC-X(7)-UCUCCCU-3'. Competition RNA gel electrophoretic mobility shift assays demonstrated that homoribopolymers of poly(rC) were effective RNA binding competitors, while poly(rA), poly(rG), and poly(rU) showed no effect. Mutagenesis of the cytidine residues contained within the LRBP-1 binding site demonstrated that all the cytidines in the bipartite sequence contribute to LRBP-1 binding specificity. Additionally, RNA gel electrophoretic mobility supershift analysis showed that LRBP-1 was not recognized by antibodies against two well-characterized poly(rC) RNA binding proteins, alphaCP-1 and alphaCP-2, implicated in the regulation of RNA stability of alpha-globin and tyrosine hydroxylase mRNAs. In summary, we show that partially purified LRBP-1 binds to a polypyrimidine sequence within nucleotides 203 and 220 of lutropin receptor mRNA with a high degree of specificity which is indicative of its role in posttranscriptional control of lutropin receptor expression.

  20. Experimental and Computational Considerations in the Study of RNA-Binding Protein-RNA Interactions.

    Science.gov (United States)

    Van Nostrand, Eric L; Huelga, Stephanie C; Yeo, Gene W

    2016-01-01

    After an RNA is transcribed, it undergoes a variety of processing steps that can change the encoded protein sequence (through alternative splicing and RNA editing), regulate the stability of the RNA, and control subcellular localization, timing, and rate of translation. The recent explosion in genomics techniques has enabled transcriptome-wide profiling of RNA processing in an unbiased manner. However, it has also brought with it both experimental challenges in developing improved methods to probe distinct processing steps, as well as computational challenges in data storage, processing, and analysis tools to enable large-scale interpretation in the genomics era. In this chapter we review experimental techniques and challenges in profiling various aspects of RNA processing, as well as recent efforts to develop analyses integrating multiple data sources and techniques to infer RNA regulatory networks.

  1. Binding of 16S rRNA to chloroplast 30S ribosomal proteins blotted on nitrocellulose

    OpenAIRE

    Rozier, Claude; Mache, Régis

    1984-01-01

    Protein-RNA associations were studied by a method using proteins blotted on a nitrocellulose sheet. This method was assayed with Escherichia Coli 30S ribosomal components. In stringent conditions (300 mM NaCl or 20° C) only 9 E. coli ribosomal proteins strongly bound to the 16S rRNA: S4, S5, S7, S9, S12, S13, S14, S19, S20. 8 of these proteins have been previously found to bind independently to the 16S rRNA. The same method was applied to determine protein-RNA interactions in spinach chloropl...

  2. A cellular protein specifically binds to the 3'-terminal sequences of hepatitis C virus intermediate negative-strand RNA

    Institute of Scientific and Technical Information of China (English)

    王巍; 邓庆丽; 黄开红; 段朝晖; 邵静; 黄志清; 黄志明

    2003-01-01

    ObjectiveTo study the mechanism of the cellular proteins involved in the process of replication of hepatitis C virus (HCV) negative-strand RNA.MethodsUltraviolet (UV) cross-linking was used to identify the cellular proteins that would bind to the 3'-end of HCV negative-strand RNA. Competition experimentwas used to confirm the specificity of this binding, in which excess nonhomologous protein and RNA transcripts were used as competitors. The required binding sequence was determined by mapping, then the binding site was predicted through secondary structure analysis.ResultsA cellular protein of 45 kD (p45) was found to bind specifically to the 3'-endof HCV negative-strand RNA by UV cross-linking. nhomologous proteins and RNA transcripts could not compete out this binding, whereas the unlabeled 3'-endof HCV negative-strand RNA could. Mapping of the protein-binding site suggested that the 3'-end 131-278nt of HCV negative-strand RNA was the possible protein-binding region. Analysis of RNA secondary structure presumed that the potential binding site was located at 194-GAAAGAAC-201. ConclusionThe cellular protein p45 could specifically bind to the secondary structure of the 3'-end of HCV intermediate negative-strand RNA, and may play an important role in HCV RNA replication.

  3. Dwarfism and impaired gut development in insulin-like growth factor II mRNA-binding protein 1-deficient mice

    DEFF Research Database (Denmark)

    Hansen, Thomas V O; Hammer, Niels A; Nielsen, Jacob

    2004-01-01

    Insulin-like growth factor II mRNA-binding protein 1 (IMP1) belongs to a family of RNA-binding proteins implicated in mRNA localization, turnover, and translational control. Mouse IMP1 is expressed during early development, and an increase in expression occurs around embryonic day 12.5 (E12.5). T...

  4. Cross-talk between PRMT1-mediated methylation and ubiquitylation on RBM15 controls RNA splicing.

    Science.gov (United States)

    Zhang, Li; Tran, Ngoc-Tung; Su, Hairui; Wang, Rui; Lu, Yuheng; Tang, Haiping; Aoyagi, Sayura; Guo, Ailan; Khodadadi-Jamayran, Alireza; Zhou, Dewang; Qian, Kun; Hricik, Todd; Côté, Jocelyn; Han, Xiaosi; Zhou, Wenping; Laha, Suparna; Abdel-Wahab, Omar; Levine, Ross L; Raffel, Glen; Liu, Yanyan; Chen, Dongquan; Li, Haitao; Townes, Tim; Wang, Hengbin; Deng, Haiteng; Zheng, Y George; Leslie, Christina; Luo, Minkui; Zhao, Xinyang

    2015-11-17

    RBM15, an RNA binding protein, determines cell-fate specification of many tissues including blood. We demonstrate that RBM15 is methylated by protein arginine methyltransferase 1 (PRMT1) at residue R578, leading to its degradation via ubiquitylation by an E3 ligase (CNOT4). Overexpression of PRMT1 in acute megakaryocytic leukemia cell lines blocks megakaryocyte terminal differentiation by downregulation of RBM15 protein level. Restoring RBM15 protein level rescues megakaryocyte terminal differentiation blocked by PRMT1 overexpression. At the molecular level, RBM15 binds to pre-messenger RNA intronic regions of genes important for megakaryopoiesis such as GATA1, RUNX1, TAL1 and c-MPL. Furthermore, preferential binding of RBM15 to specific intronic regions recruits the splicing factor SF3B1 to the same sites for alternative splicing. Therefore, PRMT1 regulates alternative RNA splicing via reducing RBM15 protein concentration. Targeting PRMT1 may be a curative therapy to restore megakaryocyte differentiation for acute megakaryocytic leukemia.

  5. Tobacco calmodulin-like protein provides secondary defense by binding to and directing degradation of virus RNA silencing suppressors

    OpenAIRE

    Nakahara, Kenji S; Masuta, Chikara; Yamada, Syouta; Shimura, Hanako; KASHIHARA, Yukiko; Wada, Tomoko S.; Meguro, Ayano; Goto, Kazunori; Tadamura, Kazuki; Sueda, Kae; Sekiguchi, Toru; Shao, Jun; Itchoda, Noriko; Matsumura, Takeshi; Igarashi, Manabu

    2012-01-01

    RNA silencing (RNAi) induced by virus-derived double-stranded RNA (dsRNA), which is in a sense regarded as a pathogen-associated molecular pattern (PAMP) of viruses, is a general plant defense mechanism. To counteract this defense, plant viruses express RNA silencing suppressors (RSSs), many of which bind to dsRNA and attenuate RNAi. We showed that the tobacco calmodulin-like protein, rgs-CaM, counterattacked viral RSSs by binding to their dsRNA-binding domains and sequestering them from inhi...

  6. Yeast mitochondrial biogenesis: a role for the PUF RNA-binding protein Puf3p in mRNA localization.

    Directory of Open Access Journals (Sweden)

    Yann Saint-Georges

    Full Text Available The asymmetric localization of mRNA plays an important role in coordinating posttranscriptional events in eukaryotic cells. We investigated the peripheral mitochondrial localization of nuclear-encoded mRNAs (MLR in various conditions in which the mRNA binding protein context and the translation efficiency were altered. We identified Puf3p, a Pumilio family RNA-binding protein, as the first trans-acting factor controlling the MLR phenomenon. This allowed the characterization of two classes of genes whose mRNAs are translated to the vicinity of mitochondria. Class I mRNAs (256 genes have a Puf3p binding motif in their 3'UTR region and many of them have their MLR properties deeply affected by PUF3 deletion. Conversely, mutations in the Puf3p binding motif alter the mitochondrial localization of BCS1 mRNA. Class II mRNAs (224 genes have no Puf3p binding site and their asymmetric localization is not affected by the absence of PUF3. In agreement with a co-translational import process, we observed that the presence of puromycin loosens the interactions between most of the MLR-mRNAs and mitochondria. Unexpectedly, cycloheximide, supposed to solidify translational complexes, turned out to destabilize a class of mRNA-mitochondria interactions. Classes I and II mRNAs, which are therefore transported to the mitochondria through different pathways, correlated with different functional modules. Indeed, Class I genes code principally for the assembly factors of respiratory chain complexes and the mitochondrial translation machinery (ribosomes and translation regulators. Class II genes encode proteins of the respiratory chain or proteins involved in metabolic pathways. Thus, MLR, which is intimately linked to translation control, and the activity of mRNA-binding proteins like Puf3p, may provide the conditions for a fine spatiotemporal control of mitochondrial protein import and mitochondrial protein complex assembly. This work therefore provides new openings

  7. Streptomycin binds to the decoding center of 16 S ribosomal RNA.

    Science.gov (United States)

    Spickler, C; Brunelle, M N; Brakier-Gingras, L

    1997-10-31

    Streptomycin, an error-inducing aminoglycoside antibiotic, binds to a single site on the small ribosomal subunit of bacteria, but this site has not yet been defined precisely. Here, we demonstrate that streptomycin binds to E. coli 16 S rRNA in the absence of ribosomal proteins, and protects a set of bases in the decoding region against dimethyl sulfate attack. The binding studies were performed in a high ionic strength buffer containing 20 mM Mg2+. The pattern of protection in the decoding region was similar to that observed when streptomycin binds to the 30 S subunit. However, streptomycin also protects the 915 region of 16 S rRNA within the 30 S subunit, whereas it did not protect the 915 region of the naked 16 S rRNA. The interaction of streptomycin with 16 S rRNA was further defined by using two fragments that correspond to the 3' minor domain of 16 S rRNA and to the decoding analog, a portion of this domain encompassing the decoding center. In the presence of streptomycin, the pattern of protection against dimethyl sulfate attack for the two fragments was similar to that seen with the full-length 16 S rRNA. This indicates that the 3' minor domain as well as the decoding analog contain the recognition signals for the binding of streptomycin. However, streptomycin could not bind to the decoding analog in the absence of Mg2+. This contrasts with neomycin, another error-inducing aminoglycoside antibiotic, that binds to the decoding analog in the absence of Mg2+, but not at 20 mM Mg2+. Our results suggest that both neomycin and streptomycin interact with the decoding center, but recognize alternative conformations of this region.

  8. Mechanism of Coupled Folding and Binding in the siRNA-PAZ Complex.

    Science.gov (United States)

    Chen, Hai-Feng

    2008-08-01

    The PAZ domain plays a key role in gene silencing pathway. The PAZ domain binds with siRNAs to form the multimeric RNA-induced silencing complex (RISC). RISC identifies mRNAs homologous to the siRNAs and promotes their degradation. It was found that binding with siRNA significantly enhances apo-PAZ folding. However, the mechanism by which folding is coupled to binding is poorly understood. Thus, the coupling relationship between binding and folding is very important for understanding the function of gene silencing. We have performed molecular dynamics (MD) of both bound and apo-PAZ to study the coupling mechanism between binding and folding in the siRNA-PAZ complex. Room-temperature MD simulations suggest that both PAZ and siRNA become more rigid and stable upon siRNA binding. Kinetic analysis of high-temperature MD simulations shows that both bound and apo-PAZ unfold via a two-state process. The unfolding pathways are different between bound and apo-PAZ: the order of helix III and helices I & II unfolding is switched. Furthermore, transition probability was used to determine the transition state ensemble for both bound and apo-PAZ. It was found that the transition state of bound PAZ is more compact than that of apo-PAZ. The predicted Φ-values suggest that the Φ-values of helix III and sheets of β3-β7 for bound PAZ are more native-like than those of apo-PAZ upon the binding of siRNA. The results can help us to understand the mechanism of gene silencing.

  9. Binding of kinetically inert metal ions to RNA: the case of platinum(II).

    Science.gov (United States)

    Chapman, Erich G; Hostetter, Alethia A; Osborn, Maire F; Miller, Amanda L; DeRose, Victoria J

    2011-01-01

    In this chapter several aspects of Pt(II) are highlighted that focus on the properties of Pt(II)-RNA adducts and the possibility that they influence RNA-based processes in cells. Cellular distribution of Pt(II) complexes results in significant platination of RNA, and localization studies find Pt(II) in the nucleus, nucleolus, and a distribution of other sites in cells. Treatment with Pt(II) compounds disrupts RNA-based processes including enzymatic processing, splicing, and translation, and this disruption may be indicative of structural changes to RNA or RNA-protein complexes. Several RNA-Pt(II) adducts have been characterized in vitro by biochemical and other methods. Evidence for Pt(II) binding in non-helical regions and for Pt(II) cross-linking of internal loops has been found. Although platinated sites have been identified, there currently exists very little in the way of detailed structural characterization of RNA-Pt(II) adducts. Some insight into the details of Pt(II) coordination to RNA, especially RNA helices, can be gained from DNA model systems. Many RNA structures, however, contain complex tertiary folds and common, purine-rich structural elements that present suitable Pt(II) nucleophiles in unique arrangements which may hold the potential for novel types of platinum-RNA adducts. Future research aimed at structural characterization of platinum-RNA adducts may provide further insights into platinum-nucleic acid binding motifs, and perhaps provide a rationale for the observed inhibition by Pt(II) complexes of splicing, translation, and enzymatic processing.

  10. Binding of circulating autoantibodies in breast cancer to native and peroxynitrite-modified RNA

    Institute of Scientific and Technical Information of China (English)

    Sheereen TARANNUM; Zarina ARIF; Khursheed ALAM

    2013-01-01

    Peroxynitrite (ONOO-) is a powerful oxidant and nitrosative agent and has in vivo existence.The half life of ONOO-at physiological pH is less than 1 s.It can react with nucleic acids,proteins,lipoproteins,saccharides,cardiolipin,etc.,and can modify their native structures.Action of ONOO-,synthesized in the authors' laboratory by a rapid quenched flow process,on structural changes of commercially available RNA was studied by ultraviolet (UV),fluorescence,and agarose gel electrophoresis.Compared to native RNA,the ONOO--modified RNA showed hyperchromicity at 260 nm.Furthermore,the ethidium bromide (EtBr) assisted emission intensities of ONOO--modified RNA samples were found to be lower than the emission intensity of native RNA-EtBr complex.Agarose gel electrophoresis of ONOO--modified RNA showed a gradual decrease in band intensities compared to native RNA,an observation clearly due to the poor intercalation of EtBr with ONOO--modified RNA.Native and ONOO--modified RNA samples were used as an antigen to detect autoantibodies in sera of patients with clinically defined breast cancer.Both direct binding and inhibition enzyme-linked immunosorbent assay (ELISA) confirmed the prevalence of native and 0.8 mmol/L ONOO--modified RNA specific autoantibodies in breast cancer patients.Moreover,the progressive retardation in the mobility of immune complexes formed with native or 0.8 mmol/L ONOO--modified RNA and affinity purified immunoglobulin G (IgG) from sera of breast cancer patients supports the findings of the direct binding and inhibition ELISAs.The peroxynitrite treatment to RNA at a higher concentration appears to have damaged or destroyed the typical epitopes on RNA and thus there was a sharp decrease in autoantibodies binding to 1.4 mmol/L ONOO--modified RNA.It may be interpreted that cellular nitrosative stress can modify and confer immunogenicity on RNA molecules.Higher concentrations of nitrogen reactive species can be detrimental to RNA.However,the emergence of

  11. Ribosomal protein S7 from Escherichia coli uses the same determinants to bind 16S ribosomal RNA and its messenger RNA.

    Science.gov (United States)

    Robert, F; Brakier-Gingras, L

    2001-02-01

    Ribosomal protein S7 from Escherichia coli binds to the lower half of the 3' major domain of 16S rRNA and initiates its folding. It also binds to its own mRNA, the str mRNA, and represses its translation. Using filter binding assays, we show in this study that the same mutations that interfere with S7 binding to 16S rRNA also weaken its affinity for its mRNA. This suggests that the same protein regions are responsible for mRNA and rRNA binding affinities, and that S7 recognizes identical sequence elements within the two RNA targets, although they have dissimilar secondary structures. Overexpression of S7 is known to inhibit bacterial growth. This phenotypic growth defect was relieved in cells overexpressing S7 mutants that bind poorly the str mRNA, confirming that growth impairment is controlled by the binding of S7 to its mRNA. Interestingly, a mutant with a short deletion at the C-terminus of S7 was more detrimental to cell growth than wild-type S7. This suggests that the C-terminal portion of S7 plays an important role in ribosome function, which is perturbed by the deletion.

  12. The RNA-binding protein HuR regulates DNA methylation through stabilization of DNMT3b mRNA.

    Science.gov (United States)

    López de Silanes, Isabel; Gorospe, Myriam; Taniguchi, Hiroaki; Abdelmohsen, Kotb; Srikantan, Subramanya; Alaminos, Miguel; Berdasco, María; Urdinguio, Rocío G; Fraga, Mario F; Jacinto, Filipe V; Esteller, Manel

    2009-05-01

    The molecular basis underlying the aberrant DNA-methylation patterns in human cancer is largely unknown. Altered DNA methyltransferase (DNMT) activity is believed to contribute, as DNMT expression levels increase during tumorigenesis. Here, we present evidence that the expression of DNMT3b is post-transcriptionally regulated by HuR, an RNA-binding protein that stabilizes and/or modulates the translation of target mRNAs. The presence of a putative HuR-recognition motif in the DNMT3b 3'UTR prompted studies to investigate if this transcript associated with HuR. The interaction between HuR and DNMT3b mRNA was studied by immunoprecipitation of endogenous HuR ribonucleoprotein complexes followed by RT-qPCR detection of DNMT3b mRNA, and by in vitro pulldown of biotinylated DNMT3b RNAs followed by western blotting detection of HuR. These studies revealed that binding of HuR stabilized the DNMT3b mRNA and increased DNMT3b expression. Unexpectedly, cisplatin treatment triggered the dissociation of the [HuR-DNMT3b mRNA] complex, in turn promoting DNMT3b mRNA decay, decreasing DNMT3b abundance, and lowering the methylation of repeated sequences and global DNA methylation. In summary, our data identify DNMT3b mRNA as a novel HuR target, present evidence that HuR affects DNMT3b expression levels post-transcriptionally, and reveal the functional consequences of the HuR-regulated DNMT3b upon DNA methylation patterns.

  13. Crystal structure of the S. solfataricus archaeal exosome reveals conformational flexibility in the RNA-binding ring.

    Directory of Open Access Journals (Sweden)

    Changrui Lu

    Full Text Available BACKGROUND: The exosome complex is an essential RNA 3'-end processing and degradation machinery. In archaeal organisms, the exosome consists of a catalytic ring and an RNA-binding ring, both of which were previously reported to assume three-fold symmetry. METHODOLOGY/PRINCIPAL FINDINGS: Here we report an asymmetric 2.9 A Sulfolobus solfataricus archaeal exosome structure in which the three-fold symmetry is broken due to combined rigid body and thermal motions mainly within the RNA-binding ring. Since increased conformational flexibility was also observed in the RNA-binding ring of the related bacterial PNPase, we speculate that this may reflect an evolutionarily conserved mechanism to accommodate diverse RNA substrates for degradation. CONCLUSION/SIGNIFICANCE: This study clearly shows the dynamic structures within the RNA-binding domains, which provides additional insights on mechanism of asymmetric RNA binding and processing.

  14. Analysis of RNA folding and ligand binding by conventional and high-throughput calorimetry.

    Science.gov (United States)

    Sokoloski, Joshua E; Bevilacqua, Philip C

    2012-01-01

    Noncoding RNAs serve myriad functions in the cell, but their biophysical properties are not well understood. Calorimetry offers direct and label-free means for characterizing the ligand-binding and thermostability properties of these RNA. We apply two main types of calorimetry--isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC)--to the characterization of these functional RNA molecules. ITC can describe ligand binding in terms of stoichiometry, affinity, and heat (enthalpy), while DSC can provide RNA stability in terms of heat capacity, melting temperature, and folding enthalpy. Here, we offer detailed experimental protocols for studying such RNA systems with commercially available conventional and high-throughput ITC and DSC instruments.

  15. Nucleic acids encoding phloem small RNA-binding proteins and transgenic plants comprising them

    Science.gov (United States)

    Lucas, William J.; Yoo, Byung-Chun; Lough, Tony J.; Varkonyi-Gasic, Erika

    2007-03-13

    The present invention provides a polynucleotide sequence encoding a component of the protein machinery involved in small RNA trafficking, Cucurbita maxima phloem small RNA-binding protein (CmPSRB 1), and the corresponding polypeptide sequence. The invention also provides genetic constructs and transgenic plants comprising the polynucleotide sequence encoding a phloem small RNA-binding protein to alter (e.g., prevent, reduce or elevate) non-cell autonomous signaling events in the plants involving small RNA metabolism. These signaling events are involved in a broad spectrum of plant physiological and biochemical processes, including, for example, systemic resistance to pathogens, responses to environmental stresses, e.g., heat, drought, salinity, and systemic gene silencing (e.g., viral infections).

  16. Protein-binding RNA aptamers affect molecular interactions distantly from their binding sites

    DEFF Research Database (Denmark)

    Dupont, Daniel M; Thuesen, Cathrine K; Bøtkjær, Kenneth A;

    2015-01-01

    Nucleic acid aptamer selection is a powerful strategy for the development of regulatory agents for molecular intervention. Accordingly, aptamers have proven their diligence in the intervention with serine protease activities, which play important roles in physiology and pathophysiology. Nonetheless...... potential, both binding to the serine protease urokinase-type plasminogen activator (uPA). We determine the subsequent impact of aptamer binding on the well-established molecular interactions (plasmin, PAI-1, uPAR, and LRP-1A) controlling uPA activities. One of the aptamers (upanap-126) binds to the area...... around the C-terminal α-helix in pro-uPA, while the other aptamer (upanap-12) binds to both the β-hairpin of the growth factor domain and the kringle domain of uPA. Based on the mapping studies, combined with data from small-angle X-ray scattering analysis, we construct a model for the upanap-12:pro...

  17. 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.

  18. The Cardiomyocyte RNA-Binding Proteome: Links to Intermediary Metabolism and Heart Disease

    Directory of Open Access Journals (Sweden)

    Yalin Liao

    2016-08-01

    Full Text Available RNA functions through the dynamic formation of complexes with RNA-binding proteins (RBPs in all clades of life. We determined the RBP repertoire of beating cardiomyocytic HL-1 cells by jointly employing two in vivo proteomic methods, mRNA interactome capture and RBDmap. Together, these yielded 1,148 RBPs, 391 of which are shared with all other available mammalian RBP repertoires, while 393 are thus far unique to cardiomyocytes. RBDmap further identified 568 regions of RNA contact within 368 RBPs. The cardiomyocyte mRNA interactome composition reflects their unique biology. Proteins with roles in cardiovascular physiology or disease, mitochondrial function, and intermediary metabolism are all highly represented. Notably, we identified 73 metabolic enzymes as RBPs. RNA-enzyme contacts frequently involve Rossmann fold domains with examples in evidence of both, mutual exclusivity of, or compatibility between RNA binding and enzymatic function. Our findings raise the prospect of previously hidden RNA-mediated regulatory interactions among cardiomyocyte gene expression, physiology, and metabolism.

  19. Chloroplast RNA-Binding Protein RBD1 Promotes Chilling Tolerance through 23S rRNA Processing in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Shuai Wang

    2016-05-01

    Full Text Available Plants have varying abilities to tolerate chilling (low but not freezing temperatures, and it is largely unknown how plants such as Arabidopsis thaliana achieve chilling tolerance. Here, we describe a genome-wide screen for genes important for chilling tolerance by their putative knockout mutants in Arabidopsis thaliana. Out of 11,000 T-DNA insertion mutant lines representing half of the genome, 54 lines associated with disruption of 49 genes had a drastic chilling sensitive phenotype. Sixteen of these genes encode proteins with chloroplast localization, suggesting a critical role of chloroplast function in chilling tolerance. Study of one of these proteins RBD1 with an RNA binding domain further reveals the importance of chloroplast translation in chilling tolerance. RBD1 is expressed in the green tissues and is localized in the chloroplast nucleoid. It binds directly to 23S rRNA and the binding is stronger under chilling than at normal growth temperatures. The rbd1 mutants are defective in generating mature 23S rRNAs and deficient in chloroplast protein synthesis especially under chilling conditions. Together, our study identifies RBD1 as a regulator of 23S rRNA processing and reveals the importance of chloroplast function especially protein translation in chilling tolerance.

  20. Probe the Binding Mode of Aristololactam-β-D-glucoside to Phenylalanine Transfer RNA in Silico

    DEFF Research Database (Denmark)

    Xiao, Xingqing; Zhao, Binwu; Yang, Li

    2016-01-01

    Understanding the interactions of drug molecules with biomacromolecules at a micro-scale level is essential to design potent drugs for the treatments of human genome diseases. To unravel the mechanism of binding of aristololactam-β-D-glucoside (ADG) and phenylalanine transfer RNA (tRNAPhe), an in......Understanding the interactions of drug molecules with biomacromolecules at a micro-scale level is essential to design potent drugs for the treatments of human genome diseases. To unravel the mechanism of binding of aristololactam-β-D-glucoside (ADG) and phenylalanine transfer RNA (t...

  1. Impaired Embryonic Development in Mice Overexpressing the RNA-Binding Protein TIAR

    OpenAIRE

    Yacine Kharraz; Pierre-Adrien Salmand; Anne Camus; Jacques Auriol; Cyril Gueydan; Véronique Kruys; Dominique Morello

    2010-01-01

    BACKGROUND: TIA-1-related (TIAR) protein is a shuttling RNA-binding protein involved in several steps of RNA metabolism. While in the nucleus TIAR participates to alternative splicing events, in the cytoplasm TIAR acts as a translational repressor on specific transcripts such as those containing AU-Rich Elements (AREs). Due to its ability to assemble abortive pre-initiation complexes coalescing into cytoplasmic granules called stress granules, TIAR is also involved in the general translationa...

  2. CED-4 is an mRNA-binding protein that delivers ced-3 mRNA to ribosomes.

    Science.gov (United States)

    Wang, Miao-xing; Itoh, Masanori; Li, Shimo; Hida, Yoko; Ohta, Kazunori; Hayakawa, Miki; Nishida, Emika; Ueda, Masashi; Islam, Saiful; Tana; Nakagawa, Toshiyuki

    2016-01-29

    Cell death abnormal (ced)-3 and ced-4 genes regulate apoptosis to maintain tissue homeostasis in Caenorhabditis elegans. Apoptosome formation and CED-4 translocation drive CED-3 activation. However, the precise role of CED-4 translocation is not yet fully understood. In this study, using a combination of immunoprecipitation and reverse transcription-polymerase chain reaction methods in cells and a glutathione-S-transferase pull down assay in a cell-free system, we show that CED-4 binds ced-3 mRNA. In the presence of ced-3 mRNA, CED-4 protein is enriched in the microsomal fraction and interacts with ribosomal protein L10a in mammalian cells, increasing the levels of CED-3. These results suggest that CED-4 forms a complex with ced-3 mRNA and delivers it to ribosomes for translation.

  3. A conserved loop in polynucleotide phosphorylase (PNPase) essential for both RNA and ADP/phosphate binding.

    Science.gov (United States)

    Carzaniga, Thomas; Mazzantini, Elisa; Nardini, Marco; Regonesi, Maria Elena; Greco, Claudio; Briani, Federica; De Gioia, Luca; Dehò, Gianni; Tortora, Paolo

    2014-02-01

    Polynucleotide phosphorylase (PNPase) reversibly catalyzes RNA phosphorolysis and polymerization of nucleoside diphosphates. Its homotrimeric structure forms a central channel where RNA is accommodated. Each protomer core is formed by two paralogous RNase PH domains: PNPase1, whose function is largely unknown, hosts a conserved FFRR loop interacting with RNA, whereas PNPase2 bears the putative catalytic site, ∼20 Å away from the FFRR loop. To date, little is known regarding PNPase catalytic mechanism. We analyzed the kinetic properties of two Escherichia coli PNPase mutants in the FFRR loop (R79A and R80A), which exhibited a dramatic increase in Km for ADP/Pi binding, but not for poly(A), suggesting that the two residues may be essential for binding ADP and Pi. However, both mutants were severely impaired in shifting RNA electrophoretic mobility, implying that the two arginines contribute also to RNA binding. Additional interactions between RNA and other PNPase domains (such as KH and S1) may preserve the enzymatic activity in R79A and R80A mutants. Inspection of enzyme structure showed that PNPase has evolved a long-range acting hydrogen bonding network that connects the FFRR loop with the catalytic site via the F380 residue. This hypothesis was supported by mutation analysis. Phylogenetic analysis of PNPase domains and RNase PH suggests that such network is a unique feature of PNPase1 domain, which coevolved with the paralogous PNPase2 domain. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  4. Single nucleotide polymorphisms in microRNA binding sites of oncogenes: implications in cancer and pharmacogenomics.

    Science.gov (United States)

    Manikandan, Mayakannan; Munirajan, Arasambattu Kannan

    2014-02-01

    Cancer, a complex genetic disease involving uncontrolled cell proliferation, is caused by inactivation of tumor suppressor genes and activation of oncogenes. A vast majority of these cancer causing genes are known targets of microRNAs (miRNAs) that bind to complementary sequences in 3' untranslated regions (UTR) of messenger RNAs and repress them from translation. Single Nucleotide Polymorphisms (SNPs) occurring naturally in such miRNA binding regions can alter the miRNA:mRNA interaction and can significantly affect gene expression. We hypothesized that 3'UTR SNPs in miRNA binding sites of proto-oncogenes could abrogate their post-transcriptional regulation, resulting in overexpression of oncogenic proteins, tumor initiation, progression, and modulation of drug response in cancer patients. Therefore, we developed a systematic computational pipeline that integrates data from well-established databases, followed stringent selection criteria and identified a panel of 30 high-confidence SNPs that may impair miRNA target sites in the 3' UTR of 54 mRNA transcripts of 24 proto-oncogenes. Further, 8 SNPs amidst them had the potential to determine therapeutic outcome in cancer patients. Functional annotation suggested that altogether these SNPs occur in proto-oncogenes enriched for kinase activities. We provide detailed in silico evidence for the functional effect of these candidate SNPs in various types of cancer.

  5. Non-canonical binding interactions of the RNA recognition motif (RRM) domains of P34 protein modulate binding within the 5S ribonucleoprotein particle (5S RNP).

    Science.gov (United States)

    Kamina, Anyango D; Williams, Noreen

    2017-01-01

    RNA binding proteins are involved in many aspects of RNA metabolism. In Trypanosoma brucei, our laboratory has identified two trypanosome-specific RNA binding proteins P34 and P37 that are involved in the maturation of the 60S subunit during ribosome biogenesis. These proteins are part of the T. brucei 5S ribonucleoprotein particle (5S RNP) and P34 binds to 5S ribosomal RNA (rRNA) and ribosomal protein L5 through its N-terminus and its RNA recognition motif (RRM) domains. We generated truncated P34 proteins to determine these domains' interactions with 5S rRNA and L5. Our analyses demonstrate that RRM1 of P34 mediates the majority of binding with 5S rRNA and the N-terminus together with RRM1 contribute the most to binding with L5. We determined that the consensus ribonucleoprotein (RNP) 1 and 2 sequences, characteristic of canonical RRM domains, are not fully conserved in the RRM domains of P34. However, the aromatic amino acids previously described to mediate base stacking interactions with their RNA target are conserved in both of the RRM domains of P34. Surprisingly, mutation of these aromatic residues did not disrupt but instead enhanced 5S rRNA binding. However, we identified four arginine residues located in RRM1 of P34 that strongly impact L5 binding. These mutational analyses of P34 suggest that the binding site for 5S rRNA and L5 are near each other and specific residues within P34 regulate the formation of the 5S RNP. These studies show the unique way that the domains of P34 mediate binding with the T. brucei 5S RNP.

  6. The Mode of Inhibitor Binding to Peptidyl-tRNA Hydrolase: Binding Studies and Structure Determination of Unbound and Bound Peptidyl-tRNA Hydrolase from Acinetobacter baumannii

    Science.gov (United States)

    Kaushik, Sanket; Singh, Nagendra; Yamini, Shavait; Singh, Avinash; Sinha, Mau; Arora, Ashish; Kaur, Punit; Sharma, Sujata; Singh, Tej P.

    2013-01-01

    The incidences of infections caused by an aerobic Gram-negative bacterium, Acinetobacter baumannii are very common in hospital environments. It usually causes soft tissue infections including urinary tract infections and pneumonia. It is difficult to treat due to acquired resistance to available antibiotics is well known. In order to design specific inhibitors against one of the important enzymes, peptidyl-tRNA hydrolase from Acinetobacter baumannii, we have determined its three-dimensional structure. Peptidyl-tRNA hydrolase (AbPth) is involved in recycling of peptidyl-tRNAs which are produced in the cell as a result of premature termination of translation process. We have also determined the structures of two complexes of AbPth with cytidine and uridine. AbPth was cloned, expressed and crystallized in unbound and in two bound states with cytidine and uridine. The binding studies carried out using fluorescence spectroscopic and surface plasmon resonance techniques revealed that both cytidine and uridine bound to AbPth at nanomolar concentrations. The structure determinations of the complexes revealed that both ligands were located in the active site cleft of AbPth. The introduction of ligands to AbPth caused a significant widening of the entrance gate to the active site region and in the process of binding, it expelled several water molecules from the active site. As a result of interactions with protein atoms, the ligands caused conformational changes in several residues to attain the induced tight fittings. Such a binding capability of this protein makes it a versatile molecule for hydrolysis of peptidyl-tRNAs having variable peptide sequences. These are the first studies that revealed the mode of inhibitor binding in Peptidyl-tRNA hydrolases which will facilitate the structure based ligand design. PMID:23844024

  7. The mode of inhibitor binding to peptidyl-tRNA hydrolase: binding studies and structure determination of unbound and bound peptidyl-tRNA hydrolase from Acinetobacter baumannii.

    Directory of Open Access Journals (Sweden)

    Sanket Kaushik

    Full Text Available The incidences of infections caused by an aerobic Gram-negative bacterium, Acinetobacter baumannii are very common in hospital environments. It usually causes soft tissue infections including urinary tract infections and pneumonia. It is difficult to treat due to acquired resistance to available antibiotics is well known. In order to design specific inhibitors against one of the important enzymes, peptidyl-tRNA hydrolase from Acinetobacter baumannii, we have determined its three-dimensional structure. Peptidyl-tRNA hydrolase (AbPth is involved in recycling of peptidyl-tRNAs which are produced in the cell as a result of premature termination of translation process. We have also determined the structures of two complexes of AbPth with cytidine and uridine. AbPth was cloned, expressed and crystallized in unbound and in two bound states with cytidine and uridine. The binding studies carried out using fluorescence spectroscopic and surface plasmon resonance techniques revealed that both cytidine and uridine bound to AbPth at nanomolar concentrations. The structure determinations of the complexes revealed that both ligands were located in the active site cleft of AbPth. The introduction of ligands to AbPth caused a significant widening of the entrance gate to the active site region and in the process of binding, it expelled several water molecules from the active site. As a result of interactions with protein atoms, the ligands caused conformational changes in several residues to attain the induced tight fittings. Such a binding capability of this protein makes it a versatile molecule for hydrolysis of peptidyl-tRNAs having variable peptide sequences. These are the first studies that revealed the mode of inhibitor binding in Peptidyl-tRNA hydrolases which will facilitate the structure based ligand design.

  8. Functional identification of catalytic metal ion binding sites within RNA.

    Directory of Open Access Journals (Sweden)

    James L Hougland

    2005-09-01

    Full Text Available The viability of living systems depends inextricably on enzymes that catalyze phosphoryl transfer reactions. For many enzymes in this class, including several ribozymes, divalent metal ions serve as obligate cofactors. Understanding how metal ions mediate catalysis requires elucidation of metal ion interactions with both the enzyme and the substrate(s. In the Tetrahymena group I intron, previous work using atomic mutagenesis and quantitative analysis of metal ion rescue behavior identified three metal ions (MA, MB, and MC that make five interactions with the ribozyme substrates in the reaction's transition state. Here, we combine substrate atomic mutagenesis with site-specific phosphorothioate substitutions in the ribozyme backbone to develop a powerful, general strategy for defining the ligands of catalytic metal ions within RNA. In applying this strategy to the Tetrahymena group I intron, we have identified the pro-SP phosphoryl oxygen at nucleotide C262 as a ribozyme ligand for MC. Our findings establish a direct connection between the ribozyme core and the functionally defined model of the chemical transition state, thereby extending the known set of transition-state interactions and providing information critical for the application of the recent group I intron crystallographic structures to the understanding of catalysis.

  9. Effect of AMP on mRNA binding by yeast NAD+-specific isocitrate dehydrogenase.

    Science.gov (United States)

    Anderson, Sondra L; Schirf, Virgil; McAlister-Henn, L

    2002-06-04

    Yeast mitochondrial NAD+-specific isocitrate dehydrogenase (IDH) has previously been shown to bind specifically to 5'-untranslated regions of yeast mitochondrial mRNAs, and transcripts containing these regions have been found to allosterically inhibit activity of the enzyme. This inhibition is relieved by AMP, an allosteric activator of this regulatory enzyme of the tricarboxylic acid cycle. We further investigated these enzyme/ligand interactions to determine if binding of RNA and AMP by IDH is competitive or independent. Gel mobility shift experiments indicated no effect of AMP on formation of an IDH/RNA complex. Similarly, sedimentation velocity ultracentrifugation experiments used to analyze interactions in solution indicated that AMP alone had little effect on the formation or stability of an RNA/IDH complex. However, when these sedimentation experiments were conducted in the presence of isocitrate, which has been shown to be essential for binding of AMP by IDH, the proportion of RNA sedimenting in a complex with IDH was significantly reduced by AMP. These results suggest that AMP can affect the binding of RNA by IDH but that this effect is apparent only in the presence of substrate. They also suggest that the catalytic activity of IDH in vivo may be subject to complex allosteric control determined by relative mitochondrial concentrations of mRNA, isocitrate, and AMP. We also found evidence for binding of 5'-untranslated regions of mitochondrial mRNAs by yeast mitochondrial NADP+-specific isocitrate dehydrogenase (IDP1) but not by the corresponding cytosolic isozyme (IDP2). However, this appears to be a nonspecific interaction since no evidence was obtained for any effect on the catalytic activity of IDP1.

  10. Specificity of recognition of mRNA 5' cap by human nuclear cap-binding complex.

    Science.gov (United States)

    Worch, Remigiusz; Niedzwiecka, Anna; Stepinski, Janusz; Mazza, Catherine; Jankowska-Anyszka, Marzena; Darzynkiewicz, Edward; Cusack, Stephen; Stolarski, Ryszard

    2005-09-01

    The heterodimeric nuclear cap-binding complex (CBC) binds to the mono-methylated 5' cap of eukaryotic RNA polymerase II transcripts such as mRNA and U snRNA. The binding is important for nuclear maturation of mRNAs and possibly in the first round of translation and nonsense-mediated decay. It is also essential for nuclear export of U snRNAs in metazoans. We report characterization by fluorescence spectroscopy of the recognition of 5' capped RNA by human CBC. The association constants (K(as)) for 17 mono- and dinucleotide cap analogs as well as for the oligomer m7GpppA(m2') pU(m2')pA(m2') cover the range from 1.8 x 10(6) M(-1) to 2.3 x 10(8) M(-1). Higher affinity for CBC is observed for the dinucleotide compared with mononucleotide analogs, especially for those containing a purine nucleoside next to m7G. The mRNA tetramer associates with CBC as tightly as the dinucleotide analogs. Replacement of Tyr138 by alanine in the CBP20 subunit of CBC reduces the cap affinity except for the mononucleotide analogs, consistent with the crystallographic observation of the second base stacking on this residue. Our spectroscopic studies showed that contrary to the other known cap-binding proteins, the first two nucleotides of a capped-RNA are indispensable for its specific recognition by CBC. Differences in the cap binding of CBC compared with the eukaryotic translation initiation factor 4E (eIF4E) are analyzed and discussed regarding replacement of CBC by eIF4E.

  11. Specific and Modular Binding Code for Cytosine Recognition in Pumilio/FBF (PUF) RNA-binding Domains

    Energy Technology Data Exchange (ETDEWEB)

    Dong, Shuyun; Wang, Yang; Cassidy-Amstutz, Caleb; Lu, Gang; Bigler, Rebecca; Jezyk, Mark R.; Li, Chunhua; Tanaka Hall, Traci M.; Wang, Zefeng (NIH); (Beijing U); (UNC)

    2011-10-28

    Pumilio/fem-3 mRNA-binding factor (PUF) proteins possess a recognition code for bases A, U, and G, allowing designed RNA sequence specificity of their modular Pumilio (PUM) repeats. However, recognition side chains in a PUM repeat for cytosine are unknown. Here we report identification of a cytosine-recognition code by screening random amino acid combinations at conserved RNA recognition positions using a yeast three-hybrid system. This C-recognition code is specific and modular as specificity can be transferred to different positions in the RNA recognition sequence. A crystal structure of a modified PUF domain reveals specific contacts between an arginine side chain and the cytosine base. We applied the C-recognition code to design PUF domains that recognize targets with multiple cytosines and to generate engineered splicing factors that modulate alternative splicing. Finally, we identified a divergent yeast PUF protein, Nop9p, that may recognize natural target RNAs with cytosine. This work deepens our understanding of natural PUF protein target recognition and expands the ability to engineer PUF domains to recognize any RNA sequence.

  12. Specific and modular binding code for cytosine recognition in Pumilio/FBF (PUF) RNA-binding domains.

    Science.gov (United States)

    Dong, Shuyun; Wang, Yang; Cassidy-Amstutz, Caleb; Lu, Gang; Bigler, Rebecca; Jezyk, Mark R; Li, Chunhua; Hall, Traci M Tanaka; Wang, Zefeng

    2011-07-29

    Pumilio/fem-3 mRNA-binding factor (PUF) proteins possess a recognition code for bases A, U, and G, allowing designed RNA sequence specificity of their modular Pumilio (PUM) repeats. However, recognition side chains in a PUM repeat for cytosine are unknown. Here we report identification of a cytosine-recognition code by screening random amino acid combinations at conserved RNA recognition positions using a yeast three-hybrid system. This C-recognition code is specific and modular as specificity can be transferred to different positions in the RNA recognition sequence. A crystal structure of a modified PUF domain reveals specific contacts between an arginine side chain and the cytosine base. We applied the C-recognition code to design PUF domains that recognize targets with multiple cytosines and to generate engineered splicing factors that modulate alternative splicing. Finally, we identified a divergent yeast PUF protein, Nop9p, that may recognize natural target RNAs with cytosine. This work deepens our understanding of natural PUF protein target recognition and expands the ability to engineer PUF domains to recognize any RNA sequence.

  13. RNA-Binding Protein Dnd1 Promotes Breast Cancer Apoptosis by Stabilizing the Bim mRNA in a miR-221 Binding Site

    Directory of Open Access Journals (Sweden)

    Feng Cheng

    2017-01-01

    Full Text Available RNA-binding proteins (RBPs and miRNAs are capable of controlling processes in normal development and cancer. Both of them could determine RNA transcripts fate from synthesis to decay. One such RBP, Dead end (Dnd1, is essential for regulating germ-cell viability and suppresses the germ-cell tumors development, yet how it exerts its functions in breast cancer has remained unresolved. The level of Dnd1 was detected in 21 cancerous tissues paired with neighboring normal tissues by qRT-PCR. We further annotated TCGA (The Cancer Genome Atlas mRNA expression profiles and found that the expression of Dnd1 and Bim is positively correlated (p=0.04. Patients with higher Dnd1 expression level had longer overall survival (p=0.0014 by KM Plotter tool. Dnd1 knockdown in MCF-7 cells decreased Bim expression levels and inhibited apoptosis. While knockdown of Dnd1 promoted the decay of Bim mRNA 3′UTR, the stability of Bim-5′UTR was not affected. In addition, mutation of miR-221-binding site in Bim-3′UTR canceled the effect of Dnd1 on Bim mRNA. Knockdown of Dnd1 in MCF-7 cells confirmed that Dnd1 antagonized miR-221-inhibitory effects on Bim expression. Overall, our findings indicate that Dnd1 facilitates apoptosis by increasing the expression of Bim via its competitive combining with miR-221 in Bim-3′UTR. The new function of Dnd1 may contribute to a vital role in breast cancer development.

  14. RNA-Binding Protein Dnd1 Promotes Breast Cancer Apoptosis by Stabilizing the Bim mRNA in a miR-221 Binding Site.

    Science.gov (United States)

    Cheng, Feng; Pan, Ying; Lu, Yi-Min; Zhu, Lei; Chen, Shuzheng

    2017-01-01

    RNA-binding proteins (RBPs) and miRNAs are capable of controlling processes in normal development and cancer. Both of them could determine RNA transcripts fate from synthesis to decay. One such RBP, Dead end (Dnd1), is essential for regulating germ-cell viability and suppresses the germ-cell tumors development, yet how it exerts its functions in breast cancer has remained unresolved. The level of Dnd1 was detected in 21 cancerous tissues paired with neighboring normal tissues by qRT-PCR. We further annotated TCGA (The Cancer Genome Atlas) mRNA expression profiles and found that the expression of Dnd1 and Bim is positively correlated (p = 0.04). Patients with higher Dnd1 expression level had longer overall survival (p = 0.0014) by KM Plotter tool. Dnd1 knockdown in MCF-7 cells decreased Bim expression levels and inhibited apoptosis. While knockdown of Dnd1 promoted the decay of Bim mRNA 3'UTR, the stability of Bim-5'UTR was not affected. In addition, mutation of miR-221-binding site in Bim-3'UTR canceled the effect of Dnd1 on Bim mRNA. Knockdown of Dnd1 in MCF-7 cells confirmed that Dnd1 antagonized miR-221-inhibitory effects on Bim expression. Overall, our findings indicate that Dnd1 facilitates apoptosis by increasing the expression of Bim via its competitive combining with miR-221 in Bim-3'UTR. The new function of Dnd1 may contribute to a vital role in breast cancer development.

  15. RNA-Binding Protein Dnd1 Promotes Breast Cancer Apoptosis by Stabilizing the Bim mRNA in a miR-221 Binding Site

    Science.gov (United States)

    Cheng, Feng; Pan, Ying; Lu, Yi-Min; Zhu, Lei

    2017-01-01

    RNA-binding proteins (RBPs) and miRNAs are capable of controlling processes in normal development and cancer. Both of them could determine RNA transcripts fate from synthesis to decay. One such RBP, Dead end (Dnd1), is essential for regulating germ-cell viability and suppresses the germ-cell tumors development, yet how it exerts its functions in breast cancer has remained unresolved. The level of Dnd1 was detected in 21 cancerous tissues paired with neighboring normal tissues by qRT-PCR. We further annotated TCGA (The Cancer Genome Atlas) mRNA expression profiles and found that the expression of Dnd1 and Bim is positively correlated (p = 0.04). Patients with higher Dnd1 expression level had longer overall survival (p = 0.0014) by KM Plotter tool. Dnd1 knockdown in MCF-7 cells decreased Bim expression levels and inhibited apoptosis. While knockdown of Dnd1 promoted the decay of Bim mRNA 3′UTR, the stability of Bim-5′UTR was not affected. In addition, mutation of miR-221-binding site in Bim-3′UTR canceled the effect of Dnd1 on Bim mRNA. Knockdown of Dnd1 in MCF-7 cells confirmed that Dnd1 antagonized miR-221-inhibitory effects on Bim expression. Overall, our findings indicate that Dnd1 facilitates apoptosis by increasing the expression of Bim via its competitive combining with miR-221 in Bim-3′UTR. The new function of Dnd1 may contribute to a vital role in breast cancer development.

  16. ATtRACT-a database of RNA-binding proteins and associated motifs.

    Science.gov (United States)

    Giudice, Girolamo; Sánchez-Cabo, Fátima; Torroja, Carlos; Lara-Pezzi, Enrique

    2016-01-01

    RNA-binding proteins (RBPs) play a crucial role in key cellular processes, including RNA transport, splicing, polyadenylation and stability. Understanding the interaction between RBPs and RNA is key to improve our knowledge of RNA processing, localization and regulation in a global manner. Despite advances in recent years, a unified non-redundant resource that includes information on experimentally validated motifs, RBPs and integrated tools to exploit this information is lacking. Here, we developed a database named ATtRACT (available athttp://attract.cnic.es) that compiles information on 370 RBPs and 1583 RBP consensus binding motifs, 192 of which are not present in any other database. To populate ATtRACT we (i) extracted and hand-curated experimentally validated data from CISBP-RNA, SpliceAid-F, RBPDB databases, (ii) integrated and updated the unavailable ASD database and (iii) extracted information from Protein-RNA complexes present in Protein Data Bank database through computational analyses. ATtRACT provides also efficient algorithms to search a specific motif and scan one or more RNA sequences at a time. It also allows discoveringde novomotifs enriched in a set of related sequences and compare them with the motifs included in the database.Database URL:http:// attract. cnic. es.

  17. NPM/ALK binds and phosphorylates the RNA/DNA-binding protein PSF in anaplastic large-cell lymphoma.

    Science.gov (United States)

    Galietta, Annamaria; Gunby, Rosalind H; Redaelli, Sara; Stano, Paola; Carniti, Cristiana; Bachi, Angela; Tucker, Philip W; Tartari, Carmen J; Huang, Ching-Jung; Colombo, Emanuela; Pulford, Karen; Puttini, Miriam; Piazza, Rocco G; Ruchatz, Holger; Villa, Antonello; Donella-Deana, Arianna; Marin, Oriano; Perrotti, Danilo; Gambacorti-Passerini, Carlo

    2007-10-01

    The oncogenic fusion tyrosine kinase nucleophosmin/anaplastic lymphoma kinase (NPM/ALK) induces cellular transformation in anaplastic large-cell lymphomas (ALCLs) carrying the t(2;5) chromosomal translocation. Protein-protein interactions involving NPM/ALK are important for the activation of downstream signaling pathways. This study was aimed at identifying novel NPM/ALK-binding proteins that might contribute to its oncogenic transformation. Using a proteomic approach, several RNA/DNA-binding proteins were found to coimmunoprecipitate with NPM/ALK, including the multifunctional polypyrimidine tract binding proteinassociated splicing factor (PSF). The interaction between NPM/ALK and PSF was dependent on an active ALK kinase domain and PSF was found to be tyrosine-phosphorylated in NPM/ALK-expressing cell lines and in primary ALK(+) ALCL samples. Furthermore, PSF was shown to be a direct substrate of purified ALK kinase domain in vitro, and PSF Tyr293 was identified as the site of phosphorylation. Y293F PSF was not phosphorylated by NPM/ALK and was not delocalized in NPM/ALK(+) cells. The expression of ALK fusion proteins induced delocalization of PSF from the nucleus to the cytoplasm and forced overexpression of PSF-inhibited proliferation and induced apoptosis in cells expressing NPM/ALK. PSF phosphorylation also increased its binding to RNA and decreased the PSF-mediated suppression of GAGE6 expression. These results identify PSF as a novel NPM/ALK-binding protein and substrate, and suggest that PSF function may be perturbed in NPM/ALK-transformed cells.

  18. Novel way of capping mRNA trimer and studies of its interaction with human nuclear cap-binding complex.

    Science.gov (United States)

    Worch, Remigiusz; Stepinski, Janusz; Niedzwiecka, Anna; Jankowska-Anyszka, Marzena; Mazza, Catherine; Cusack, Stephen; Stolarski, Ryszard; Darzynkiewicz, Edward

    2005-01-01

    Binding of mRNA 5' cap by the nuclear cap-binding complex (CBC) is crucial for a wide variety of mRNA metabolic events. The interaction involving the CBP20 subunit of CBC is mediated by numerous hydrogen bonds and by stacking of the tyrosine sidechains with two first bases of the capped mRNA. To examine a possible role of a longer mRNA chain in the CBC-cap recognition, we have synthesized an mRNA tetramer using a novel way of capping an RNA trimer and determined its affinity for CBC by fluorescence titration.

  19. Novel predicted RNA-binding domains associated with the translation machinery.

    Science.gov (United States)

    Aravind, L; Koonin, E V

    1999-03-01

    Two previously undetected domains were identified in a variety of RNA-binding proteins, particularly RNA-modifying enzymes, using methods for sequence profile analysis. A small domain consisting of 60-65 amino acid residues was detected in the ribosomal protein S4, two families of pseudouridine synthases, a novel family of predicted RNA methylases, a yeast protein containing a pseudouridine synthetase and a deaminase domain, bacterial tyrosyl-tRNA synthetases, and a number of uncharacterized, small proteins that may be involved in translation regulation. Another novel domain, designated PUA domain, after PseudoUridine synthase and Archaeosine transglycosylase, was detected in archaeal and eukaryotic pseudouridine synthases, archaeal archaeosine synthases, a family of predicted ATPases that may be involved in RNA modification, a family of predicted archaeal and bacterial rRNA methylases. Additionally, the PUA domain was detected in a family of eukaryotic proteins that also contain a domain homologous to the translation initiation factor eIF1/SUI1; these proteins may comprise a novel type of translation factors. Unexpectedly, the PUA domain was detected also in bacterial and yeast glutamate kinases; this is compatible with the demonstrated role of these enzymes in the regulation of the expression of other genes. We propose that the S4 domain and the PUA domain bind RNA molecules with complex folded structures, adding to the growing collection of nucleic acid-binding domains associated with DNA and RNA modification enzymes. The evolution of the translation machinery components containing the S4, PUA, and SUI1 domains must have included several events of lateral gene transfer and gene loss as well as lineage-specific domain fusions.

  20. Proteome-wide search reveals unexpected RNA-binding proteins in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Nikoleta G Tsvetanova

    Full Text Available The vast landscape of RNA-protein interactions at the heart of post-transcriptional regulation remains largely unexplored. Indeed it is likely that, even in yeast, a substantial fraction of the regulatory RNA-binding proteins (RBPs remain to be discovered. Systematic experimental methods can play a key role in discovering these RBPs--most of the known yeast RBPs lack RNA-binding domains that might enable this activity to be predicted. We describe here a proteome-wide approach to identify RNA-protein interactions based on in vitro binding of RNA samples to yeast protein microarrays that represent over 80% of the yeast proteome. We used this procedure to screen for novel RBPs and RNA-protein interactions. A complementary mass spectrometry technique also identified proteins that associate with yeast mRNAs. Both the protein microarray and mass spectrometry methods successfully identify previously annotated RBPs, suggesting that other proteins identified in these assays might be novel RBPs. Of 35 putative novel RBPs identified by either or both of these methods, 12, including 75% of the eight most highly-ranked candidates, reproducibly associated with specific cellular RNAs. Surprisingly, most of the 12 newly discovered RBPs were enzymes. Functional characteristics of the RNA targets of some of the novel RBPs suggest coordinated post-transcriptional regulation of subunits of protein complexes and a possible link between mRNA trafficking and vesicle transport. Our results suggest that many more RBPs still remain to be identified and provide a set of candidates for further investigation.

  1. A library screening approach identifies naturally occurring RNA sequences for a G-quadruplex binding ligand.

    Science.gov (United States)

    Mirihana Arachchilage, Gayan; Morris, Mark J; Basu, Soumitra

    2014-02-07

    An RNA G-quadruplex library was synthesised and screened against kanamycin A as the ligand. Naturally occurring G-quadruplex forming sequences that differentially bind to kanamycin A were identified and characterized. This provides a simple and effective strategy for identification of potential intracellular G-quadruplex targets for a ligand.

  2. RNA-binding proteins in mouse male germline stem cells: a mammalian perspective.

    Science.gov (United States)

    Qi, Huayu

    2016-01-01

    Adult stem cells that reside in particular types of tissues are responsible for tissue homeostasis and regeneration. Cellular functions of adult stem cells are intricately related to the gene expression programs in those cells. Past research has demonstrated that regulation of gene expression at the transcriptional level can decisively alter cell fate of stem cells. However, cellular contents of mRNAs are sometimes not equivalent to proteins, the functional units of cells. It is increasingly realized that post-transcriptional and translational regulation of gene expression are also fundamental for stem cell functions. Compared to differentiated somatic cells, effects on cellular status manifested by varied expression of RNA-binding proteins and global protein synthesis have been demonstrated in several stem cell systems. Through the cooperation of both cis-elements of mRNAs and trans-acting RNA-binding proteins that are intimately associated with them, regulation of localization, stability, and translational status of mRNAs directly influences the self-renewal and differentiation of stem cells. Previous studies have uncovered some of the molecular mechanisms that underlie the functions of RNA-binding proteins in stem cells in invertebrate species. However, their roles in adult stem cells in mammals are just beginning to be unveiled. This review highlights some of the RNA-binding proteins that play important functions during the maintenance and differentiation of mouse male germline stem cells, the adult stem cells in the male reproductive organ.

  3. Identification of Spermidine Binding Site in T-box Riboswitch Antiterminator RNA.

    Science.gov (United States)

    Liu, Jia; Zeng, Chunxi; Hogan, Vivian; Zhou, Shu; Monwar, Md Masud; Hines, Jennifer V

    2016-02-01

    The T-box transcription antitermination riboswitch controls bacterial gene expression by structurally responding to uncharged, cognate tRNA. Previous studies indicated that cofactors, such as the polyamine spermidine, might serve a specific functional role in enhancing riboswitch efficacy. As riboswitch function depends on key RNA structural changes involving the antiterminator element, the interaction of spermidine with the T-box riboswitch antiterminator element was investigated. Spermidine binds antiterminator model RNA with high affinity (micromolar Kd ) based on isothermal titration calorimetry and fluorescence-monitored binding assays. NMR titration studies, molecular modeling, and inline and enzymatic probing studies indicate that spermidine binds at the 3' portion of the highly conserved seven-nucleotide bulge in the antiterminator. Together, these results support the conclusion that spermidine binds the T-box antiterminator RNA preferentially in a location important for antiterminator function. The implications of these findings are significant both for better understanding of the T-box riboswitch mechanism and for antiterminator-targeted drug discovery efforts.

  4. Focus on PNA Flexibility and RNA Binding using Molecular Dynamics and Metadynamics

    Science.gov (United States)

    Verona, Massimiliano Donato; Verdolino, Vincenzo; Palazzesi, Ferruccio; Corradini, Roberto

    2017-02-01

    Peptide Nucleic Acids (PNAs) can efficiently target DNA or RNA acting as chemical tools for gene regulation. Their backbone modification and functionalization is often used to increase the affinity for a particular sequence improving selectivity. The understanding of the trading forces that lead the single strand PNA to bind the DNA or RNA sequence is preparatory for any further rational design, but a clear and unique description of this process is still not complete. In this paper we report further insights into this subject, by a computational investigation aiming at the characterization of the conformations of a single strand PNA and how these can be correlated to its capability in binding DNA/RNA. Employing Metadynamics we were able to better define conformational pre-organizations of the single strand PNA and γ-modified PNA otherwise unrevealed through classical molecular dynamics. Our simulations driven on backbone modified PNAs lead to the conclusion that this γ-functionalization affects the single strand preorganization and targeting properties to the DNA/RNA, in agreement with circular dichroism (CD) spectra obtained for this class of compounds. MD simulations on PNA:RNA dissociation and association mechanisms allowed to reveal the critical role of central bases and preorganization in the binding process.

  5. The extended AT-hook is a novel RNA binding motif.

    Science.gov (United States)

    Filarsky, Michael; Zillner, Karina; Araya, Ingrid; Villar-Garea, Ana; Merkl, Rainer; Längst, Gernot; Németh, Attila

    2015-01-01

    The AT-hook has been defined as a DNA binding peptide motif that contains a glycine-arginine-proline (G-R-P) tripeptide core flanked by basic amino acids. Recent reports documented variations in the sequence of AT-hooks and revealed RNA binding activity of some canonical AT-hooks, suggesting a higher structural and functional variability of this protein domain than previously anticipated. Here we describe the discovery and characterization of the extended AT-hook peptide motif (eAT-hook), in which basic amino acids appear symmetrical mainly at a distance of 12-15 amino acids from the G-R-P core. We identified 80 human and 60 mouse eAT-hook proteins and biochemically characterized the eAT-hooks of Tip5/BAZ2A, PTOV1 and GPBP1. Microscale thermophoresis and electrophoretic mobility shift assays reveal the nucleic acid binding features of this peptide motif, and show that eAT-hooks bind RNA with one order of magnitude higher affinity than DNA. In addition, cellular localization studies suggest a role for the N-terminal eAT-hook of PTOV1 in nucleocytoplasmic shuttling. In summary, our findings classify the eAT-hook as a novel nucleic acid binding motif, which potentially mediates various RNA-dependent cellular processes.

  6. 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...... and involved in the human neurological diseases amyotrophic lateral sclerosis and fronto-temporal lobar degeneration. Results 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 (Ch......IP-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...

  7. Fragile X mental retardation protein: A paradigm for translational control by RNA-binding proteins.

    Science.gov (United States)

    Chen, Eileen; Joseph, Simpson

    2015-07-01

    Translational control is a common mechanism used to regulate gene expression and occur in bacteria to mammals. Typically in translational control, an RNA-binding protein binds to a unique sequence in the mRNA to regulate protein synthesis by the ribosomes. Alternatively, a protein may bind to or modify a translation factor to globally regulate protein synthesis by the cell. Here, we review translational control by the fragile X mental retardation protein (FMRP), the absence of which causes the neurological disease, fragile X syndrome (FXS). Copyright © 2015 Elsevier B.V. and Société française de biochimie et biologie Moléculaire (SFBBM). All rights reserved.

  8. Discovery of widespread GTP-binding motifs in genomic DNA and RNA.

    Science.gov (United States)

    Curtis, Edward A; Liu, David R

    2013-04-18

    Biological RNAs that bind small molecules have been implicated in a variety of regulatory and catalytic processes. Inspired by these examples, we used in vitro selection to search a pool of genome-encoded RNA fragments for naturally occurring GTP aptamers. Several aptamer classes were identified, including one (the "G motif") with a G-quadruplex structure. Further analysis revealed that most RNA and DNA G-quadruplexes bind GTP. The G motif is abundant in eukaryotes, and the human genome contains ~75,000 examples with dissociation constants comparable to the GTP concentration of a eukaryotic cell (~300 μM). G-quadruplexes play roles in diverse cellular processes, and our findings raise the possibility that GTP may play a role in the function of these elements. Consistent with this possibility, the sequence requirements of several classes of regulatory G-quadruplexes parallel those of GTP binding.

  9. RNA-binding domain in the nucleocapsid protein of gill-associated nidovirus of penaeid shrimp.

    Directory of Open Access Journals (Sweden)

    Chumporn Soowannayan

    Full Text Available Gill-associated virus (GAV infects Penaeus monodon shrimp and is the type species okavirus in the Roniviridae, the only invertebrate nidoviruses known currently. Electrophoretic mobility shift assays (EMSAs using His(6-tagged full-length and truncated proteins were employed to examine the nucleic acid binding properties of the GAV nucleocapsid (N protein in vitro. The EMSAs showed full-length N protein to bind to all synthetic single-stranded (ssRNAs tested independent of their sequence. The ssRNAs included (+ and (- sense regions of the GAV genome as well as a (+ sense region of the M RNA segment of Mourilyan virus, a crustacean bunya-like virus. GAV N protein also bound to double-stranded (dsRNAs prepared to GAV ORF1b gene regions and to bacteriophage M13 genomic ssDNA. EMSAs using the five N protein constructs with variable-length N-terminal and/or C-terminal truncations localized the RNA binding domain to a 50 amino acid (aa N-terminal sequence spanning Met(11 to Arg(60. Similarly to other RNA binding proteins, the first 16 aa portion of this sequence was proline/arginine rich. To examine this domain in more detail, the 18 aa peptide (M(11PVRRPLPPQPPRNARLI(29 encompassing this sequence was synthesized and found to bind nucleic acids similarly to the full-length N protein in EMSAs. The data indicate a fundamental role for the GAV N protein proline/arginine-rich domain in nucleating genomic ssRNA to form nucleocapsids. Moreover, as the synthetic peptide formed higher-order complexes in the presence of RNA, the domain might also play some role in protein/protein interactions stabilizing the helical structure of GAV nucleocapsids.

  10. Spatial assembly and RNA binding stoichiometry of a LINE-1 protein essential for retrotransposition.

    Science.gov (United States)

    Basame, Solomon; Wai-lun Li, Patrick; Howard, Grant; Branciforte, Dan; Keller, David; Martin, Sandra L

    2006-03-24

    LINE-1, or L1, is a highly successful retrotransposon in mammals, comprising 17% and 19% of the human and mouse genomes, respectively. L1 retrotransposition and hence amplification requires the protein products of its two open reading frames, ORF1 and ORF2. The sequence of the ORF1 protein (ORF1p) is not related to any protein with known function. ORF1p has RNA binding and nucleic acid chaperone activities that are both required for retrotransposition. Earlier studies have shown that ORF1p forms a homotrimer with an asymmetric dumbbell shape, in which a rod separates a large end from a small end. Here, we determine the topological arrangement of monomers within the homotrimer by comparing atomic force microscopy (AFM) images of the full ORF1p with those of truncations containing just the N or C-terminal regions. In addition, AFM images of ORF1p bound to RNA at high protein/RNA molar ratios show that ORF1p can form tightly packed clusters on RNA, with binding occurring at the C-terminal domain. The number of bound ORF1p trimers increases with increasing length of the RNA, revealing that the binding site size is about 50 nt, a value confirmed by nitrocellulose filter binding under stoichiometric conditions. These results are consistent with a role for ORF1p during L1 retrotransposition that includes both coating the RNA and acting as a nucleic acid chaperone. Furthermore, these in vitro L1 ribonucleoprotein particles provide insight into the structure of the L1 retrotransposition intermediate.

  11. Limited portability of G-patch domains in regulators of the Prp43 RNA helicase required for pre-mRNA splicing and ribosomal RNA maturation in Saccharomyces cerevisiae.

    Science.gov (United States)

    Banerjee, Daipayan; McDaniel, Peter M; Rymond, Brian C

    2015-05-01

    The Prp43 DExD/H-box protein is required for progression of the biochemically distinct pre-messenger RNA and ribosomal RNA (rRNA) maturation pathways. In Saccharomyces cerevisiae, the Spp382/Ntr1, Sqs1/Pfa1, and Pxr1/Gno1 proteins are implicated as cofactors necessary for Prp43 helicase activation during spliceosome dissociation (Spp382) and rRNA processing (Sqs1 and Pxr1). While otherwise dissimilar in primary sequence, these Prp43-binding proteins each contain a short glycine-rich G-patch motif required for function and thought to act in protein or nucleic acid recognition. Here yeast two-hybrid, domain-swap, and site-directed mutagenesis approaches are used to investigate G-patch domain activity and portability. Our results reveal that the Spp382, Sqs1, and Pxr1 G-patches differ in Prp43 two-hybrid response and in the ability to reconstitute the Spp382 and Pxr1 RNA processing factors. G-patch protein reconstitution did not correlate with the apparent strength of the Prp43 two-hybrid response, suggesting that this domain has function beyond that of a Prp43 tether. Indeed, while critical for Pxr1 activity, the Pxr1 G-patch appears to contribute little to the yeast two-hybrid interaction. Conversely, deletion of the primary Prp43 binding site within Pxr1 (amino acids 102-149) does not impede rRNA processing but affects small nucleolar RNA (snoRNA) biogenesis, resulting in the accumulation of slightly extended forms of select snoRNAs, a phenotype unexpectedly shared by the prp43 loss-of-function mutant. These and related observations reveal differences in how the Spp382, Sqs1, and Pxr1 proteins interact with Prp43 and provide evidence linking G-patch identity with pathway-specific DExD/H-box helicase activity.

  12. Binding Sites of miR-1273 Family on the mRNA of Target Genes

    Directory of Open Access Journals (Sweden)

    Anatoly Ivashchenko

    2014-01-01

    Full Text Available This study examined binding sites of 2,578 miRNAs in the mRNAs of 12,175 human genes using the MirTarget program. It found that the miRNAs of miR-1273 family have between 33 and 1,074 mRNA target genes, with a free hybridization energy of 90% or more of its maximum value. The miR-1273 family consists of miR-1273a, miR-1273c, miR-1273d, miR-1273e, miR-1273f, miR-1273g-3p, miR-1273g-5p, miR-1273h-3p, and miR-1273h-5p. Unique miRNAs (miR-1273e, miR-1273f, and miR-1273g-3p have more than 400 target genes. We established 99 mRNA nucleotide sequences that contain arranged binding sites for the miR-1273 family. High conservation of each miRNA binding site in the mRNA of the target genes was found. The arranged binding sites of the miR-1273 family are located in the 5′UTR, CDS, or 3′UTR of many mRNAs. Five repeating sites containing some of the miR-1273 family’s binding sites were found in the 3′UTR of several target genes. The oligonucleotide sequences of miR-1273 binding sites located in CDSs code for homologous amino acid sequences in the proteins of target genes. The biological role of unique miRNAs was also discussed.

  13. Binding of 16S rRNA to chloroplast 30S ribosomal proteins blotted on nitrocellulose.

    Science.gov (United States)

    Rozier, C; Mache, R

    1984-10-11

    Protein-RNA associations were studied by a method using proteins blotted on a nitrocellulose sheet. This method was assayed with Escherichia Coli 30S ribosomal components. In stringent conditions (300 mM NaCl or 20 degrees C) only 9 E. coli ribosomal proteins strongly bound to the 16S rRNA: S4, S5, S7, S9, S12, S13, S14, S19, S20. 8 of these proteins have been previously found to bind independently to the 16S rRNA. The same method was applied to determine protein-RNA interactions in spinach chloroplast 30S ribosomal subunits. A set of only 7 proteins was bound to chloroplast rRNA in stringent conditions: chloroplast S6, S10, S11, S14, S15, S17 and S22. They also bound to E. coli 16S rRNA. This set includes 4 chloroplast-synthesized proteins: S6, S11, S15 and S22. The core particles obtained after treatment by LiCl of chloroplast 30S ribosomal subunit contained 3 proteins (S6, S10 and S14) which are included in the set of 7 binding proteins. This set of proteins probably play a part in the early steps of the assembly of the chloroplast 30S ribosomal subunit.

  14. Disease-associated mutation in SRSF2 misregulates splicing by altering RNA-binding affinities.

    Science.gov (United States)

    Zhang, Jian; Lieu, Yen K; Ali, Abdullah M; Penson, Alex; Reggio, Kathryn S; Rabadan, Raul; Raza, Azra; Mukherjee, Siddhartha; Manley, James L

    2015-08-25

    Serine/arginine-rich splicing factor 2 (SRSF2) is an RNA-binding protein that plays important roles in splicing of mRNA precursors. SRSF2 mutations are frequently found in patients with myelodysplastic syndromes and certain leukemias, but how these mutations affect SRSF2 function has only begun to be examined. We used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease to introduce the P95H mutation to SRSF2 in K562 leukemia cells, generating an isogenic model so that splicing alterations can be attributed solely to mutant SRSF2. We found that SRSF2 (P95H) misregulates 548 splicing events (RNA gel shift assays showed that a mutant SRSF2 derivative bound more tightly than its wild-type counterpart to RNA sites containing UCCAG but bound less tightly to UGGAG sites. Thus in most cases the pattern of exon inclusion or exclusion correlated with stronger or weaker RNA binding, respectively. We further show that the P95H mutation does not affect other functions of SRSF2, i.e., protein-protein interactions with key splicing factors. Our results thus demonstrate that the P95H mutation positively or negatively alters the binding affinity of SRSF2 for cognate RNA sites in target transcripts, leading to misregulation of exon inclusion. Our findings shed light on the mechanism of the disease-associated SRSF2 mutation in splicing regulation and also reveal a group of misspliced mRNA isoforms for potential therapeutic targeting.

  15. TmiRUSite and TmiROSite scripts: searching for mRNA fragments with miRNA binding sites with encoded amino acid residues

    OpenAIRE

    Berillo, Olga; Régnier, Mireille; Ivashchenko, Anatoly

    2014-01-01

    microRNAs are small RNA molecules that inhibit the translation of target genes. microRNA binding sites are located in the untranslated regions as well as in the coding domains. We describe TmiRUSite and TmiROSite scripts developed using python as tools for the extraction of nucleotide sequences for miRNA binding sites with their encoded amino acid residue sequences. The scripts allow for retrieving a set of additional sequences at left and at right from the binding site. The scripts presents ...

  16. TmiRUSite and TmiROSite scripts: searching for mRNA fragments with miRNA binding sites with encoded amino acid residues

    OpenAIRE

    Berillo, Olga; Régnier, Mireille; Ivashchenko, Anatoly

    2014-01-01

    microRNAs are small RNA molecules that inhibit the translation of target genes. microRNA binding sites are located in the untranslated regions as well as in the coding domains. We describe TmiRUSite and TmiROSite scripts developed using python as tools for the extraction of nucleotide sequences for miRNA binding sites with their encoded amino acid residue sequences. The scripts allow for retrieving a set of additional sequences at left and at right from the binding site. The scripts presents ...

  17. Protein CTC from Aquifex aeolicus possesses a full-sized 5S rRNA-binding domain.

    Science.gov (United States)

    Korobeinikova, A V; Shestakov, S A; Korepanov, A P; Garber, M B; Gongadze, G M

    2009-03-01

    Ribosomal 5S RNA is the only identified target for proteins of the CTC family. All known proteins of this family, except for CTC from Aquifex aeolicus, contain a full-sized 5S rRNA-binding domain. In the present study a mistake in the published A. aeolicus genome is corrected. It has been demonstrated that the ctc gene of this organism encodes the protein with a full-length 5S rRNA-binding domain. This protein binds specifically to the bacterial 5S rRNA. Thereby, our data show that CTC A. aeolicus is not an exception from the other known CTC proteins.

  18. Intracellular localization and interaction of mRNA binding proteins as detected by FRET

    Directory of Open Access Journals (Sweden)

    Port J

    2010-09-01

    Full Text Available Abstract Background A number of RNA binding proteins (BPs bind to A+U rich elements (AREs, commonly present within 3'UTRs of highly regulated RNAs. Individual RNA-BPs proteins can modulate RNA stability, RNA localization, and/or translational efficiency. Although biochemical studies have demonstrated selectivity of ARE-BPs for individual RNAs, less certain is the in vivo composition of RNA-BP multiprotein complexes and how their composition is affected by signaling events and intracellular localization. Using FRET, we previously demonstrated that two ARE-BPs, HuR and AUF1, form stable homomeric and heteromeric associations in the nucleus and cytoplasm. In the current study, we use immuno-FRET of endogenous proteins to examine the intracellular localization and interactions of HuR and AUF1 as well as KSRP, TIA-1, and Hedls. These results were compared to those obtained with their exogenously expressed, fluorescently labeled counterparts. Results All ARE-BPs examined were found to colocalize and to form stable associations with selected other RNA-BPs in one or more cellular locations variably including the nucleus, cytoplasm (in general, or in stress granules or P bodies. Interestingly, FRET based interaction of the translational suppressor, TIA-1, and the decapping protein, Hedls, was found to occur at the interface of stress granules and P bodies, dynamic sites of intracellular RNA storage and/or turnover. To explore the physical interactions of RNA-BPs with ARE containing RNAs, in vitro transcribed Cy3-labeled RNA was transfected into cells. Interestingly, Cy3-RNA was found to coalesce in P body like punctate structures and, by FRET, was found to interact with the RNA decapping proteins, Hedls and Dcp1. Conclusions Biochemical methodologies, such as co-immunoprecipitation, and cell biological approaches such as standard confocal microscopy are useful in demonstrating the possibility of proteins and/or proteins and RNAs interacting. However, as

  19. The RNA-binding protein HuR regulates DNA methylation through stabilization of DNMT3b mRNA

    OpenAIRE

    Lopez de Silanes, I.; Gorospe, M.; Taniguchi, H; Abdelmohsen, K; Srikantan, S.; Alaminos, M.; Berdasco, M.; Urdinguio, R. G.; Fraga, M. F.; Jacinto, F. V.; Esteller, M.

    2009-01-01

    The molecular basis underlying the aberrant DNA-methylation patterns in human cancer is largely unknown. Altered DNA methyltransferase (DNMT) activity is believed to contribute, as DNMT expression levels increase during tumorigenesis. Here, we present evidence that the expression of DNMT3b is post-transcriptionally regulated by HuR, an RNA-binding protein that stabilizes and/or modulates the translation of target mRNAs. The presence of a putative HuR-recognition motif in the DNMT3b 3???UTR pr...

  20. In vivo cross-linking followed by polyA enrichment to identify yeast mRNA binding proteins.

    Science.gov (United States)

    Mitchell, Sarah F; Parker, Roy

    2015-01-01

    mRNA binding proteins regulate gene expression by controlling the processing, localization, decay, and translation of messenger RNAs (mRNAs). To fully understand this process, it is necessary to identify the complete set of mRNA binding proteins. This work describes a method for the systematic identification of yeast mRNA binding proteins. This method applies in vivo UV cross-linking, affinity pull-down of polyA(+) mRNAs, and analysis by mass spectrometry to identify proteins that directly bind to mRNAs.

  1. The RNA-binding protein ELAV regulates Hox RNA processing, expression and function within the Drosophila nervous system.

    Science.gov (United States)

    Rogulja-Ortmann, Ana; Picao-Osorio, Joao; Villava, Casandra; Patraquim, Pedro; Lafuente, Elvira; Aspden, Julie; Thomsen, Stefan; Technau, Gerhard M; Alonso, Claudio R

    2014-05-01

    The regulated head-to-tail expression of Hox genes provides a coordinate system for the activation of specific programmes of cell differentiation according to axial level. Recent work indicates that Hox expression can be regulated via RNA processing but the underlying mechanisms and biological significance of this form of regulation remain poorly understood. Here we explore these issues within the developing Drosophila central nervous system (CNS). We show that the pan-neural RNA-binding protein (RBP) ELAV (Hu antigen) regulates the RNA processing patterns of the Hox gene Ultrabithorax (Ubx) within the embryonic CNS. Using a combination of biochemical, genetic and imaging approaches we demonstrate that ELAV binds to discrete elements within Ubx RNAs and that its genetic removal reduces Ubx protein expression in the CNS leading to the respecification of cellular subroutines under Ubx control, thus defining for the first time a specific cellular role of ELAV within the developing CNS. Artificial provision of ELAV in glial cells (a cell type that lacks ELAV) promotes Ubx expression, suggesting that ELAV-dependent regulation might contribute to cell type-specific Hox expression patterns within the CNS. Finally, we note that expression of abdominal A and Abdominal B is reduced in elav mutant embryos, whereas other Hox genes (Antennapedia) are not affected. Based on these results and the evolutionary conservation of ELAV and Hox genes we propose that the modulation of Hox RNA processing by ELAV serves to adapt the morphogenesis of the CNS to axial level by regulating Hox expression and consequently activating local programmes of neural differentiation.

  2. BORIS/CTCFL is an RNA-binding protein that associates with polysomes.

    Science.gov (United States)

    Ogunkolade, Babatunji W; Jones, Tania A; Aarum, Johan; Szary, Jaroslaw; Owen, Nicholas; Ottaviani, Diego; Mumin, Muhammad A; Patel, Shyam; Pieri, Christopher A; Silver, Andrew R; Sheer, Denise

    2013-11-26

    BORIS (CTCFL), a paralogue of the multifunctional and ubiquitously expressed transcription factor CTCF, is best known for its role in transcriptional regulation. In the nucleus, BORIS is particularly enriched in the nucleolus, a crucial compartment for ribosomal RNA and RNA metabolism. However, little is known about cytoplasmic BORIS, which represents the major pool of BORIS protein. We show, firstly, that BORIS has a putative nuclear export signal in the C-terminal domain. Furthermore, BORIS associates with mRNA in both neural stem cells and young neurons. The majority of the BORIS-associated transcripts are different in the two cell types. Finally, by using polysome profiling we show that BORIS is associated with actively translating ribosomes. We have demonstrated the RNA binding properties of cellular BORIS and its association with actively translating ribosomes. We suggest that BORIS is involved in gene expression at both the transcriptional and post-transcriptional levels.

  3. ATRX directs binding of PRC2 to Xist RNA and Polycomb targets.

    Science.gov (United States)

    Sarma, Kavitha; Cifuentes-Rojas, Catherine; Ergun, Ayla; Del Rosario, Amanda; Jeon, Yesu; White, Forest; Sadreyev, Ruslan; Lee, Jeannie T

    2014-11-06

    X chromosome inactivation (XCI) depends on the long noncoding RNA Xist and its recruitment of Polycomb Repressive Complex 2 (PRC2). PRC2 is also targeted to other sites throughout the genome to effect transcriptional repression. Using XCI as a model, we apply an unbiased proteomics approach to isolate Xist and PRC2 regulators and identified ATRX. ATRX unexpectedly functions as a high-affinity RNA-binding protein that directly interacts with RepA/Xist RNA to promote loading of PRC2 in vivo. Without ATRX, PRC2 cannot load onto Xist RNA nor spread in cis along the X chromosome. Moreover, epigenomic profiling reveals that genome-wide targeting of PRC2 depends on ATRX, as loss of ATRX leads to spatial redistribution of PRC2 and derepression of Polycomb responsive genes. Thus, ATRX is a required specificity determinant for PRC2 targeting and function.

  4. APC/C-mediated degradation of dsRNA-binding protein 4 (DRB4 involved in RNA silencing.

    Directory of Open Access Journals (Sweden)

    Katia Marrocco

    Full Text Available BACKGROUND: Selective protein degradation via the ubiquitin-26S proteasome is a major mechanism underlying DNA replication and cell division in all Eukaryotes. In particular, the APC/C (Anaphase Promoting Complex or Cyclosome is a master ubiquitin protein ligase (E3 that targets regulatory proteins for degradation allowing sister chromatid separation and exit from mitosis. Interestingly, recent work also indicates that the APC/C remains active in differentiated animal and plant cells. However, its role in post-mitotic cells remains elusive and only a few substrates have been characterized. METHODOLOGY/PRINCIPAL FINDINGS: In order to identify novel APC/C substrates, we performed a yeast two-hybrid screen using as the bait Arabidopsis APC10/DOC1, one core subunit of the APC/C, which is required for substrate recruitment. This screen identified DRB4, a double-stranded RNA binding protein involved in the biogenesis of different classes of small RNA (sRNA. This protein interaction was further confirmed in vitro and in plant cells. Moreover, APC10 interacts with DRB4 through the second dsRNA binding motif (dsRBD2 of DRB4, which is also required for its homodimerization and binding to its Dicer partner DCL4. We further showed that DRB4 protein accumulates when the proteasome is inactivated and, most importantly, we found that DRB4 stability depends on APC/C activity. Hence, depletion of Arabidopsis APC/C activity by RNAi leads to a strong accumulation of endogenous DRB4, far beyond its normal level of accumulation. However, we could not detect any defects in sRNA production in lines where DRB4 was overexpressed. CONCLUSIONS/SIGNIFICANCE: Our work identified a first plant substrate of the APC/C, which is not a regulator of the cell cycle. Though we cannot exclude that APC/C-dependent degradation of DRB4 has some regulatory roles under specific growth conditions, our work rather points to a housekeeping function of APC/C in maintaining precise cellular

  5. Deformability in the cleavage site of primary microRNA is not sensed by the double-stranded RNA binding domains in the microprocessor component DGCR8.

    Science.gov (United States)

    Quarles, Kaycee A; Chadalavada, Durga; Showalter, Scott A

    2015-06-01

    The prevalence of double-stranded RNA (dsRNA) in eukaryotic cells has only recently been appreciated. Of interest here, RNA silencing begins with dsRNA substrates that are bound by the dsRNA-binding domains (dsRBDs) of their processing proteins. Specifically, processing of microRNA (miRNA) in the nucleus minimally requires the enzyme Drosha and its dsRBD-containing cofactor protein, DGCR8. The smallest recombinant construct of DGCR8 that is sufficient for in vitro dsRNA binding, referred to as DGCR8-Core, consists of its two dsRBDs and a C-terminal tail. As dsRBDs rarely recognize the nucleotide sequence of dsRNA, it is reasonable to hypothesize that DGCR8 function is dependent on the recognition of specific structural features in the miRNA precursor. Previously, we demonstrated that noncanonical structural elements that promote RNA flexibility within the stem of miRNA precursors are necessary for efficient in vitro cleavage by reconstituted Microprocessor complexes. Here, we combine gel shift assays with in vitro processing assays to demonstrate that neither the N-terminal dsRBD of DGCR8 in isolation nor the DGCR8-Core construct is sensitive to the presence of noncanonical structural elements within the stem of miRNA precursors, or to single-stranded segments flanking the stem. Extending DGCR8-Core to include an N-terminal heme-binding region does not change our conclusions. Thus, our data suggest that although the DGCR8-Core region is necessary for dsRNA binding and recruitment to the Microprocessor, it is not sufficient to establish the previously observed connection between RNA flexibility and processing efficiency.

  6. Anti-Japanese-encephalitis-viral effects of kaempferol and daidzin and their RNA-binding characteristics.

    Directory of Open Access Journals (Sweden)

    Ting Zhang

    Full Text Available BACKGROUND: New therapeutic tools and molecular targets are needed for treatment of Japanese encephalitis virus (JEV infections. JEV requires an α-1 translational frameshift to synthesize the NS1' protein required for viral neuroinvasiveness. Several flavonoids have been shown to possess antiviral activity in vitro against a wide spectrum of viruses. To date, the antiviral activities of flavonol kaempferol (Kae and isoflavonoid daidzin (Dai against JEV have not been described. METHODOLOGY/PRINCIPAL FINDINGS: The 50% cytotoxic concentration (CC(50 and 50% effective concentration (EC(50 against JEV were investigated in BHK21 cells by MTS reduction. Activity against viral genomic RNA and proteins was measured by real-time RT-PCR and western blotting. The frameshift site RNA-binding characterization was also determined by electrospray ionization mass spectrometry, isothermal titration calorimetry and autodocking analysis. EC(50 values of Kae and Dai were 12.6 and 25.9 µM against JEV in cells pretreated before infection, whereas in cells infected before treatment, EC(50 was 21.5 and 40.4 µM, respectively. Kae exhibited more potent activity against JEV and RNA binding in cells following internalization through direct inhibition of viral replication and protein expression, indicating that its antiviral activity was principally due to direct virucidal effects. The JEV frameshift site RNA (fsRNA was selected as a target for assaying Kae and Dai. ITC of fsRNA revealed an apparent K(b value for Kae that was nine fold stronger than that for Dai. This binding was confirmed and localized to the RNA using ESI-MS and autodock analysis. Kae could form non-covalent complexes with fsRNA more easily than Dai could. CONCLUSIONS/SIGNIFICANCE: Kae demonstrates more potent antiviral activity against JEV than does Dai. The mode of action of Kae as an anti-JEV agent seems to be related to its ability to inactivate virus by binding with JEV fsRNA.

  7. The RNA-binding Protein TDP-43 Selectively Disrupts MicroRNA-1/206 Incorporation into the RNA-induced Silencing Complex*♦

    Science.gov (United States)

    King, Isabelle N.; Yartseva, Valeria; Salas, Donaldo; Kumar, Abhishek; Heidersbach, Amy; Ando, D. Michael; Stallings, Nancy R.; Elliott, Jeffrey L.; Srivastava, Deepak; Ivey, Kathryn N.

    2014-01-01

    MicroRNA (miRNA) maturation is regulated by interaction of particular miRNA precursors with specific RNA-binding proteins. Following their biogenesis, mature miRNAs are incorporated into the RNA-induced silencing complex (RISC) where they interact with mRNAs to negatively regulate protein production. However, little is known about how mature miRNAs are regulated at the level of their activity. To address this, we screened for proteins differentially bound to the mature form of the miR-1 or miR-133 miRNA families. These muscle-enriched, co-transcribed miRNA pairs cooperate to suppress smooth muscle gene expression in the heart. However, they also have opposing roles, with the miR-1 family, composed of miR-1 and miR-206, promoting myogenic differentiation, whereas miR-133 maintains the progenitor state. Here, we describe a physical interaction between TDP-43, an RNA-binding protein that forms aggregates in the neuromuscular disease, amyotrophic lateral sclerosis, and the miR-1, but not miR-133, family. Deficiency of the TDP-43 Drosophila ortholog enhanced dmiR-1 activity in vivo. In mammalian cells, TDP-43 limited the activity of both miR-1 and miR-206, but not the miR-133 family, by disrupting their RISC association. Consistent with TDP-43 dampening miR-1/206 activity, protein levels of the miR-1/206 targets, IGF-1 and HDAC4, were elevated in TDP-43 transgenic mouse muscle. This occurred without corresponding Igf-1 or Hdac4 mRNA increases and despite higher miR-1 and miR-206 expression. Our findings reveal that TDP-43 negatively regulates the activity of the miR-1 family of miRNAs by limiting their bioavailability for RISC loading and suggest a processing-independent mechanism for differential regulation of miRNA activity. PMID:24719334

  8. Binding of Y-box proteins to RNA: involvement of different protein domains.

    Science.gov (United States)

    Ladomery, M; Sommerville, J

    1994-01-01

    Eukaryotic Y-box proteins are reported to interact with a wide variety of nucleic acid structures to act as transcription factors and mRNA masking proteins. The modular structure of Y-box proteins includes a highly conserved N-terminal cold-shock domain (CSD, equivalent to the bacterial cold-shock proteins) plus four basic C-terminal domains containing arginine clusters and aromatic residues. In addition, the basic domains are separated by acidic regions which contain several potential sites for serine/threonine phosphorylation. The interaction of Y-box proteins, isolated from Xenopus oocytes (FRGY2 type), with RNA molecules has been studied by UV crosslinking and protein fragmentation. We have identified two distinct binding activities. The CSD interacts preferentially with the polypurines poly(A,G) and poly(G) but not poly(A), this activity being sensitive to 5 mM MgCl2 but not to 5 mM spermidine. In the presence of 1 mM MgCl2 or 1 mM spermidine, the basic domains interact preferentially with poly(C,U), this activity being sensitive to 0.5 M NaCl. Binding of the basic domains is also sensitive to low concentrations of heparin. The basic domains can be crosslinked individually to labelled RNA. These results are discussed with reference to the various specificities noted in the binding of Y-box proteins to RNA and DNA. Images PMID:7530842

  9. Metal ion binding and function in natural and artificial small RNA enzymes from a structural perspective.

    Science.gov (United States)

    Wedekind, Joseph E

    2011-01-01

    Ribozymes are often perceived as part of an antiquated catalytic arsenal hearkening back to a pre-biotic RNA World that was eventually supplanted by proteins. However, recent genome-wide searches have revealed a plethora of new catalytic RNA motifs that appear to be variations on well-known themes. This suggests that ribozymes have continued to evolve in order to fulfill specific, RNA-essential biological niches. Although such ribozymes are small and catalyze one-step phosphodiester-bond scission reactions, ongoing structure and function analyses at the lab bench have demonstrated that RNA has the capacity for a diverse number of reactions such as carbon-carbon bond formation, and tRNA aminoacylation. Here we describe the fundamental structure and metal binding properties of four naturally occurring RNA enzymes: the hammerhead, hairpin, hepatitis delta virus, and glmS metabolite sensing ribozyme. In addition, we discuss the fold and ion coordination of three artificial ribozymes developed to probe the boundaries of RNA catalysis; these include the leadzyme, the flexizyme, and the Diels-Alder ribozyme. Our approach is to relate structure to function with the knowledge of ideal metal-ion coordination geometry that we have derived herein from surveys of high-resolution small molecule structures. An emergent theme is that natural and artificial ribozymes that catalyze single-step reactions often possess a pre-formed active site. Multivalent ions facilitate RNA active site formation, but can also provide Lewis acid functionality that is necessary for catalysis. When metal ion binding isn't possible, ribozymes make due by ionizing their bases, or by recruiting cofactors that augment their chemical functionality.

  10. In Vitro Assays for RNA Binding and Protein Priming of Hepatitis B Virus Polymerase.

    Science.gov (United States)

    Clark, Daniel N; Jones, Scott A; Hu, Jianming

    2017-01-01

    The hepatitis B virus (HBV) polymerase synthesizes the viral DNA genome from the pre-genomic RNA (pgRNA) template through reverse transcription. Initiation of viral DNA synthesis is accomplished via a novel protein priming mechanism, so named because the polymerase itself acts as a primer, whereby the initiating nucleotide becomes covalently linked to a tyrosine residue on the viral polymerase. Protein priming, in turn, depends on specific recognition of the packaging signal on pgRNA called epsilon. These early events in viral DNA synthesis can now be dissected in vitro as described here.The polymerase is expressed in mammalian cells and purified by immunoprecipitation. The purified protein is associated with host cell factors, is enzymatically active, and its priming activity is epsilon dependent. A minimal epsilon RNA construct from pgRNA is co-expressed with the polymerase in cells. This RNA binds to and co-immunoprecipitates with the polymerase. Modifications can be made to either the epsilon RNA or the polymerase protein by manipulating the expression plasmids. Also, the priming reaction itself can be modified to assay for the initiation or subsequent DNA synthesis during protein priming, the susceptibility of the polymerase to chemical inhibitors, and the precise identification of the DNA products upon their release from the polymerase. The identity of associated host factors can also be evaluated. This protocol closely mirrors our current understanding of the RNA binding and protein priming steps of the HBV replication cycle, and it is amenable to modification. It should therefore facilitate both basic research and drug discovery.

  11. APUM5, encoding a Pumilio RNA binding protein, negatively regulates abiotic stress responsive gene expression

    Science.gov (United States)

    2014-01-01

    Background A mutant screening was carried out previously to look for new genes related to the Cucumber mosaic virus infection response in Arabidopsis. A Pumilio RNA binding protein-coding gene, Arabidopsis Pumilio RNA binding protein 5 (APUM5), was obtained from this screening. Results APUM5 transcriptional profiling was carried out using a bioinformatics tool. We found that APUM5 was associated with both biotic and abiotic stress responses. However, bacterial and fungal pathogen infection susceptibility was not changed in APUM5 transgenic plants compared to that in wild type plants although APUM5 expression was induced upon pathogen infection. In contrast, APUM5 was involved in the abiotic stress response. 35S-APUM5 transgenic plants showed hypersensitive phenotypes under salt and drought stresses during germination, primary root elongation at the seedling stage, and at the vegetative stage in soil. We also showed that some abiotic stress-responsive genes were negatively regulated in 35S-APUM5 transgenic plants. The APUM5-Pumilio homology domain (PHD) protein bound to the 3′ untranslated region (UTR) of the abiotic stress-responsive genes which contained putative Pumilio RNA binding motifs at the 3′ UTR. Conclusions These results suggest that APUM5 may be a new post-transcriptional regulator of the abiotic stress response by direct binding of target genes 3′ UTRs. PMID:24666827

  12. Crystal structure of Hfq from Bacillus subtilis in complex with SELEX-derived RNA aptamer: insight into RNA-binding properties of bacterial Hfq

    Science.gov (United States)

    Someya, Tatsuhiko; Baba, Seiki; Fujimoto, Mai; Kawai, Gota; Kumasaka, Takashi; Nakamura, Kouji

    2012-01-01

    Bacterial Hfq is a protein that plays an important role in the regulation of genes in cooperation with sRNAs. Escherichia coli Hfq (EcHfq) has two or more sites that bind RNA(s) including U-rich and/or the poly(A) tail of mRNA. However, functional and structural information about Bacillus subtilis Hfq (BsHfq) including the RNA sequences that specifically bind to it remain unknown. Here, we describe RNA aptamers including fragment (AG)3A that are recognized by BsHfq and crystal structures of the BsHfq–(AG)3A complex at 2.2 Å resolution. Mutational and structural studies revealed that the RNA fragment binds to the distal site, one of the two binding sites on Hfq, and identified amino acid residues that are critical for sequence-specific interactions between BsHfq and (AG)3A. In particular, R32 appears to interact with G bases in (AG)3A. Poly(A) also binds to the distal site of EcHfq, but the overall RNA structure and protein–RNA interaction patterns engaged in the R32 residues of BsHfq–(AG)3A differ from those of EcHfq–poly(A). These findings provide novel insight into how the Hfq homologue recognizes RNA. PMID:22053080

  13. The human nuclear poly(a-binding protein promotes RNA hyperadenylation and decay.

    Directory of Open Access Journals (Sweden)

    Stefan M Bresson

    Full Text Available Control of nuclear RNA stability is essential for proper gene expression, but the mechanisms governing RNA degradation in mammalian nuclei are poorly defined. In this study, we uncover a mammalian RNA decay pathway that depends on the nuclear poly(A-binding protein (PABPN1, the poly(A polymerases (PAPs, PAPα and PAPγ, and the exosome subunits RRP6 and DIS3. Using a targeted knockdown approach and nuclear RNA reporters, we show that PABPN1 and PAPα, redundantly with PAPγ, generate hyperadenylated decay substrates that are recognized by the exosome and degraded. Poly(A tail extension appears to be necessary for decay, as cordycepin treatment or point mutations in the PAP-stimulating domain of PABPN1 leads to the accumulation of stable transcripts with shorter poly(A tails than controls. Mechanistically, these data suggest that PABPN1-dependent promotion of PAP activity can stimulate nuclear RNA decay. Importantly, efficiently exported RNAs are unaffected by this decay pathway, supporting an mRNA quality control function for this pathway. Finally, analyses of both bulk poly(A tails and specific endogenous transcripts reveals that a subset of nuclear RNAs are hyperadenylated in a PABPN1-dependent fashion, and this hyperadenylation can be either uncoupled or coupled with decay. Our results highlight a complex relationship between PABPN1, PAPα/γ, and nuclear RNA decay, and we suggest that these activities may play broader roles in the regulation of human gene expression.

  14. Localized frustration and binding-induced conformational change in recognition of 5S RNA by TFIIIA zinc finger.

    Science.gov (United States)

    Tan, Cheng; Li, Wenfei; Wang, Wei

    2013-12-19

    Protein TFIIIA is composed of nine tandemly arranged Cys2His2 zinc fingers. It can bind either to the 5S RNA gene as a transcription factor or to the 5S RNA transcript as a chaperone. Although structural and biochemical data provided valuable information on the recognition between the TFIIIIA and the 5S DNA/RNA, the involved conformational motions and energetic factors contributing to the binding affinity and specificity remain unclear. In this work, we conducted MD simulations and MM/GBSA calculations to investigate the binding-induced conformational changes in the recognition of the 5S RNA by the central three zinc fingers of TFIIIA and the energetic factors that influence the binding affinity and specificity at an atomistic level. Our results revealed drastic interdomain conformational changes between these three zinc fingers, involving the exposure/burial of several crucial DNA/RNA binding residues, which can be related to the competition between DNA and RNA for the binding of TFIIIA. We also showed that the specific recognition between finger 4/finger 6 and the 5S RNA introduces frustrations to the nonspecific interactions between finger 5 and the 5S RNA, which may be important to achieve optimal binding affinity and specificity.

  15. Clusters of basic amino acids contribute to RNA binding and nucleolar localization of ribosomal protein L22.

    Directory of Open Access Journals (Sweden)

    Jennifer L Houmani

    Full Text Available The ribosomal protein L22 is a component of the 60S eukaryotic ribosomal subunit. As an RNA-binding protein, it has been shown to interact with both cellular and viral RNAs including 28S rRNA and the Epstein-Barr virus encoded RNA, EBER-1. L22 is localized to the cell nucleus where it accumulates in nucleoli. Although previous studies demonstrated that a specific amino acid sequence is required for nucleolar localization, the RNA-binding domain has not been identified. Here, we investigated the hypothesis that the nucleolar accumulation of L22 is linked to its ability to bind RNA. To address this hypothesis, mutated L22 proteins were generated to assess the contribution of specific amino acids to RNA binding and protein localization. Using RNA-protein binding assays, we demonstrate that basic amino acids 80-93 are required for high affinity binding of 28S rRNA and EBER-1 by L22. Fluorescence localization studies using GFP-tagged mutated L22 proteins further reveal that basic amino acids 80-93 are critical for nucleolar accumulation and for incorporation into ribosomes. Our data support the growing consensus that the nucleolar accumulation of ribosomal proteins may not be mediated by a defined localization signal, but rather by specific interaction with established nucleolar components such as rRNA.

  16. Plakophilin-associated RNA-binding proteins in prostate cancer and their implications in tumor progression and metastasis

    NARCIS (Netherlands)

    Yang, Cheng; Stroebel, Philipp; Marx, Alexander; Hofmann, Ilse

    2013-01-01

    Both plakophilins (PKP) 1 and 3 play a role in the progression of prostate cancer. The RNA-binding proteins (RBPs) GAP-SH3-binding protein (G3BP), fragile-X-related protein 1 (FXR1), poly(A)-binding protein, cytoplasmic 1 (PABPC1), and up-frameshift factor 1 (UPF1) are associated with PKP3. All thes

  17. Cyanobacteria contain a structural homologue of the Hfq protein with altered RNA-binding properties

    DEFF Research Database (Denmark)

    Bøggild, Andreas; Overgaard, Martin; Valentin-Hansen, Poul

    2009-01-01

    regulating mRNA turnover in eukaryotes. However, bacterial Hfq proteins are homohexameric, whereas eukaryotic Sm/Lsm proteins are heteroheptameric. Recently, Hfq proteins with poor sequence conservation were identified in archaea and cyanobacteria. In this article, we describe crystal structures of the Hfq...... proteins from the cyanobacteria Synechocystis sp. PCC 6803 and Anabaena PCC 7120 at 1.3 and 2.3 A resolution, respectively, and show that they retain the classic Sm fold despite low sequence conservation. In addition, the intersubunit contacts and RNA-binding site are divergent, and we show biochemically...

  18. Cyanobacteria contain a structural homologue of the Hfq protein with altered RNA binding properties

    DEFF Research Database (Denmark)

    Bøggild, Andreas; Overgaard, Martin; Valentin-Hansen, Poul

    2009-01-01

    regulating mRNA turnover in eukaryotes. However, bacterial Hfq proteins are homohexameric, whereas eukaryotic Sm/Lsm proteins are heteroheptameric. Recently, Hfq proteins with poor sequence conservation were identified in archaea and cyanobacteria. In this article, we describe crystal structures of the Hfq...... proteins from the cyanobacteria Synechocystis sp. PCC 6803 and Anabaena PCC 7120 at 1.3 and 2.3 A resolution, respectively, and show that they retain the classic Sm fold despite low sequence conservation. In addition, the intersubunit contacts and RNA-binding site are divergent, and we show biochemically...

  19. Quinacrine impairs enterovirus 71 RNA replication by preventing binding of polypyrimidine-tract binding protein with internal ribosome entry sites.

    Directory of Open Access Journals (Sweden)

    Jianmin Wang

    Full Text Available Since the 1980s, epidemics of enterovirus 71 (EV71 and other enteroviruses have occurred in Asian countries and regions, causing a wide range of human diseases. No effective therapy is available for the treatment of these infections. Internal ribosome entry sites (IRESs are indispensable for the initiation of translation in enteroviruses. Several cellular factors, as well as the ribosome, are recruited to the conserved IRES during this process. Quinacrine intercalates into the RNA architecture and inhibits RNA transcription and protein synthesis, and a recent study showed that quinacrine inhibited encephalomyocarditis virus and poliovirus IRES-mediated translation in vitro without disrupting internal cellular IRES. Here, we report that quinacrine was highly active against EV71, protecting cells from EV71 infection. Replication of viral RNA, expression of viral capsid protein, and production of virus were all strongly inhibited by quinacrine. Interaction of the polypyrimidine tract-binding protein (PTB with the conserved IRES was prevented by quinacrine. Coxsackieviruses and echovirus were also inhibited by quinacrine in cultured cells. These results indicate that quinacrine may serve as a potential protective agent for use in the treatment of patients with chronic enterovirus infection.

  20. IN VITRO SELECTION AND CHARACTERIZATION OF CELLULOSE-BINDING RNA APTAMERS USING ISOTHERMAL AMPLIFICATION

    Science.gov (United States)

    Boese, B. J.; Corbino, K.; Breaker, R. R.

    2017-01-01

    We sought to create new cellulose-binding RNA aptamers for use as modular components in the engineering of complex functional nucleic acids. We designed our in vitro selection strategy to incorporate self-sustained sequence replication (3SR), which is an isothermal nucleic acid amplification protocol that allows for the rapid amplification of RNAs with little manipulation. The best performing aptamer representative was chosen for reselection and further optimization. The aptamer exhibits robust affinity for cellulose in both the powdered and paper form, but did not show any significant affinity for closely related polysaccharides. The minimal cellulose-binding RNA aptamer also can be grafted onto other RNAs to permit the isolation of RNAs from complex biochemical mixtures via cellulose affinity chromatography. This was demonstrated by fusing the aptamer to a glmS ribozyme sequence, and selectively eluting ribozyme cleavage products from cellulose using the glucosamine 6-phosphate to activate glmS ribozyme function. PMID:18696364

  1. 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.

  2. Efficient and dynamic nuclear localization of green fluorescent protein via RNA binding

    Energy Technology Data Exchange (ETDEWEB)

    Kitamura, Akira; Nakayama, Yusaku; Kinjo, Masataka, E-mail: kinjo@sci.hokudai.ac.jp

    2015-07-31

    Classical nuclear localization signal (NLS) sequences have been used for artificial localization of green fluorescent protein (GFP) in the nucleus as a positioning marker or for measurement of the nuclear-cytoplasmic shuttling rate in living cells. However, the detailed mechanism of nuclear retention of GFP-NLS remains unclear. Here, we show that a candidate mechanism for the strong nuclear retention of GFP-NLS is via the RNA-binding ability of the NLS sequence. GFP tagged with a classical NLS derived from Simian virus 40 (GFP-NLS{sup SV40}) localized not only in the nucleoplasm, but also to the nucleolus, the nuclear subdomain in which ribosome biogenesis takes place. GFP-NLS{sup SV40} in the nucleolus was mobile, and intriguingly, the diffusion coefficient, which indicates the speed of diffusing molecules, was 1.5-fold slower than in the nucleoplasm. Fluorescence correlation spectroscopy (FCS) analysis showed that GFP-NLS{sup SV40} formed oligomers via RNA binding, the estimated molecular weight of which was larger than the limit for passive nuclear export into the cytoplasm. These findings suggest that the nuclear localization of GFP-NLS{sup SV40} likely results from oligomerization mediated via RNA binding. The analytical technique used here can be applied for elucidating the details of other nuclear localization mechanisms, including those of several types of nuclear proteins. In addition, GFP-NLS{sup SV40} can be used as an excellent marker for studying both the nucleoplasm and nucleolus in living cells. - Highlights: • Nuclear localization signal-tagged GFP (GFP-NLS) showed clear nuclear localization. • The GFP-NLS dynamically localized not only in the nucleoplasm, but also to the nucleolus. • The nuclear localization of GFP-NLS results from transient oligomerization mediated via RNA binding. • Our NLS-tagging procedure is ideal for use in artificial sequestration of proteins in the nucleus.

  3. Gemin5: A Multitasking RNA-Binding Protein Involved in Translation Control

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    David Piñeiro

    2015-04-01

    Full Text Available Gemin5 is a RNA-binding protein (RBP that was first identified as a peripheral component of the survival of motor neurons (SMN complex. This predominantly cytoplasmic protein recognises the small nuclear RNAs (snRNAs through its WD repeat domains, allowing assembly of the SMN complex into small nuclear ribonucleoproteins (snRNPs. Additionally, the amino-terminal end of the protein has been reported to possess cap-binding capacity and to interact with the eukaryotic initiation factor 4E (eIF4E. Gemin5 was also shown to downregulate translation, to be a substrate of the picornavirus L protease and to interact with viral internal ribosome entry site (IRES elements via a bipartite non-canonical RNA-binding site located at its carboxy-terminal end. These features link Gemin5 with translation control events. Thus, beyond its role in snRNPs biogenesis, Gemin5 appears to be a multitasking protein cooperating in various RNA-guided processes. In this review, we will summarise current knowledge of Gemin5 functions. We will discuss the involvement of the protein on translation control and propose a model to explain how the proteolysis fragments of this RBP in picornavirus-infected cells could modulate protein synthesis.

  4. The core microprocessor component DiGeorge syndrome critical region 8 (DGCR8) is a nonspecific RNA-binding protein.

    Science.gov (United States)

    Roth, Braden M; Ishimaru, Daniella; Hennig, Mirko

    2013-09-13

    MicroRNA (miRNA) biogenesis follows a conserved succession of processing steps, beginning with the recognition and liberation of an miRNA-containing precursor miRNA hairpin from a large primary miRNA transcript (pri-miRNA) by the Microprocessor, which consists of the nuclear RNase III Drosha and the double-stranded RNA-binding domain protein DGCR8 (DiGeorge syndrome critical region protein 8). Current models suggest that specific recognition is driven by DGCR8 detection of single-stranded elements of the pri-miRNA stem-loop followed by Drosha recruitment and pri-miRNA cleavage. Because countless RNA transcripts feature single-stranded-dsRNA junctions and DGCR8 can bind hundreds of mRNAs, we explored correlations between RNA binding properties of DGCR8 and specific pri-miRNA substrate processing. We found that DGCR8 bound single-stranded, double-stranded, and random hairpin transcripts with similar affinity. Further investigation of DGCR8/pri-mir-16 interactions by NMR detected intermediate exchange regimes over a wide range of stoichiometric ratios. Diffusion analysis of DGCR8/pri-mir-16 interactions by pulsed field gradient NMR lent further support to dynamic complex formation involving free components in exchange with complexes of varying stoichiometry, although in vitro processing assays showed exclusive cleavage of pri-mir-16 variants bearing single-stranded flanking regions. Our results indicate that DGCR8 binds RNA nonspecifically. Therefore, a sequential model of DGCR8 recognition followed by Drosha recruitment is unlikely. Known RNA substrate requirements are broad and include 70-nucleotide hairpins with unpaired flanking regions. Thus, specific RNA processing is likely facilitated by preformed DGCR8-Drosha heterodimers that can discriminate between authentic substrates and other hairpins.

  5. The Nuclear PolyA-Binding Protein Nab2p Is Essential for mRNA Production.

    Science.gov (United States)

    Schmid, Manfred; Olszewski, Pawel; Pelechano, Vicent; Gupta, Ishaan; Steinmetz, Lars M; Jensen, Torben Heick

    2015-07-07

    Polyadenylation of mRNA is a key step in eukaryotic gene expression. However, despite the major impact of poly(A) tails on mRNA metabolism, the precise roles of poly(A)-binding proteins (PABPs) in nuclear mRNA biogenesis remain elusive. Here, we demonstrate that rapid nuclear depletion of the S. cerevisiae PABP Nab2p leads to a global loss of cellular mRNA, but not of RNA lacking poly(A) tails. Disappearance of mRNA is a nuclear event, but not due to decreased transcription. Instead, the absence of Nab2p results in robust nuclear mRNA decay by the ribonucleolytic RNA exosome in a polyadenylation-dependent process. We conclude that Nab2p is required to protect early mRNA and therefore constitutes a crucial nuclear mRNA biogenesis factor. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  6. The Nuclear PolyA-Binding Protein Nab2p Is Essential for mRNA Production

    DEFF Research Database (Denmark)

    Schmid, Manfred; Olszewski, Pawel; Pelechano, Vicent;

    2015-01-01

    Polyadenylation of mRNA is a key step in eukaryotic gene expression. However, despite the major impact of poly(A) tails on mRNA metabolism, the precise roles of poly(A)-binding proteins (PABPs) in nuclear mRNA biogenesis remain elusive. Here, we demonstrate that rapid nuclear depletion of the S....... cerevisiae PABP Nab2p leads to a global loss of cellular mRNA, but not of RNA lacking poly(A) tails. Disappearance of mRNA is a nuclear event, but not due to decreased transcription. Instead, the absence of Nab2p results in robust nuclear mRNA decay by the ribonucleolytic RNA exosome in a polyadenylation......-dependent process. We conclude that Nab2p is required to protect early mRNA and therefore constitutes a crucial nuclear mRNA biogenesis factor....

  7. New Insights into the Functions of Transcription Factors that Bind the RNA Polymerase Secondary Channel.

    Science.gov (United States)

    Zenkin, Nikolay; Yuzenkova, Yulia

    2015-06-25

    Transcription elongation is regulated at several different levels, including control by various accessory transcription elongation factors. A distinct group of these factors interacts with the RNA polymerase secondary channel, an opening at the enzyme surface that leads to its active center. Despite investigation for several years, the activities and in vivo roles of some of these factors remain obscure. Here, we review the recent progress in understanding the functions of the secondary channel binding factors in bacteria. In particular, we highlight the surprising role of global regulator DksA in fidelity of RNA synthesis and the resolution of RNA polymerase traffic jams by the Gre factor. These findings indicate a potential link between transcription fidelity and collisions of the transcription and replication machineries.

  8. Bacterial 5S rRNA-binding proteins of the CTC family.

    Science.gov (United States)

    Gongadze, G M; Korepanov, A P; Korobeinikova, A V; Garber, M B

    2008-12-01

    The presence of CTC family proteins is a unique feature of bacterial cells. In the CTC family, there are true ribosomal proteins (found in ribosomes of exponentially growing cells), and at the same time there are also proteins temporarily associated with the ribosome (they are produced by the cells under stress only and incorporate into the ribosome). One feature is common for these proteins - they specifically bind to 5S rRNA. In this review, the history of investigations of the best known representatives of this family is described briefly. Structural organization of the CTC family proteins and their occurrence among known taxonomic bacterial groups are discussed. Structural features of 5S rRNA and CTC protein are described that predetermine their specific interaction. Taking into account the position of a CTC protein and its intermolecular contacts in the ribosome, a possible role of its complex with 5S rRNA in ribosome functioning is discussed.

  9. New Insights into the Functions of Transcription Factors that Bind the RNA Polymerase Secondary Channel

    Directory of Open Access Journals (Sweden)

    Nikolay Zenkin

    2015-06-01

    Full Text Available Transcription elongation is regulated at several different levels, including control by various accessory transcription elongation factors. A distinct group of these factors interacts with the RNA polymerase secondary channel, an opening at the enzyme surface that leads to its active center. Despite investigation for several years, the activities and in vivo roles of some of these factors remain obscure. Here, we review the recent progress in understanding the functions of the secondary channel binding factors in bacteria. In particular, we highlight the surprising role of global regulator DksA in fidelity of RNA synthesis and the resolution of RNA polymerase traffic jams by the Gre factor. These findings indicate a potential link between transcription fidelity and collisions of the transcription and replication machineries.

  10. Tobacco calmodulin-like protein provides secondary defense by binding to and directing degradation of virus RNA silencing suppressors.

    Science.gov (United States)

    Nakahara, Kenji S; Masuta, Chikara; Yamada, Syouta; Shimura, Hanako; Kashihara, Yukiko; Wada, Tomoko S; Meguro, Ayano; Goto, Kazunori; Tadamura, Kazuki; Sueda, Kae; Sekiguchi, Toru; Shao, Jun; Itchoda, Noriko; Matsumura, Takeshi; Igarashi, Manabu; Ito, Kimihito; Carthew, Richard W; Uyeda, Ichiro

    2012-06-19

    RNA silencing (RNAi) induced by virus-derived double-stranded RNA (dsRNA), which is in a sense regarded as a pathogen-associated molecular pattern (PAMP) of viruses, is a general plant defense mechanism. To counteract this defense, plant viruses express RNA silencing suppressors (RSSs), many of which bind to dsRNA and attenuate RNAi. We showed that the tobacco calmodulin-like protein, rgs-CaM, counterattacked viral RSSs by binding to their dsRNA-binding domains and sequestering them from inhibiting RNAi. Autophagy-like protein degradation seemed to operate to degrade RSSs with the sacrifice of rgs-CaM. These RSSs could thus be regarded as secondary viral PAMPs. This study uncovered a unique defense system in which an rgs-CaM-mediated countermeasure against viral RSSs enhanced host antiviral RNAi in tobacco.

  11. Cytoplasmic translocation of polypyrimidine tract-binding protein and its binding to viral RNA during Japanese encephalitis virus infection inhibits virus replication.

    Directory of Open Access Journals (Sweden)

    Deepika Bhullar

    Full Text Available Japanese encephalitis virus (JEV has a single-stranded, positive-sense RNA genome containing a single open reading frame flanked by the 5'- and 3'-non-coding regions (NCRs. The virus genome replicates via a negative-sense RNA intermediate. The NCRs and their complementary sequences in the negative-sense RNA are the sites for assembly of the RNA replicase complex thereby regulating the RNA synthesis and virus replication. In this study, we show that the 55-kDa polypyrimidine tract-binding protein (PTB interacts in vitro with both the 5'-NCR of the positive-sense genomic RNA--5NCR(+, and its complementary sequence in the negative-sense replication intermediate RNA--3NCR(-. The interaction of viral RNA with PTB was validated in infected cells by JEV RNA co-immunoprecipitation and JEV RNA-PTB colocalization experiments. Interestingly, we observed phosphorylation-coupled translocation of nuclear PTB to cytoplasmic foci that co-localized with JEV RNA early during JEV infection. Our studies employing the PTB silencing and over-expression in cultured cells established an inhibitory role of PTB in JEV replication. Using RNA-protein binding assay we show that PTB competitively inhibits association of JEV 3NCR(- RNA with viral RNA-dependent RNA polymerase (NS5 protein, an event required for the synthesis of the plus-sense genomic RNA. cAMP is known to promote the Protein kinase A (PKA-mediated PTB phosphorylation. We show that cells treated with a cAMP analogue had an enhanced level of phosphorylated PTB in the cytoplasm and a significantly suppressed JEV replication. Data presented here show a novel, cAMP-induced, PTB-mediated, innate host response that could effectively suppress JEV replication in mammalian cells.

  12. Regulatory Interactions of Csr Components: the RNA Binding Protein CsrA Activates csrB Transcription in Escherichia coli

    OpenAIRE

    Gudapaty, Seshagirirao; Suzuki, Kazushi; Wang, Xin; Babitzke, Paul; Romeo, Tony

    2002-01-01

    The global regulator CsrA (carbon storage regulator) of Escherichia coli is a small RNA binding protein that represses various metabolic pathways and processes that are induced in the stationary phase of growth, while it activates certain exponential phase functions. Both repression and activation by CsrA involve posttranscriptional mechanisms, in which CsrA binding to mRNA leads to decreased or increased transcript stability, respectively. CsrA also binds to a small untranslated RNA, CsrB, f...

  13. Identification of Endogenous mRNA-Binding Proteins in Yeast Using Crosslinking and PolyA Enrichment.

    Science.gov (United States)

    Mitchell, Sarah F; Parker, Roy

    2016-01-01

    The maturation, localization, stability, and translation of messenger RNAs (mRNAs) are regulated by a wide variety of mRNA-binding proteins. Identification of the complete set of mRNA-binding proteins is a key step in understanding the regulation of gene expression. Herein, we describe a method for identifying yeast mRNA-binding proteins in a systematic manner using UV crosslinking, purification of polyA(+) mRNAs under denaturing conditions, and mass spectrometry to identify covalently bound proteins.

  14. Prediction of RNA binding residues: an extensive analysis based on structure and function to select the best predictor.

    Science.gov (United States)

    Nagarajan, R; Gromiha, M Michael

    2014-01-01

    Protein-RNA complexes play key roles in several cellular processes by the interactions of amino acids with RNA. To understand the recognition mechanism, it is important to identify the specific amino acids involved in RNA binding. Various computational methods have been developed for predicting RNA binding residues from protein sequence. However, their performances mainly depend on the training dataset, feature selection for developing a model and learning capacity of the model. Hence, it is important to reveal the correspondence between the performance of methods and properties of RNA-binding proteins (RBPs). In this work, we have collected all available RNA binding residues prediction methods and revealed their performances on unbiased, stringent and diverse datasets for RBPs with less than 25% sequence identity based on structural class, fold, superfamily, family, protein function, RNA type, RNA strand and RNA conformation. The best methods for each type of RBPs and the type of RBPs, which require further refinement in prediction, have been brought out. We also analyzed the performance of these methods for the disordered regions, structures which are not included in the training dataset and recently solved structures. The reliability of prediction is better than randomly choosing any method or combination of methods. This approach would be a valuable resource for biologists to choose the best method based on the type of RBPs for designing their experiments and the tool is freely accessible online at www.iitm.ac.in/bioinfo/RNA-protein/.

  15. Prediction of RNA binding residues: an extensive analysis based on structure and function to select the best predictor.

    Directory of Open Access Journals (Sweden)

    R Nagarajan

    Full Text Available Protein-RNA complexes play key roles in several cellular processes by the interactions of amino acids with RNA. To understand the recognition mechanism, it is important to identify the specific amino acids involved in RNA binding. Various computational methods have been developed for predicting RNA binding residues from protein sequence. However, their performances mainly depend on the training dataset, feature selection for developing a model and learning capacity of the model. Hence, it is important to reveal the correspondence between the performance of methods and properties of RNA-binding proteins (RBPs. In this work, we have collected all available RNA binding residues prediction methods and revealed their performances on unbiased, stringent and diverse datasets for RBPs with less than 25% sequence identity based on structural class, fold, superfamily, family, protein function, RNA type, RNA strand and RNA conformation. The best methods for each type of RBPs and the type of RBPs, which require further refinement in prediction, have been brought out. We also analyzed the performance of these methods for the disordered regions, structures which are not included in the training dataset and recently solved structures. The reliability of prediction is better than randomly choosing any method or combination of methods. This approach would be a valuable resource for biologists to choose the best method based on the type of RBPs for designing their experiments and the tool is freely accessible online at www.iitm.ac.in/bioinfo/RNA-protein/.

  16. Binding of the 9-O-N-aryl/arylalkyl amino carbonyl methyl substituted berberine analogs to tRNA(phe..

    Directory of Open Access Journals (Sweden)

    Anirban Basu

    Full Text Available BACKGROUND: Three new analogs of berberine with aryl/ arylalkyl amino carbonyl methyl substituent at the 9-position of the isoquinoline chromophore along with berberrubine were studied for their binding to tRNA(phe by wide variety of biophysical techniques like spectrophotometry, spectrofluorimetry, circular dichroism, thermal melting, viscosity and isothermal titration calorimetry. METHODOLOGY/ PRINCIPAL FINDINGS: Scatchard binding isotherms revealed that the cooperative binding mode of berberine was propagated in the analogs also. Thermal melting studies showed that all the 9-O-N-aryl/arylalkyl amino carbonyl methyl substituted berberine analogs stabilized the tRNA(phe more in comparison to berberine. Circular dichroism studies showed that these analogs perturbed the structure of tRNA(phe more in comparison to berberine. Ferrocyanide quenching studies and viscosity results proved the intercalative binding mode of these analogs into the helical organization of tRNA(phe. The binding was entropy driven for the analogs in sharp contrast to the enthalpy driven binding of berberine. The introduction of the aryl/arylalkyl amino carbonyl methyl substituent at the 9-position thus switched the enthalpy driven binding of berberine to entropy dominated binding. Salt and temperature dependent calorimetric studies established the involvement of multiple weak noncovalent interactions in the binding process. CONCLUSIONS/ SIGNIFICANCE: The results showed that 9-O-N-aryl/arylalkyl amino carbonyl methyl substituted berberine analogs exhibited almost ten folds higher binding affinity to tRNA(phe compared to berberine whereas the binding of berberrubine was dramatically reduced by about twenty fold in comparison to berberine. The spacer length of the substitution at the 9-position of the isoquinoline chromophore appears to be critical in modulating the binding affinities towards tRNA(phe.

  17. Sequential dimerization of human zipcode-binding protein IMP1 on RNA: a cooperative mechanism providing RNP stability

    DEFF Research Database (Denmark)

    Nielsen, J.; Kristensen, M. A.; Willemoes, Martin;

    2004-01-01

    zipcode-binding protein IMP1 on targets in the 3'-UTR from Igf-II mRNA and in H19 RNA. In both cases, two molecules of IMP1 bound to RNA by a sequential, cooperative mechanism, characterized by an initial fast step, followed by a slow second step. The first step created an obligatory assembly intermediate...... of low stability, whereas the second step was the discriminatory event that converted a putative RNA target into a ‘locked' stable RNP. The ability to dimerize was also observed between members of the IMP family of zipcode-binding proteins, providing a multitude of further interaction possibilities...

  18. AtMBD6, a methyl CpG binding domain protein, maintains gene silencing in Arabidopsis by interacting with RNA binding proteins.

    Science.gov (United States)

    Parida, Adwaita Prasad; Sharma, Amrapali; Sharma, Arun Kumar

    2017-03-01

    DNA methylation, mediated by double-stranded RNA, is a conserved epigenetic phenomenon that protects a genome from transposons, silences unwanted genes and has a paramount function in plant or animal development. Methyl CpG binding domain proteins are members of a class of proteins that bind to methylated DNA. The Arabidopsis thaliana genome encodes 13 methyl CpG binding domain (MBD) proteins, but the molecular/biological functions of most of these proteins are still not clear. In the present study, we identified four proteins that interact with AtMBD6. Interestingly, three of them contain RNA binding domains and are co-localized with AtMBD6 in the nucleus. The interacting partners includes AtRPS2C (a 40S ribosomal protein), AtNTF2 (nuclear transport factor 2) and AtAGO4 (Argonoute 4). The fourth protein that physically interacts with AtMBD6 is a histone-modifying enzyme, histone deacetylase 6 (AtHDA6), which is a known component of the RNA-mediated gene silencing system. Analysis of genomic DNA methylation in the atmbd6, atrps2c and atntf2 mutants, using methylation-sensitive PCR detected decreased DNA methylation at miRNA/siRNA producing loci, pseudogenes and other targets of RNA-directed DNA methylation. Our results indicate that AtMBD6 is involved in RNA-mediated gene silencing and it binds to RNA binding proteins like AtRPS2C, AtAGO4 and AtNTF2. AtMBD6 also interacts with histone deacetylase AtHDA6 that might have a role in chromatin condensation at the targets of RdDM.

  19. The actin binding cytoskeletal protein Moesin is involved in nuclear mRNA export.

    Science.gov (United States)

    Kristó, Ildikó; Bajusz, Csaba; Borsos, Barbara N; Pankotai, Tibor; Dopie, Joseph; Jankovics, Ferenc; Vartiainen, Maria K; Erdélyi, Miklós; Vilmos, Péter

    2017-10-01

    Current models imply that the evolutionarily conserved, actin-binding Ezrin-Radixin-Moesin (ERM) proteins perform their activities at the plasma membrane by anchoring membrane proteins to the cortical actin network. Here we show that beside its cytoplasmic functions, the single ERM protein of Drosophila, Moesin, has a novel role in the nucleus. The activation of transcription by heat shock or hormonal treatment increases the amount of nuclear Moesin, indicating biological function for the protein in the nucleus. The distribution of Moesin in the nucleus suggests a function in transcription and the depletion of mRNA export factors Nup98 or its interacting partner, Rae1, leads to the nuclear accumulation of Moesin, suggesting that the nuclear function of the protein is linked to mRNA export. Moesin localizes to mRNP particles through the interaction with the mRNA export factor PCID2 and knock down of Moesin leads to the accumulation of mRNA in the nucleus. Based on our results we propose that, beyond its well-known, manifold functions in the cytoplasm, the ERM protein of Drosophila is a new, functional component of the nucleus where it participates in mRNA export. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. RNA Binds to Tau Fibrils and Sustains Template-Assisted Growth.

    Science.gov (United States)

    Dinkel, Paul D; Holden, Michael R; Matin, Nadira; Margittai, Martin

    2015-08-04

    Tau fibrils are the main proteinacious components of neurofibrillary lesions in Alzheimer disease. Although RNA molecules are sequestered into these lesions, their relationship to Tau fibrils is only poorly understood. Such understanding, however, is important, as short fibrils can transfer between neurons and nonproteinacious factors including RNA could play a defining role in modulating the latter process. Here, we used sedimentation assays combined with electron paramagnetic resonance (EPR), fluorescence, and absorbance spectroscopy to determine the effects of RNA on Tau fibril structure and growth. We observe that, in the presence of RNA, three-repeat (3R) and four-repeat (4R) Tau form fibrils with parallel, in-register arrangement of β-strands and exhibit an asymmetric seeding barrier in which 4R Tau grows onto 3R Tau seeds but not vice versa. These structural features are similar to those previously observed for heparin-induced fibrils, indicating that basic conformational properties are conserved, despite their being molecular differences of the nucleating agents. Furthermore, RNA sustains template-assisted growth and binds to the fibril surface and can be exchanged by heparin. These findings suggest that, in addition to mediating fibrillization, cofactors decorating the surface of Tau fibrils may modulate biological interactions and thereby influence the spreading of Tau pathology in the human brain.

  1. RNA-binding proteins related to stress response and differentiation in protozoa.

    Science.gov (United States)

    Alves, Lysangela Ronalte; Goldenberg, Samuel

    2016-02-26

    RNA-binding proteins (RBPs) are key regulators of gene expression. There are several distinct families of RBPs and they are involved in the cellular response to environmental changes, cell differentiation and cell death. The RBPs can differentially combine with RNA molecules and form ribonucleoprotein (RNP) complexes, defining the function and fate of RNA molecules in the cell. RBPs display diverse domains that allow them to be categorized into distinct families. They play important roles in the cellular response to physiological stress, in cell differentiation, and, it is believed, in the cellular localization of certain mRNAs. In several protozoa, a physiological stress (nutritional, temperature or pH) triggers differentiation to a distinct developmental stage. Most of the RBPs characterized in protozoa arise from trypanosomatids. In these protozoa gene expression regulation is mostly post-transcriptional, which suggests that some RBPs might display regulatory functions distinct from those described for other eukaryotes. mRNA stability can be altered as a response to stress. Transcripts are sequestered to RNA granules that ultimately modulate their availability to the translation machinery, storage or degradation, depending on the associated proteins. These aggregates of mRNPs containing mRNAs that are not being translated colocalize in cytoplasmic foci, and their numbers and size vary according to cell conditions such as oxidative stress, nutritional status and treatment with drugs that inhibit translation.

  2. RNA-binding proteins related to stress response and differentiation in protozoa

    Institute of Scientific and Technical Information of China (English)

    Lysangela Ronalte Alves; Samuel Goldenberg

    2016-01-01

    RNA-binding proteins(RBPs) are key regulators of gene expression. There are several distinct families of RBPs and they are involved in the cellular response to environmental changes, cell differentiation and cell death. The RBPs can differentially combine with RNA molecules and form ribonucleoprotein(RNP) complexes, defining the function and fate of RNA molecules in the cell. RBPs display diverse domains that allow them to be categorized into distinct families. They play important roles in the cellular response to physiological stress, in cell differentiation, and, it is believed, in the cellular localization of certain mRNAs. In several protozoa, a physiological stress(nutritional, temperature or pH) triggers differentiation to a distinct developmental stage. Most of the RBPs characterized in protozoa arise from trypanosomatids. In these protozoa gene expression regulation is mostly post-transcriptional, which suggests that some RBPs might display regulatory functions distinct from those described for other eukaryotes. mRNA stability can be altered as a response to stress. Transcripts are sequestered to RNA granules that ultimately modulate their availability to the translation machinery, storage or degradation, depending on the associated proteins. These aggregates of mRNPs containing mRNAs that are not being translated colocalize in cytoplasmic foci, and their numbers and size vary according to cell conditions such as oxidative stress, nutritional status and treatment with drugs that inhibit translation.

  3. Regulation of the growth hormone (GH) receptor and GH-binding protein mRNA

    Energy Technology Data Exchange (ETDEWEB)

    Kaji, Hidesuke; Ohashi, Shin-Ichirou; Abe, Hiromi; Chihara, Kazuo [Kobe Univ. School of Medicine, Kobe (Japan)

    1994-12-31

    In fasting rats, a transient increase in growth hormone-binding protein (GHBP) mRNA levels was observed after 1 day, in muscle, heart, and liver, but not in fat tissues. The liver GH receptor (GHR) mRNA level was significantly increased after 1 day (but not after 5 days) of bovine GH (bGH) treatment in fed rats. Both the liver GHR mRNA level and the net increment of plasma IGF-I markedly decreased after 5 days of bGH administration in fasting rats. These findings suggest that GHR and GHBP mRNAs in the liver are expressed in a different way and that the expression of GHBP mRNA is regulated differently between tissues, at least in rats. The results also suggest that refractoriness to GH in a sustained fasting state might be beneficial in preventing anabolic effects of GH. In humans, GHR mRNA in lymphocytes, from subjects with either GH-deficiency or acromegaly, could be detected by the reverse transcription-polymerase chain reaction method. In one patient with partial GH insensitivity, a heterozygous missense mutation (P561T) was identified in the cytoplasmic domain of GHR. 15 refs., 4 figs.

  4. In vitro RNase and nucleic acid binding activities implicate coilin in U snRNA processing.

    Directory of Open Access Journals (Sweden)

    Hanna J Broome

    Full Text Available Coilin is known as the marker protein for Cajal bodies (CBs, subnuclear domains important for the biogenesis of small nuclear ribonucleoproteins (snRNPs which function in pre-mRNA splicing. CBs associate non-randomly with U1 and U2 gene loci, which produce the small nuclear RNA (snRNA component of the respective snRNP. Despite recognition as the CB marker protein, coilin is primarily nucleoplasmic, and the function of this fraction is not fully characterized. Here we show that coilin binds double stranded DNA and has RNase activity in vitro. U1 and U2 snRNAs undergo a processing event of the primary transcript prior to incorporation in the snRNP. We find that coilin displays RNase activity within the CU region of the U2 snRNA primary transcript in vitro, and that coilin knockdown results in accumulation of the 3' pre-processed U1 and U2 snRNA. These findings present new characteristics of coilin in vitro, and suggest additional functions of the protein in vivo.

  5. Molecular dynamics studies of the nucleoprotein of influenza A virus: role of the protein flexibility in RNA binding.

    Directory of Open Access Journals (Sweden)

    Bogdan Tarus

    Full Text Available The influenza viruses contain a segmented, negative stranded RNA genome. Each RNA segment is covered by multiple copies of the nucleoprotein (NP. X-ray structures have shown that NP contains well-structured domains juxtaposed with regions of missing electron densities corresponding to loops. In this study, we tested if these flexible loops gated or promoted RNA binding and RNA-induced oligomerization of NP. We first performed molecular dynamics simulations of wt NP monomer and trimer in comparison with the R361A protein mutated in the RNA binding groove, using the H1N1 NP as the initial structure. Calculation of the root-mean-square fluctuations highlighted the presence of two flexible loops in NP trimer: loop 1 (73-90, loop 2 (200-214. In NP, loops 1 and 2 formed a 10-15 Å-wide pinch giving access to the RNA binding groove. Loop 1 was stabilized by interactions with K113 of the adjacent β-sheet 1 (91-112 that interacted with the RNA grove (linker 360-373 via multiple hydrophobic contacts. In R361A, a salt bridge formed between E80 of loop 1 and R208 of loop 2 driven by hydrophobic contacts between L79 and W207, due to a decreased flexibility of loop 2 and loop 1 unfolding. Thus, RNA could not access its binding groove in R361A; accordingly, R361A had a much lower affinity for RNA than NP. Disruption of the E80-R208 interaction in the triple mutant R361A-E80A-E81A increased its RNA binding affinity and restored its oligomerization back to wt levels in contrast with impaired levels of R361A. Our data suggest that the flexibility of loops 1 and 2 is required for RNA sampling and binding which likely involve conformational change(s of the nucleoprotein.

  6. Replication protein of tobacco mosaic virus cotranslationally binds the 5′ untranslated region of genomic RNA to enable viral replication

    Science.gov (United States)

    Kawamura-Nagaya, Kazue; Ishibashi, Kazuhiro; Huang, Ying-Ping; Miyashita, Shuhei; Ishikawa, Masayuki

    2014-01-01

    Genomic RNA of positive-strand RNA viruses replicate via complementary (i.e., negative-strand) RNA in membrane-bound replication complexes. Before replication complex formation, virus-encoded replication proteins specifically recognize genomic RNA molecules and recruit them to sites of replication. Moreover, in many of these viruses, selection of replication templates by the replication proteins occurs preferentially in cis. This property is advantageous to the viruses in several aspects of viral replication and evolution, but the underlying molecular mechanisms have not been characterized. Here, we used an in vitro translation system to show that a 126-kDa replication protein of tobacco mosaic virus (TMV), a positive-strand RNA virus, binds a 5′-terminal ∼70-nucleotide region of TMV RNA cotranslationally, but not posttranslationally. TMV mutants that carried nucleotide changes in the 5′-terminal region and showed a defect in the binding were unable to synthesize negative-strand RNA, indicating that this binding is essential for template selection. A C-terminally truncated 126-kDa protein, but not the full-length 126-kDa protein, was able to posttranslationally bind TMV RNA in vitro, suggesting that binding of the 126-kDa protein to the 70-nucleotide region occurs during translation and before synthesis of the C-terminal inhibitory domain. We also show that binding of the 126-kDa protein prevents further translation of the bound TMV RNA. These data provide a mechanistic explanation of how the 126-kDa protein selects replication templates in cis and how fatal collision between translating ribosomes and negative-strand RNA-synthesizing polymerases on the genomic RNA is avoided. PMID:24711385

  7. Weak binding affinity of human 4EHP for mRNA cap analogs.

    Science.gov (United States)

    Zuberek, Joanna; Kubacka, Dorota; Jablonowska, Agnieszka; Jemielity, Jacek; Stepinski, Janusz; Sonenberg, Nahum; Darzynkiewicz, Edward

    2007-05-01

    Ribosome recruitment to the majority of eukaryotic mRNAs is facilitated by the interaction of the cap binding protein, eIF4E, with the mRNA 5' cap structure. eIF4E stimulates translation through its interaction with a scaffolding protein, eIF4G, which helps to recruit the ribosome. Metazoans also contain a homolog of eIF4E, termed 4EHP, which binds the cap structure, but not eIF4G, and thus cannot stimulate translation, but it instead inhibits the translation of only one known, and possibly subset mRNAs. To understand why 4EHP does not inhibit general translation, we studied the binding affinity of 4EHP for cap analogs using two methods: fluorescence titration and stopped-flow measurements. We show that 4EHP binds cap analogs m(7)GpppG and m(7)GTP with 30 and 100 lower affinity than eIF4E. Thus, 4EHP cannot compete with eIF4E for binding to the cap structure of most mRNAs.

  8. AKAP3 synthesis is mediated by RNA binding proteins and PKA signaling during mouse spermiogenesis.

    Science.gov (United States)

    Xu, Kaibiao; Yang, Lele; Zhao, Danyun; Wu, Yaoyao; Qi, Huayu

    2014-06-01

    Mammalian spermatogenesis is regulated by coordinated gene expression in a spatiotemporal manner. The spatiotemporal regulation of major sperm proteins plays important roles during normal development of the male gamete, of which the underlying molecular mechanisms are poorly understood. A-kinase anchoring protein 3 (AKAP3) is one of the major components of the fibrous sheath of the sperm tail that is formed during spermiogenesis. In the present study, we analyzed the expression of sperm-specific Akap3 and the potential regulatory factors of its protein synthesis during mouse spermiogenesis. Results showed that the transcription of Akap3 precedes its protein synthesis by about 2 wk. Nascent AKAP3 was found to form protein complex with PKA and RNA binding proteins (RBPs), including PIWIL1, PABPC1, and NONO, as revealed by coimmunoprecipitation and protein mass spectrometry. RNA electrophoretic gel mobility shift assay showed that these RBPs bind sperm-specific mRNAs, of which proteins are synthesized during the elongating stage of spermiogenesis. Biochemical and cell biological experiments demonstrated that PIWIL1, PABPC1, and NONO interact with each other and colocalize in spermatids' RNA granule, the chromatoid body. In addition, NONO was found in extracytoplasmic granules in round spermatids, whereas PIWIL1 and PABPC1 were diffusely localized in cytoplasm of elongating spermatids, indicating their participation at different steps of mRNA metabolism during spermatogenesis. Interestingly, type I PKA subunits colocalize with PIWIL1 and PABPC1 in the cytoplasm of elongating spermatids and cosediment with the RBPs in polysomal fractions on sucrose gradients. Further biochemical analyses revealed that activation of PKA positively regulates AKAP3 protein synthesis without changing its mRNA level in elongating spermatids. Taken together, these results indicate that PKA signaling directly participates in the regulation of protein translation in postmeiotic male germ cells

  9. Polypyrimidine tract-binding proteins of potato mediate tuberization through an interaction with StBEL5 RNA.

    Science.gov (United States)

    Cho, Sung Ki; Sharma, Pooja; Butler, Nathaniel M; Kang, Il-Ho; Shah, Shweta; Rao, A Gururaj; Hannapel, David J

    2015-11-01

    Polypyrimidine tract-binding (PTB) proteins are a family of RNA-binding proteins that function in a wide range of RNA metabolic processes by binding to motifs rich in uracils and cytosines. A PTB protein of pumpkin was identified as the core protein of an RNA-protein complex that trafficks RNA. The biological function of the PTB-RNA complex, however, has not been demonstrated. In potato, six PTB proteins have been identified, and two, designated StPTB1 and StPTB6, are similar to the phloem-mobile pumpkin type. RNA binding assays confirmed the interaction of StPTB1 and StPTB6 with discrete pyrimidine-rich sequences of the 3'-untranslated regions of the phloem-mobile mRNA, StBEL5. The promoter of StPTB1 was active in companion cells of phloem in both stem and petioles. Expression of both types was evident in phloem cells of roots and in stolons during tuber formation. RNA accumulation of both PTB proteins was induced by short days in leaves in correlation with enhanced accumulation of StBEL5 RNA. StPTB suppression lines exhibited reduced tuber yields and decreased StBEL5 RNA accumulation, whereas StPTB overexpression lines displayed an increase in tuber production correlated with the enhanced production in stolons of steady-state levels of StBEL5 transcripts and RNA of key tuber identity genes. In StPTB overexpression lines, both the stability and long-distance transport of StBEL5 transcripts were enhanced, whereas in suppression lines stability and transport decreased. Using a transgenic approach, it is shown that the StPTB family of RNA-binding proteins regulate specific stages of development through an interaction with phloem-mobile transcripts of StBEL5.

  10. The structure of the C-terminal domain of the largest editosome interaction protein and its role in promoting RNA binding by RNA-editing ligase L2.

    Science.gov (United States)

    Park, Young-Jun; Budiarto, Tanya; Wu, Meiting; Pardon, Els; Steyaert, Jan; Hol, Wim G J

    2012-08-01

    Trypanosomatids, such as the sleeping sickness parasite Trypanosoma brucei, contain a ∼ 20S RNA-editing complex, also called the editosome, which is required for U-insertion/deletion editing of mitochondrial mRNAs. The editosome contains a core of 12 proteins including the large interaction protein A1, the small interaction protein A6, and the editing RNA ligase L2. Using biochemical and structural data, we identified distinct domains of T. brucei A1 which specifically recognize A6 and L2. We provide evidence that an N-terminal domain of A1 interacts with the C-terminal domain of L2. The C-terminal domain of A1 appears to be required for the interaction with A6 and also plays a key role in RNA binding by the RNA-editing ligase L2 in trans. Three crystal structures of the C-terminal domain of A1 have been elucidated, each in complex with a nanobody as a crystallization chaperone. These structures permitted the identification of putative dsRNA recognition sites. Mutational analysis of conserved residues of the C-terminal domain identified Arg703, Arg731 and Arg734 as key requirements for RNA binding. The data show that the editing RNA ligase activity is modulated by a novel mechanism, i.e. by the trans-acting RNA binding C-terminal domain of A1.

  11. RNA Binding Proteins RZ-1B and RZ-1C Play Critical Roles in Regulating Pre-mRNA Splicing and Gene Expression during Development in Arabidopsis.

    Science.gov (United States)

    Wu, Zhe; Zhu, Danling; Lin, Xiaoya; Miao, Jin; Gu, Lianfeng; Deng, Xian; Yang, Qian; Sun, Kangtai; Zhu, Danmeng; Cao, Xiaofeng; Tsuge, Tomohiko; Dean, Caroline; Aoyama, Takashi; Gu, Hongya; Qu, Li-Jia

    2016-01-01

    Nuclear-localized RNA binding proteins are involved in various aspects of RNA metabolism, which in turn modulates gene expression. However, the functions of nuclear-localized RNA binding proteins in plants are poorly understood. Here, we report the functions of two proteins containing RNA recognition motifs, RZ-1B and RZ-1C, in Arabidopsis thaliana. RZ-1B and RZ-1C were localized to nuclear speckles and interacted with a spectrum of serine/arginine-rich (SR) proteins through their C termini. RZ-1C preferentially bound to purine-rich RNA sequences in vitro through its N-terminal RNA recognition motif. Disrupting the RNA binding activity of RZ-1C with SR proteins through overexpression of the C terminus of RZ-1C conferred defective phenotypes similar to those observed in rz-1b rz-1c double mutants, including delayed seed germination, reduced stature, and serrated leaves. Loss of function of RZ-1B and RZ-1C was accompanied by defective splicing of many genes and global perturbation of gene expression. In addition, we found that RZ-1C directly targeted FLOWERING LOCUS C (FLC), promoting efficient splicing of FLC introns and likely also repressing FLC transcription. Our findings highlight the critical role of RZ-1B/1C in regulating RNA splicing, gene expression, and many key aspects of plant development via interaction with proteins including SR proteins.

  12. Marlin-1, a novel RNA-binding protein associates with GABA receptors.

    Science.gov (United States)

    Couve, Andrés; Restituito, Sophie; Brandon, Julia M; Charles, Kelly J; Bawagan, Hinayana; Freeman, Katie B; Pangalos, Menelas N; Calver, Andrew R; Moss, Stephen J

    2004-04-02

    GABA(B) receptors are heterodimeric G protein-coupled receptors that mediate slow synaptic inhibition in the central nervous system. Whereas heterodimerization between GABA(B) receptor GABA(B)R1 and GABA(B)R2 subunits is essential for functional expression, how neurons coordinate the assembly of these critical receptors remains to be established. Here we have identified Marlin-1, a novel GABA(B) receptor-binding protein that associates specifically with the GABA(B)R1 subunit in yeast, tissue culture cells, and neurons. Marlin-1 is expressed in the brain and exhibits a granular distribution in cultured hippocampal neurons. Marlin-1 binds different RNA species including the 3'-untranslated regions of both the GABA(B)R1 and GABA(B)R2 mRNAs in vitro and also associates with RNA in cultured neurons. Inhibition of Marlin-1 expression via small RNA interference technology results in enhanced intracellular levels of the GABA(B)R2 receptor subunit without affecting the level of GABA(B)R1. Together our results suggest that Marlin-1 functions to regulate the cellular levels of GABA(B) R2 subunits, which may have significant effects on the production of functional GABA(B) receptor heterodimers. Therefore, our observations provide an added level of regulation for the control of GABA(B) receptor expression and for the efficacy of inhibitory synaptic transmission.

  13. Dynamic nucleocytoplasmic shuttling of an Arabidopsis SR splicing factor: role of the RNA-binding domains.

    Science.gov (United States)

    Rausin, Glwadys; Tillemans, Vinciane; Stankovic, Nancy; Hanikenne, Marc; Motte, Patrick

    2010-05-01

    Serine/arginine-rich (SR) proteins are essential nuclear-localized splicing factors. We have investigated the dynamic subcellular distribution of the Arabidopsis (Arabidopsis thaliana) RSZp22 protein, a homolog of the human 9G8 SR factor. Little is known about the determinants underlying the control of plant SR protein dynamics, and so far most studies relied on ectopic transient overexpression. Here, we provide a detailed analysis of the RSZp22 expression profile and describe its nucleocytoplasmic shuttling properties in specific cell types. Comparison of transient ectopic- and stable tissue-specific expression highlights the advantages of both approaches for nuclear protein dynamic studies. By site-directed mutagenesis of RSZp22 RNA-binding sequences, we show that functional RNA recognition motif RNP1 and zinc-knuckle are dispensable for the exclusive protein nuclear localization and speckle-like distribution. Fluorescence resonance energy transfer imaging also revealed that these motifs are implicated in RSZp22 molecular interactions. Furthermore, the RNA-binding motif mutants are defective for their export through the CRM1/XPO1/Exportin-1 receptor pathway but retain nucleocytoplasmic mobility. Moreover, our data suggest that CRM1 is a putative export receptor for mRNPs in plants.

  14. Hyaluronan- and RNA-binding deubiquitinating enzymes of USP17 family members associated with cell viability

    Directory of Open Access Journals (Sweden)

    Kim Dongku

    2006-11-01

    Full Text Available Abstract Background Protein degradation by the ubiquitin system plays a crucial role in numerous cellular signaling pathways. Deubiquitination, a reversal of ubiquitination, has been recognized as an important regulatory step in the ubiquitin-dependent degradation pathway. Results While identifying putative ubiquitin specific protease (USP enzymes that contain a conserved Asp (I domain in humans, 4 USP17 subfamily members, highly homologous to DUB-3, have been found (USP17K, USP17L, USP17M, and USP17N, from human chorionic villi. Expression analysis showed that USP17 transcripts are highly expressed in the heart, liver, and pancreas and are expressed moderately in various human cancerous cell lines. Amino acid sequence analysis revealed that they contain the highly conserved Cys, His, and Asp domains which are responsible for the deubiquitinating activity. Biochemical enzyme assays indicated that they have deubiquitinating activity. Interestingly, the sequence analysis showed that these proteins, with exception of USP17N, contain the putative hyaluronan/RNA binding motifs, and cetylpyridinium chloride (CPC-precipitation analysis confirmed the association between these proteins and intracellular hyaluronan and RNA. Conclusion Here, we report that the overexpression of these proteins, with exception of USP17N, leads to apoptosis, suggesting that the hyaluronan and RNA binding motifs in these enzymes play an important role in regulating signal transduction involved in cell death.

  15. Post-transcriptional gene regulation by RNA-binding proteins in vascular endothelial dysfunction.

    Science.gov (United States)

    Xin, HongBo; Deng, KeYu; Fu, MinGui

    2014-08-01

    Endothelial cell dysfunction is a term which implies the dysregulation of normal endothelial cell functions, including impairment of the barrier functions, control of vascular tone, disturbance of proliferative and migratory capacity of endothelial cells, as well as control of leukocyte trafficking. Endothelial dysfunction is an early step in vascular inflammatory diseases such as atherosclerosis, diabetic vascular complications, sepsis-induced or severe virus infection-induced organ injuries. The expressions of inflammatory cytokines and vascular adhesion molecules induced by various stimuli, such as modified lipids, smoking, advanced glycation end products and bacteria toxin, significantly contribute to the development of endothelial dysfunction. The transcriptional regulation of inflammatory cytokines and vascular adhesion molecules has been well-studied. However, the regulation of those gene expressions at post-transcriptional level is emerging. RNA-binding proteins have emerged as critical regulators of gene expression acting predominantly at the post-transcriptional level in microRNA-dependent or independent manners. This review summarizes the latest insights into the roles of RNA-binding proteins in controlling vascular endothelial cell functions and their contribution to the pathogenesis of vascular inflammatory diseases.

  16. The mechanism by which influenza A virus nucleoprotein forms oligomers and binds RNA

    Energy Technology Data Exchange (ETDEWEB)

    Ye, Qiaozhen; Krug, Robert M.; Tao, Yizhi Jane

    2006-12-06

    Influenza A viruses pose a serious threat to world public health, particularly the currently circulating avian H5N1 viruses. The influenza viral nucleoprotein forms the protein scaffold of the helical genomic ribonucleoprotein complexes, and has a critical role in viral RNA replication. Here we report a 3.2 Angstrom crystal structure of this nucleoprotein, the overall shape of which resembles a crescent with a head and a body domain, with a protein fold different compared with that of the rhabdovirus nucleoprotein. Oligomerization of the influenza virus nucleoprotein is mediated by a flexible tail loop that is inserted inside a neighboring molecule. This flexibility in the tail loop enables the nucleoprotein to form loose polymers as well as rigid helices, both of which are important for nucleoprotein functions. Single residue mutations in the tail loop result in the complete loss of nucleoprotein oligomerization. An RNA-binding groove, which is found between the head and body domains at the exterior of the nucleoprotein oligomer, is lined with highly conserved basic residues widely distributed in the primary sequence. The nucleoprotein structure shows that only one of two proposed nuclear localization signals are accessible, and suggests that the body domain of nucleoprotein contains the binding site for the viral polymerase. Our results identify the tail loop binding pocket as a potential target for antiviral development.

  17. The actinobacterial transcription factor RbpA binds to the principal sigma subunit of RNA polymerase.

    Science.gov (United States)

    Tabib-Salazar, Aline; Liu, Bing; Doughty, Philip; Lewis, Richard A; Ghosh, Somadri; Parsy, Marie-Laure; Simpson, Peter J; O'Dwyer, Kathleen; Matthews, Steve J; Paget, Mark S

    2013-06-01

    RbpA is a small non-DNA-binding transcription factor that associates with RNA polymerase holoenzyme and stimulates transcription in actinobacteria, including Streptomyces coelicolor and Mycobacterium tuberculosis. RbpA seems to show specificity for the vegetative form of RNA polymerase as opposed to alternative forms of the enzyme. Here, we explain the basis of this specificity by showing that RbpA binds directly to the principal σ subunit in these organisms, but not to more diverged alternative σ factors. Nuclear magnetic resonance spectroscopy revealed that, although differing in their requirement for structural zinc, the RbpA orthologues from S. coelicolor and M. tuberculosis share a common structural core domain, with extensive, apparently disordered, N- and C-terminal regions. The RbpA-σ interaction is mediated by the C-terminal region of RbpA and σ domain 2, and S. coelicolor RbpA mutants that are defective in binding σ are unable to stimulate transcription in vitro and are inactive in vivo. Given that RbpA is essential in M. tuberculosis and critical for growth in S. coelicolor, these data support a model in which RbpA plays a key role in the σ cycle in actinobacteria.

  18. RNA-binding proteins involved in post-transcriptional regulation in bacteria

    Directory of Open Access Journals (Sweden)

    Elke eVan Assche

    2015-03-01

    Full Text Available Post-transcriptional regulation is a very important mechanism to control gene expression in changing environments. In the past decade, a lot of interest has been directed towards the role of small RNAs in bacterial post-transcriptional regulation. However, small RNAs are not the only molecules controlling gene expression at this level, RNA-binding proteins play an important role as well. CsrA and Hfq are the two best studied bacterial proteins of this type, but recently, additional proteins involved in post-transcriptional control have been identified. This review focuses on the general working mechanisms of post-transcriptionally active RNA-binding proteins, which include (i adaptation of the susceptibility of mRNAs and sRNAs to RNases, (ii modulating the accessibility of the ribosome binding site of mRNAs, (iii recruiting and assisting in the interaction of mRNAs with other molecules and (iv regulating transcription terminator / antiterminator formation, and gives an overview of both the well-studied and the newly identified proteins that are involved in post-transcriptional regulatory processes. Additionally, the post-transcriptional mechanisms by which the expression or the activity of these proteins is regulated, are described. For many of the newly identified proteins, however, mechanistic questions remain. Most likely, more post-transcriptionally active proteins will be identified in the future.

  19. A strategy to analyze the phenotypic consequences of inhibiting the association of an RNA-binding protein with a specific RNA.

    Science.gov (United States)

    Cibois, Marie; Gautier-Courteille, Carole; Vallée, Audrey; Paillard, Luc

    2010-01-01

    Targeted inactivations of RNA-binding proteins (RNA-BPs) can lead to huge phenotypical defects. These defects are due to the deregulation of certain mRNAs. However, we generally do not know, among the hundreds of mRNAs that are normally controlled by one RNA-BP, which are responsible for the observed phenotypes. Here, we designed an antisense oligonucleotide ("target protector") that masks the binding site of the RNA-BP CUG-binding protein 1 (CUGBP1) on the mRNA Suppressor of Hairless [Su(H)] that encodes a key player of Notch signaling. We showed that injecting this oligonucleotide into Xenopus embryos specifically inhibited the binding of CUGBP1 to the mRNA. This caused the derepression of Su(H) mRNA, the overexpression of Su(H) protein, and a phenotypic defect, loss of somitic segmentation, similar to that caused by a knockdown of CUGBP1. To demonstrate a causal relationship between Su(H) derepression and the segmentation defects, a rescue experiment was designed. Embryonic development was restored when the translation of Su(H) mRNA was re-repressed and the level of Su(H) protein was reduced to a normal level. This "target protector and rescue assay" demonstrates that the phenotypic defects associated with CUGBP1 inactivation in Xenopus are essentially due to the deregulation of Su(H) mRNA. Similar approaches may be largely used to uncover the links between the phenotype caused by the inactivation of an RNA-BP and the identity of the RNAs associated with that protein.

  20. Dynein light chain binding to a 3′-untranslated sequence mediates parathyroid hormone mRNA association with microtubules

    Science.gov (United States)

    Epstein, Eyal; Sela-Brown, Alin; Ringel, Israel; Kilav, Rachel; King, Stephen M.; Benashski, Sharon E.; Yisraeli, Joel K.; Silver, Justin; Naveh-Many, Tally

    2000-01-01

    The 3′-untranslated region (UTR) of mRNAs binds proteins that determine mRNA stability and localization. The 3′-UTR of parathyroid hormone (PTH) mRNA specifically binds cytoplasmic proteins. We screened an expression library for proteins that bind the PTH mRNA 3′-UTR, and the sequence of 1 clone was identical to that of the dynein light chain LC8, a component of the dynein complexes that translocate cytoplasmic components along microtubules. Recombinant LC8 binds PTH mRNA 3′-UTR, as shown by RNA electrophoretic mobility shift assay. We showed that PTH mRNA colocalizes with microtubules in the parathyroid gland, as well as with a purified microtubule preparation from calf brain, and that this association was mediated by LC8. To our knowledge, this is the first report of a dynein complex protein binding an mRNA. The dynein complex may be the motor that is responsible for transporting mRNAs to specific locations in the cytoplasm and for the consequent is asymmetric distribution of translated proteins in the cell. PMID:10683380

  1. Significance of the first transcribed nucleoside of capped RNA for ligand-induced folding of the cap-binding complex

    Science.gov (United States)

    Worch, Remigiusz; Niedzwiecka, Anna; Stepinski, Janusz; Jankowska-Anyszka, Marzena; Mazza, Catherine; Darzynkiewicz, Edward; Cusack, Stephen; Stolarski, Ryszard

    2005-05-01

    Many proteins, including those that bind RNA, change conformation upon binding a ligand, a phenomenon known as induced fit. CBP20, the small subunit of the nuclear cap-binding complex (CBC), recognizes specifically the 5' cap of eukaryotic mRNA and snRNA. The N- and C-terminal regions of the CBP20 subunit of the human nuclear cap-binding complex only acquire a proper fold in complex with capped RNA. The cap is composed of 7-methylguanosine linked by a 5'-to-5' triphosphate bridge to the first transcribed nucleoside of the RNA. The significance of the latter for the capped RNA-CBC association and local folding of CBC has been characterized by emission spectroscopy. Fluorescence titration of CBC has been performed for three selected, mono- and dinucleotide mRNA 5' cap analogues. The measured values of the equilibrium association constant and the corresponding Gibbs free energy depend on the type of the first transcribed nucleoside (purine or pyrimidine), and decrease ~10-fold in the case of a mononucleotide analogue, 7-methylguanosine triphosphate. However, the total quenching of the intrinsic protein fluorescence is similar for each analogue. Changes of the solvent-accessible CBC hydrophobic surface of CBC on binding of the structurally different cap analogues have been followed using bis-ANS fluorescent probe.

  2. Pentamidine binds to tRNA through non-specific hydrophobic interactions and inhibits aminoacylation and translation.

    Science.gov (United States)

    Sun, Tao; Zhang, Yi

    2008-03-01

    The selective and potent inhibition of mitochondrial translation in Saccharomyces cerevisiae by pentamidine suggests a novel antimicrobial action for this drug. Electrophoresis mobility shift assay, T1 ribonuclease footprinting, hydroxyl radical footprinting and isothermal titration calorimetry collectively demonstrated that pentamidine non-specifically binds to two distinct classes of sites on tRNA. The binding was driven by favorable entropy changes indicative of a large hydrophobic interaction, suggesting that the aromatic rings of pentamidine are inserted into the stacked base pairs of tRNA helices. Pentamidine binding disrupts the tRNA secondary structure and masks the anticodon loop in the tertiary structure. Consistently, we showed that pentamidine specifically inhibits tRNA aminoacylation but not the cognate amino acid adenylation. Pentamidine inhibited protein translation in vitro with an EC(50) equivalent to that binds to tRNA and inhibits tRNA aminoacylation in vitro, but drastically higher than that inhibits translation in vivo, supporting the established notion that the antimicrobial activity of pentamidine is largely due to its selective accumulation by the pathogen rather than by the host cell. Therefore, interrupting tRNA aminoacylation by the entropy-driven non-specific binding is an important mechanism of pentamidine in inhibiting protein translation, providing new insights into the development of antimicrobial drugs.

  3. Human cyclophilin 33 (hCyP33) in T-cell binds specifically to poly(A)~+RNA (mRNA)

    Institute of Scientific and Technical Information of China (English)

    张万起; 袁直; 宓怀风; 元云峰; 何炳林; 王亦农

    2002-01-01

    Human cyclophilin 33 (hCyP33), found in 1996, consists of an RNA-binding domain in N-terminus, a cyclophilin domain in C-terminus and a connected part between the two domains. RNA-binding proteins concern functions, such as splicing, modification and transport, after transcription in eukaryotic cells. Cyclophilins (CyPs) possess enzymatic activity, namely peptidyl-proryl cis-trans isomerase (PPlase). They are involved in folding, transport and interaction of proteins. Cyclosporin A (CsA), an immunosuppressant used by organ transplantation, binds to CyPs and suppresses their enzymatic activity. However, up to now it is unknown that which cellular and physiological roles hCyP33, which possesses the above-mentioned both functions, plays. In this paper the binding specificity of hCyP33 to different cellular RNA is investigated by means of ion-exchange chromatography and affinity adsorption. The results show that it binds specifically to poly(A) tailed mRNA, namely poly(A)+RNA.

  4. Human cyclophilin 33 (hCyP33) in T-cell binds specifically to poly(A)+RNA (mRNA)

    Institute of Scientific and Technical Information of China (English)

    张万起; 元云峰; 王亦农; 袁直; 宓怀风; 何炳林

    2002-01-01

    Human cyclophilin 33 (hCyP33), found in 1996, consists of an RNA-binding domain in N-terminus, a cyclophilin domain in C-terminus and a connected part between the two domains. RNA-binding proteins concern functions, such as splicing, modification and transport, after transcription in eukaryotic cells. Cyclophilins (CyPs) possess enzymatic activity, namely peptidyl-proryl cis-trans isomerase (PPIase). They are involved in folding, transport and interaction of proteins. Cyclosporin A (CsA), an immunosuppressant used by organ transplantation, binds to CyPs and suppresses their enzymatic activity. However, up to now it is unknown that which cellular and physiological roles hCyP33, which possesses the above-mentioned both functions, plays. In this paper the binding specificity of hCyP33 to different cellular RNA is investigated by means of ion-exchange chromatography and affinity adsorption. The results show that it binds specifically to poly(A) tailed mRNA, namely poly(A)+RNA.

  5. Methyltransferase-like protein 16 binds the 3'-terminal triple helix of MALAT1 long noncoding RNA.

    Science.gov (United States)

    Brown, Jessica A; Kinzig, Charles G; DeGregorio, Suzanne J; Steitz, Joan A

    2016-12-06

    Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a cancer-promoting long noncoding RNA, accumulates in cells by using a 3'-triple-helical RNA stability element for nuclear expression (ENE). The ENE, a stem-loop structure containing a U-rich internal loop, interacts with a downstream A-rich tract (ENE+A) to form a blunt-ended triple helix composed of nine U•A-U triples interrupted by a C•G-C triple and C-G doublet. This unique structure prompted us to explore the possibility of protein binding. Native gel-shift assays revealed a shift in radiolabeled MALAT1 ENE+A RNA upon addition of HEK293T cell lysate. Competitive gel-shift assays suggested that protein binding depends not only on the triple-helical structure but also its nucleotide composition. Selection from the lysate using a biotinylated-RNA probe followed by mass spectrometry identified methyltransferase-like protein 16 (METTL16), a putative RNA methyltransferase, as an interacting protein of the MALAT1 ENE+A. Gel-shift assays confirmed the METTL16-MALAT1 ENE+A interaction in vitro: Binding was observed with recombinant METTL16, but diminished in lysate depleted of METTL16, and a supershift was detected after adding anti-METTL16 antibody. Importantly, RNA immunoprecipitation after in vivo UV cross-linking and an in situ proximity ligation assay for RNA-protein interactions confirmed an association between METTL16 and MALAT1 in cells. METTL16 is an abundant (∼5 × 10(5) molecules per cell) nuclear protein in HeLa cells. Its identification as a triple-stranded RNA binding protein supports the formation of RNA triple helices inside cells and suggests the existence of a class of triple-stranded RNA binding proteins, which may enable the discovery of additional cellular RNA triple helices.

  6. Galectin-3 is a non-classic RNA binding protein that stabilizes the mucin MUC4 mRNA in the cytoplasm of cancer cells

    Science.gov (United States)

    Coppin, Lucie; Vincent, Audrey; Frénois, Frédéric; Duchêne, Belinda; Lahdaoui, Fatima; Stechly, Laurence; Renaud, Florence; Villenet, Céline; Seuningen, Isabelle Van; Leteurtre, Emmanuelle; Dion, Johann; Grandjean, Cyrille; Poirier, Françoise; Figeac, Martin; Delacour, Delphine; Porchet, Nicole; Pigny, Pascal

    2017-01-01

    Pancreatic cancer cells express high levels of MUC1, MUC4 and MUC16 mRNAs that encode membrane-bound mucins. These mRNAs share unusual features such as a long half-life. However, it remains unknown how mucin mRNA stability is regulated. Galectin-3 (Gal-3) is an endogenous lectin playing important biological functions in epithelial cells. Gal-3 is encoded by LGALS3 which is up-regulated in pancreatic cancer. Despite the absence of a RNA-recognition motif, Gal-3 interacts indirectly with pre-mRNAs in the nucleus and promotes constitutive splicing. However a broader role of Gal-3 in mRNA fate is unexplored. We report herein that Gal-3 increases MUC4 mRNA stability through an intermediate, hnRNP-L which binds to a conserved CA repeat element in the 3′UTR in a Gal-3 dependent manner and also controls Muc4 mRNA levels in epithelial tissues of Gal3−/− mice. Gal-3 interacts with hnRNP-L in the cytoplasm, especially during cell mitosis, but only partly associates with protein markers of P-Bodies or Stress Granules. By RNA-IP plus RNA-seq analysis and imaging, we demonstrate that Gal-3 binds to mature spliced MUC4 mRNA in the perinuclear region, probably in hnRNP-L-containing RNA granules. Our findings highlight a new role for Gal-3 as a non-classic RNA-binding protein that regulates MUC4 mRNA post-transcriptionally. PMID:28262838

  7. A non-proteolytic role for ubiquitin in deadenylation of MHC-I mRNA by the RNA-binding E3-ligase MEX-3C

    Science.gov (United States)

    Cano, Florencia; Rapiteanu, Radu; Sebastiaan Winkler, G.; Lehner, Paul J.

    2015-01-01

    The regulation of protein and mRNA turnover is essential for many cellular processes. We recently showed that ubiquitin—traditionally linked to protein degradation—directly regulates the degradation of mRNAs through the action of a newly identified family of RNA-binding E3 ubiquitin ligases. How ubiquitin regulates mRNA decay remains unclear. Here, we identify a new role for ubiquitin in regulating deadenylation, the initial and often rate-limiting step in mRNA degradation. MEX-3C, a canonical member of this family of RNA-binding ubiquitin ligases, associates with the cytoplasmic deadenylation complexes and ubiquitinates CNOT7(Caf1), the main catalytic subunit of the CCR4-NOT deadenylation machinery. We establish a new role for ubiquitin in regulating MHC-I mRNA deadenylation as ubiquitination of CNOT7 by MEX-3C regulates its deadenylation activity and is required for MHC-I mRNA degradation. Since neither proteasome nor lysosome inhibitors rescued MEX-3C-mediated MHC-I mRNA degradation, our findings suggest a new non-proteolytic function for ubiquitin in the regulation of mRNA decay. PMID:26471122

  8. Allosteric regulation of helicase core activities of the DEAD-box helicase YxiN by RNA binding to its RNA recognition motif.

    Science.gov (United States)

    Samatanga, Brighton; Andreou, Alexandra Z; Klostermeier, Dagmar

    2017-01-23

    DEAD-box proteins share a structurally similar core of two RecA-like domains (RecA_N and RecA_C) that contain the conserved motifs for ATP-dependent RNA unwinding. In many DEAD-box proteins the helicase core is flanked by ancillary domains. To understand the regulation of the DEAD-box helicase YxiN by its C-terminal RNA recognition motif (RRM), we investigated the effect of RNA binding to the RRM on its position relative to the core, and on core activities. RRM/RNA complex formation substantially shifts the RRM from a position close to the RecA_C to the proximity of RecA_N, independent of RNA contacts with the core. RNA binding to the RRM is communicated to the core, and stimulates ATP hydrolysis and RNA unwinding. The conformational space of the core depends on the identity of the RRM-bound RNA. Allosteric regulation of core activities by RNA-induced movement of ancillary domains may constitute a general regulatory mechanism of DEAD-box protein activity.

  9. The QKI-6 and QKI-7 RNA binding proteins block proliferation and promote Schwann cell myelination.

    Directory of Open Access Journals (Sweden)

    Daniel Larocque

    Full Text Available BACKGROUND: The quaking viable (qk(v mice have uncompacted myelin in their central and peripheral nervous system (CNS, PNS. The qk gene encodes 3 major alternatively spliced isoforms that contain unique sequence at their C-terminus dictating their cellular localization. QKI-5 is a nuclear isoform, whereas QKI-6 and QKI-7 are cytoplasmic isoforms. The qk(v mice harbor an enhancer/promoter deletion that prevents the expression of isoforms QKI-6 and QKI-7 in myelinating cells resulting in a dysmyelination phenotype. It was shown that QKI regulates the differentiation of oligodendrocytes, the myelinating cells of the CNS, however, little is known about the role of the QKI proteins, or RNA binding proteins in PNS myelination. METHODOLOGY/PRINCIPAL FINDINGS: To define the role of the QKI proteins in PNS myelination, we ectopically expressed QKI-6 and QKI-7 in primary rat Schwann cell/neuron from dorsal root ganglia cocultures. We show that the QKI isoforms blocked proliferation and promoted Schwann cell differentiation and myelination. In addition, these events were coordinated with elevated proteins levels of p27(KIP1 and myelin basic protein (MBP, markers of Schwann cell differentiation. QKI-6 and QKI-7 expressing co-cultures contained myelinated fibers that had directionality and contained significantly thicker myelin, as assessed by electron microscopy. Moreover, QKI-deficient Schwann cells had reduced levels of MBP, p27(KIP1 and Krox-20 mRNAs, as assessed by quantitative RT-PCR. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that the QKI-6 and QKI-7 RNA binding proteins are positive regulators of PNS myelination and show that the QKI RNA binding proteins play a key role in Schwann cell differentiation and myelination.

  10. RNA-binding protein IGF2BP3 targeting of oncogenic transcripts promotes hematopoietic progenitor proliferation.

    Science.gov (United States)

    Palanichamy, Jayanth Kumar; Tran, Tiffany M; Howard, Jonathan M; Contreras, Jorge R; Fernando, Thilini R; Sterne-Weiler, Timothy; Katzman, Sol; Toloue, Masoud; Yan, Weihong; Basso, Giuseppe; Pigazzi, Martina; Sanford, Jeremy R; Rao, Dinesh S

    2016-04-01

    Posttranscriptional control of gene expression is important for defining both normal and pathological cellular phenotypes. In vitro, RNA-binding proteins (RBPs) have recently been shown to play important roles in posttranscriptional regulation; however, the contribution of RBPs to cell specification is not well understood. Here, we determined that the RBP insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) is specifically overexpressed in mixed lineage leukemia-rearranged (MLL-rearranged) B-acute lymphoblastic leukemia (B-ALL), which constitutes a subtype of this malignancy associated with poor prognosis and high risk of relapse. IGF2BP3 was required for the survival of B-ALL cell lines, as knockdown led to decreased proliferation and increased apoptosis. Enforced expression of IGF2BP3 provided murine BM cells with a strong survival advantage, led to proliferation of hematopoietic stem and progenitor cells, and skewed hematopoietic development to the B cell/myeloid lineage. Cross-link immunoprecipitation and high throughput sequencing uncovered the IGF2BP3-regulated transcriptome, which includes oncogenes MYC and CDK6 as direct targets. IGF2BP3 regulated transcripts via targeting elements within 3' untranslated regions (3'UTR), and enforced IGF2BP3 expression in mice resulted in enhanced expression of Myc and Cdk6 in BM. Together, our data suggest that IGF2BP3-mediated targeting of oncogenic transcripts may represent a critical pathogenetic mechanism in MLL-rearranged B-ALL and support IGF2BP3 and its cognate RNA-binding partners as potential therapeutic targets in this disease.

  11. Single TRAM domain RNA-binding proteins in Archaea: functional insight from Ctr3 from the Antarctic methanogen Methanococcoides burtonii.

    Science.gov (United States)

    Taha; Siddiqui, K S; Campanaro, S; Najnin, T; Deshpande, N; Williams, T J; Aldrich-Wright, J; Wilkins, M; Curmi, P M G; Cavicchioli, R

    2016-09-01

    TRAM domain proteins present in Archaea and Bacteria have a β-barrel shape with anti-parallel β-sheets that form a nucleic acid binding surface; a structure also present in cold shock proteins (Csps). Aside from protein structures, experimental data defining the function of TRAM domains is lacking. Here, we explore the possible functional properties of a single TRAM domain protein, Ctr3 (cold-responsive TRAM domain protein 3) from the Antarctic archaeon Methanococcoides burtonii that has increased abundance during low temperature growth. Ribonucleic acid (RNA) bound by Ctr3 in vitro was determined using RNA-seq. Ctr3-bound M. burtonii RNA with a preference for transfer (t)RNA and 5S ribosomal RNA, and a potential binding motif was identified. In tRNA, the motif represented the C loop; a region that is conserved in tRNA from all domains of life and appears to be solvent exposed, potentially providing access for Ctr3 to bind. Ctr3 and Csps are structurally similar and are both inferred to function in low temperature translation. The broad representation of single TRAM domain proteins within Archaea compared with their apparent absence in Bacteria, and scarcity of Csps in Archaea but prevalence in Bacteria, suggests they represent distinct evolutionary lineages of functionally equivalent RNA-binding proteins.

  12. Binding site for Xenopus ribosomal protein L5 and accompanying structural changes in 5S rRNA.

    Science.gov (United States)

    Scripture, J Benjamin; Huber, Paul W

    2011-05-10

    The structure of the eukaryotic L5-5S rRNA complex was investigated in protection and interference experiments and is compared with the corresponding structure (L18-5S rRNA) in the Haloarcula marismortui 50S subunit. In close correspondence with the archaeal structure, the contact sites for the eukaryotic ribosomal protein are located primarily in helix III and loop C and secondarily in loop A and helix V. While the former is unique to L5, the latter is also a critical contact site for transcription factor IIIA (TFIIIA), accounting for the mutually exclusive binding of these two proteins to 5S RNA. The binding of L5 causes structural changes in loops B and C that expose nucleotides that contact the Xenopus L11 ortholog in H. marismortui. This induced change in the structure of the RNA reveals the origins of the cooperative binding to 5S rRNA that has been observed for the bacterial counterparts of these proteins. The native structure of helix IV and loop D antagonizes binding of L5, indicating that this region of the RNA is dynamic and also influenced by the protein. Examination of the crystal structures of Thermus thermophilus ribosomes in the pre- and post-translocation states identified changes in loop D and in the surrounding region of 23S rRNA that support the proposal that 5S rRNA acts to transmit information between different functional domains of the large subunit.

  13. The Nuclear PolyA-Binding Protein Nab2p Is Essential for mRNA Production

    DEFF Research Database (Denmark)

    Schmid, Manfred; Olszewski, Pawel; Pelechano, Vicent

    2015-01-01

    Polyadenylation of mRNA is a key step in eukaryotic gene expression. However, despite the major impact of poly(A) tails on mRNA metabolism, the precise roles of poly(A)-binding proteins (PABPs) in nuclear mRNA biogenesis remain elusive. Here, we demonstrate that rapid nuclear depletion of the S....... cerevisiae PABP Nab2p leads to a global loss of cellular mRNA, but not of RNA lacking poly(A) tails. Disappearance of mRNA is a nuclear event, but not due to decreased transcription. Instead, the absence of Nab2p results in robust nuclear mRNA decay by the ribonucleolytic RNA exosome in a polyadenylation...

  14. A poly(A) binding protein-specific sequence motif: MRTENGKSKGFGFVC binding to mRNA poly(A) and polynucleotides and its role on mRNA translation.

    Science.gov (United States)

    Rubin, H N; Halim, M N; Leavis, P C

    1994-06-01

    A consensus sequence (GKSKGFGFV) was recognized in all the sequenced poly(A) binding proteins. We synthesized a 15-amino acid peptide (corresponding to 354-368 in the yeast poly(A) binding protein) which includes the consensus sequence to test its binding affinity to different nucleotides, polynucleotides and mRNA with or without a poly(A) tail. Biochemical and biophysical studies revealed that the 15-amino acid peptide has a strong binding affinity to poly(A) alone or poly(A) attached at the 3' end of mRNA. Circular dichroism spectroscopy demonstrated that the secondary structure of the 15-mer is consistent with that expected based on the structure of the native RNP domain. Furthermore, among the various mononucleotides performed in the present studies, ATP was preferentially found to bind to the 15-mer. To further examine the biological significance of the binding of the 15-mer to the poly(A) tail of mRNA, in vitro translation of the mRNA poly(A)+ in the presence of the 15-mer drastically increased globin synthesis by almost 2-fold, while translation of the deadenylated mRNA in the presence of the 15-mer almost did not alter the rate of incorporation of radiolabeled leucine into globin.

  15. Both Maintenance and Avoidance of RNA-Binding Protein Interactions Constrain Coding Sequence Evolution.

    Science.gov (United States)

    Savisaar, Rosina; Hurst, Laurence D

    2017-05-01

    While the principal force directing coding sequence (CDS) evolution is selection on protein function, to ensure correct gene expression CDSs must also maintain interactions with RNA-binding proteins (RBPs). Understanding how our genes are shaped by these RNA-level pressures is necessary for diagnostics and for improving transgenes. However, the evolutionary impact of the need to maintain RBP interactions remains unresolved. Are coding sequences constrained by the need to specify RBP binding motifs? If so, what proportion of mutations are affected? Might sequence evolution also be constrained by the need not to specify motifs that might attract unwanted binding, for instance because it would interfere with exon definition? Here, we have scanned human CDSs for motifs that have been experimentally determined to be recognized by RBPs. We observe two sets of motifs-those that are enriched over nucleotide-controlled null and those that are depleted. Importantly, the depleted set is enriched for motifs recognized by non-CDS binding RBPs. Supporting the functional relevance of our observations, we find that motifs that are more enriched are also slower-evolving. The net effect of this selection to preserve is a reduction in the over-all rate of synonymous evolution of 2-3% in both primates and rodents. Stronger motif depletion, on the other hand, is associated with stronger selection against motif gain in evolution. The challenge faced by our CDSs is therefore not only one of attracting the right RBPs but also of avoiding the wrong ones, all while also evolving under selection pressures related to protein structure. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  16. Conserved regulation of MAP kinase expression by PUF RNA-binding proteins.

    Directory of Open Access Journals (Sweden)

    Myon-Hee Lee

    2007-12-01

    Full Text Available Mitogen-activated protein kinase (MAPK and PUF (for Pumilio and FBF [fem-3 binding factor] RNA-binding proteins control many cellular processes critical for animal development and tissue homeostasis. In the present work, we report that PUF proteins act directly on MAPK/ERK-encoding mRNAs to downregulate their expression in both the Caenorhabditis elegans germline and human embryonic stem cells. In C. elegans, FBF/PUF binds regulatory elements in the mpk-1 3' untranslated region (3' UTR and coprecipitates with mpk-1 mRNA; moreover, mpk-1 expression increases dramatically in FBF mutants. In human embryonic stem cells, PUM2/PUF binds 3'UTR elements in both Erk2 and p38alpha mRNAs, and PUM2 represses reporter constructs carrying either Erk2 or p38alpha 3' UTRs. Therefore, the PUF control of MAPK expression is conserved. Its biological function was explored in nematodes, where FBF promotes the self-renewal of germline stem cells, and MPK-1 promotes oocyte maturation and germ cell apoptosis. We found that FBF acts redundantly with LIP-1, the C. elegans homolog of MAPK phosphatase (MKP, to restrict MAPK activity and prevent apoptosis. In mammals, activated MAPK can promote apoptosis of cancer cells and restrict stem cell self-renewal, and MKP is upregulated in cancer cells. We propose that the dual negative regulation of MAPK by both PUF repression and MKP inhibition may be a conserved mechanism that influences both stem cell maintenance and tumor progression.

  17. Naphthyridine-Benzoazaquinolone: Evaluation of a Tricyclic System for the Binding to (CAG)n Repeat DNA and RNA.

    Science.gov (United States)

    Li, Jinxing; Sakata, Akihiro; He, Hanping; Bai, Li-Ping; Murata, Asako; Dohno, Chikara; Nakatani, Kazuhiko

    2016-07-05

    The expansion of CAG repeats in the human genome causes the neurological disorder Huntington's disease. The small-molecule naphthyridine-azaquinolone NA we reported earlier bound to the CAG/CAG motif in the hairpin structure of the CAG repeat DNA. In order to investigate and improve NA-binding to the CAG repeat DNA and RNA, we conducted systematic structure-binding studies of NA to CAG repeats. Among the five new NA derivatives we synthesized, surface plasmon resonance (SPR) assay showed that all of the derivatives modified from amide linkages in NA to a carbamate linkage failed to bind to CAG repeat DNA and RNA. One derivative, NBzA, modified by incorporating an additional ring to the azaquinolone was found to bind to both d(CAG)9 and r(CAG)9 . NBzA binding to d(CAG)9 was similar to NA binding in terms of large changes in the SPR assay and circular dichroism (CD) as well as pairwise binding, as assessed by electron spray ionization time-of-flight (ESI-TOF) mass spectrometry. For the binding to r(CAG)9 , both NA and NBzA showed stepwise binding in ESI-TOF MS, and NBzA-binding to r(CAG)9 induced more extensive conformational change than NA-binding. The tricyclic system in NBzA did not show significant effects on the binding, selectivity, and translation, but provides a large chemical space for further modification to gain higher affinity and selectivity. These studies revealed that the linker structure in NA and NBzA was suitable for the binding to CAG DNA and RNA, and that the tricyclic benzoazaquinolone did not interfere with the binding.

  18. Does unpaired adenosine-66 from helix II of Escherichia coli 5S RNA bind to protein L18?

    DEFF Research Database (Denmark)

    Christiansen, J; Douthwaite, S R; Christensen, A

    1985-01-01

    Adenosine-66 is unpaired within helix II of Escherichia coli 5S RNA and lies in the binding site of ribosomal protein L18. It has been proposed as a recognition site for protein L18. We have investigated further the structural importance of this nucleotide by deleting it. The 5S RNA gene of the rrn...... plasmid derived from pKK3535. Binding studies with protein L18 revealed that the protein bound much more weakly to the mutated 5S RNA. We consider the most likely explanation of this result is that L18 interacts with adenosine-66, and we present a tentative model for an interaction between the unpaired...

  19. Short Peptide Nucleic Acids Bind Strongly to Homopurine Tract of Double Helical RNA at pH 5.5

    OpenAIRE

    Li, Ming; Zengeya, Thomas; Rozners, Eriks

    2010-01-01

    The important role that non-coding RNA plays in cell biology makes it an attractive target for molecular recognition. However, the discovery of small molecules that bind double helical RNA selectively and may serve as biochemical probes and potential drug leads has been relatively slow. Herein, we show that peptide nucleic acids, as short as six nucleobases, bind very strongly (Ka > 107) and sequence selectively to a homopurine tract of double helical RNA at pH 5.5. The isothermal titration c...

  20. Identification of an RNA-binding protein that is phosphorylated by PTH and potentially mediates PTH-induced destabilization of Npt2a mRNA.

    Science.gov (United States)

    Murray, Rebecca D; Merchant, Michael L; Hardin, Ericka; Clark, Barbara; Khundmiri, Syed J; Lederer, Eleanor D

    2016-02-01

    Parathyroid hormone (PTH) is a key regulator of the expression and function of the type IIa sodium-phosphate cotransporter (Npt2a), the protein responsible for regulated renal phosphate reabsorption. We previously showed that PTH induces rapid decay of Npt2a mRNA through posttranscriptional mechanisms. We hypothesized that PTH-induced changes in RNA-binding protein (RBP) activity mediate the degradation of Npt2a mRNA. To address this aim, we treated opossum kidney (OK) cells, a PTH-sensitive proximal tubule cell culture model, with 100 nM PTH for 30 min and 2 h, followed by mass spectrometry characterization of the PTH-stimulated phosphoproteome. We identified 1,182 proteins differentially phosphorylated in response to PTH, including 68 RBPs. Preliminary analysis identified a phospho-RBP, hnRNPK-homology-type-splicing regulatory protein (KSRP), with predicted binding sites for the 3'-untranslated region (UTR) of Npt2a mRNA. Western blot analysis confirmed expression of KSRP in OK cells and showed PTH-dependent translocation to the nucleus. Immunoprecipitation of KSRP from control and PTH-treated cells followed by RNA isolation and RT-quantitative PCR analysis identified Npt2a mRNA from both control and PTH-treated KSRP pulldowns. Knockdown of KSRP followed by PTH treatment showed that KSRP is required for mediating PTH-stimulated reduction in sodium/hydrogen exchanger 3 mRNA, but not Npt2a mRNA. We conclude that 1) PTH is a major regulator of both transcription and translation, and 2) KSRP binds Npt2a mRNA but its role in PTH regulation of Npt2a mRNA is not clear.

  1. Polypyrimidine tract binding protein inhibits IgM pre-mRNA splicing by diverting U2 snRNA base-pairing away from the branch point.

    Science.gov (United States)

    Zheng, Xuexiu; Cho, Sunghee; Moon, Heegyum; Loh, Tiing Jen; Oh, Huyn Kyung; Green, Michael R; Shen, Haihong

    2014-04-01

    The mouse immunoglobulin (IgM) pre-mRNA contains a splicing inhibitor that bears multiple binding sites for the splicing repressor polypyrimidine tract binding protein (PTB). Here we show that the inhibitor directs assembly of an ATP-dependent complex that contains PTB and U1 and U2 small nuclear RNAs (snRNAs). Unexpectedly, although U2 snRNA is present in the inhibitor complex, it is not base-paired to the branch point. We present evidence that inhibitor-bound PTB contacts U2 snRNA to promote base-pairing to an adjacent branch point-like sequence within the inhibitor, thereby preventing the U2 snRNA-branch point interaction and resulting in splicing repression. Our studies reveal a novel mechanism by which PTB represses splicing.

  2. Genome-Wide microRNA Binding Site Variation between Extinct Wild Aurochs and Modern Cattle Identifies Candidate microRNA-Regulated Domestication Genes

    Science.gov (United States)

    Braud, Martin; Magee, David A.; Park, Stephen D. E.; Sonstegard, Tad S.; Waters, Sinead M.; MacHugh, David E.; Spillane, Charles

    2017-01-01

    The domestication of cattle from the now-extinct wild aurochs (Bos primigenius) involved selection for physiological and behavioral traits, with underlying genetic factors that remain largely unknown. Non-coding microRNAs have emerged as key regulators of the spatio-temporal expression of target genes controlling mammalian growth and development, including in livestock species. During the domestication process, selection of mutational changes in miRNAs and/or miRNA binding sites could have provided a mechanism to generate some of the traits that differentiate domesticated cattle from wild aurochs. To investigate this, we analyzed the open reading frame DNA sequence of 19,994 orthologous protein-coding gene pairs from extant Bos taurus genomes and a single extinct B. primigenius genome. We identified miRNA binding site polymorphisms in the 3′ UTRs of 1,620 of these orthologous genes. These 1,620 genes with altered miRNA binding sites between the B. taurus and B. primigenius lineages represent candidate domestication genes. Using a novel Score Site ratio metric we have ranked these miRNA-regulated genes according to the extent of divergence between miRNA binding site presence, frequency and copy number between the orthologous genes from B. taurus and B. primigenius. This provides an unbiased approach to identify cattle genes that have undergone the most changes in miRNA binding (i.e., regulation) between the wild aurochs and modern-day cattle breeds. In addition, we demonstrate that these 1,620 candidate domestication genes are enriched for roles in pigmentation, fertility, neurobiology, metabolism, immunity and production traits (including milk quality and feed efficiency). Our findings suggest that directional selection of miRNA regulatory variants was important in the domestication and subsequent artificial selection that gave rise to modern taurine cattle. PMID:28197171

  3. Targeting folded RNA: A branched peptide boronic acid that binds to a large surface area of HIV-1 RRE RNA†

    Science.gov (United States)

    Zhang, Wenyu; Bryson, David I.; Crumpton, Jason B.; Wynn, Jessica; Santos, Webster L.

    2013-01-01

    On-bead high throughput screening of a medium sized (1000–2000 Da) branched peptide boronic acid (BPBA) library consisting of 46,656 unique sequences against HIV-1 RRE RNA generated peptides with binding affinities in the low micromolar range. In particular, BPBA1 had a Kd of 1.4 µM with RRE IIB, preference for RNA over DNA (27 fold), and selectivity of up to >75 fold against a panel of RRE IIB variants. Structure-activity studies suggest that the boronic acid moiety and “branching” in peptides are key structural features for efficient binding and selectivity for the folded RNA target. BPBA1 was efficiently taken up by HeLa and A2780 cells. RNA-footprinting studies revealed that the BPBA1 binding site encompasses a large surface area that spans both the upper stem as well as the internal loop regions of RRE IIB. PMID:23925474

  4. Requirement of the RNA-binding protein SmpB during intracellular growth of Listeria monocytogenes.

    Science.gov (United States)

    Mraheil, Mobarak Abu; Frantz, Renate; Teubner, Lisa; Wendt, Heiko; Linne, Uwe; Wingerath, Jessica; Wirth, Thomas; Chakraborty, Trinad

    2017-04-01

    Bacterial trans-translation is the main quality control mechanism employed to relieve stalled ribosomes. Trans-translation is mediated by the small protein B (SmpB) and transfer-mRNA (tmRNA) ribonucleoprotein complex, which interacts with translational complexes stalled at the 3' end of non-stop mRNAs to release the stalled ribosomes thereby targeting the nascent polypeptides and truncated mRNAs for degradation. The trans-translation system exists with a few exceptions in all bacteria. In the present study, we assessed the contribution of SmpB to the growth and virulence of Listeria monocytogenes, a human intracellular food-borne pathogen that colonizes host tissues to cause severe invasive infections. A smpB knockout significantly decreased the intracellular growth rate of L. monocytogenes during infection of murine macrophages. In addition, the mutant strain was attenuated for virulence when examined with the Galleria mellonella larvae killing assay and the organ colonisation model of mice following infection. Proteomic analysis of whole cell extracts of ΔsmpB deletion mutant revealed elevated protein levels of several proteins involved in ribosome assembly and interaction with tRNA substrates. These included the elongation factor Tu [EF-Tu] which promotes the GTP-dependent binding of aminoacyl-tRNA to the A-site of ribosomes during protein biosynthesis as well as the CysK which is known to interact with bacterial toxins that cleave tRNA substrates. The data presented here shed light on the role of SmpB and trans-translation during intracellular growth of L. monocytogenes. Copyright © 2017 Elsevier GmbH. All rights reserved.

  5. The thumb subdomain of yeast mitochondrial RNA polymerase is involved in processivity, transcript fidelity and mitochondrial transcription factor binding.

    Science.gov (United States)

    Velazquez, Gilberto; Sousa, Rui; Brieba, Luis G

    2015-01-01

    Single subunit RNA polymerases have evolved 2 mechanisms to synthesize long transcripts without falling off a DNA template: binding of nascent RNA and interactions with an RNA:DNA hybrid. Mitochondrial RNA polymerases share a common ancestor with T-odd bacteriophage single subunit RNA polymerases. Herein we characterized the role of the thumb subdomain of the yeast mtRNA polymerase gene (RPO41) in complex stability, processivity, and fidelity. We found that deletion and point mutants of the thumb subdomain of yeast mtRNA polymerase increase the synthesis of abortive transcripts and the probability that the polymerase will disengage from the template during the formation of the late initial transcription and elongation complexes. Mutations in the thumb subdomain increase the amount of slippage products from a homopolymeric template and, unexpectedly, thumb subdomain deletions decrease the binding affinity for mitochondrial transcription factor (Mtf1). The latter suggests that the thumb subdomain is part of an extended binding surface area involved in binding Mtf1.

  6. G quadruplex RNA structures in PSD-95 mRNA: potential regulators of miR-125a seed binding site accessibility.

    Science.gov (United States)

    Stefanovic, Snezana; Bassell, Gary J; Mihailescu, Mihaela Rita

    2015-01-01

    Fragile X syndrome (FXS) is the most common inherited form of intellectual disability caused by the CGG trinucleotide expansion in the 3'-untranslated region of the FMR1 gene on the X chromosome, that silences the expression of the Fragile X mental retardation protein (FMRP). FMRP has been shown to bind to a G-rich region within the PSD-95 mRNA which encodes for the postsynaptic density protein 95 (PSD-95), and together with the microRNA miR-125a, to play an important role in the reversible inhibition of the PSD-95 mRNA translation in neurons. The loss of FMRP in Fmr1 KO mice disables this translation control in the production of the PSD-95 protein. Interestingly, the miR-125a binding site on PSD-95 mRNA is embedded in the G-rich region bound by FMRP and postulated to adopt one or more G quadruplex structures. In this study, we have used different biophysical techniques to validate and characterize the formation of parallel G quadruplex structures and binding of miR-125a to its complementary sequence located within the 3' UTR of PSD-95 mRNA. Our results indicate that the PSD-95 mRNA G-rich region folds into alternate G quadruplex conformations that coexist in equilibrium. miR-125a forms a stable complex with PSD-95 mRNA, as evident by characteristic Watson-Crick base-pairing that coexists with one of the G quadruplex forms, suggesting a novel mechanism for G quadruplex structures to regulate the access of miR-125a to its binding site. © 2014 Stefanovic et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  7. The Msi Family of RNA-Binding Proteins Function Redundantly as Intestinal Oncoproteins

    Directory of Open Access Journals (Sweden)

    Ning Li

    2015-12-01

    Full Text Available Members of the Msi family of RNA-binding proteins have recently emerged as potent oncoproteins in a range of malignancies. MSI2 is highly expressed in hematopoietic cancers, where it is required for disease maintenance. In contrast to the hematopoietic system, colorectal cancers can express both Msi family members, MSI1 and MSI2. Here, we demonstrate that, in the intestinal epithelium, Msi1 and Msi2 have analogous oncogenic effects. Further, comparison of Msi1/2-induced gene expression programs and transcriptome-wide analyses of Msi1/2-RNA-binding targets reveal significant functional overlap, including induction of the PDK-Akt-mTORC1 axis. Ultimately, we demonstrate that concomitant loss of function of both MSI family members is sufficient to abrogate the growth of human colorectal cancer cells, and Msi gene deletion inhibits tumorigenesis in several mouse models of intestinal cancer. Our findings demonstrate that MSI1 and MSI2 act as functionally redundant oncoproteins required for the ontogeny of intestinal cancers.

  8. A ribonuclease-resistant region of 5S RNA and its relation to the RNA binding sites of proteins L18 and L25

    DEFF Research Database (Denmark)

    Douthwaite, S; Garrett, R A; Wagner, R

    1979-01-01

    An RNA fragment, constituting three subfragments of nucleotide sequences 1-11, 69-87 and 89-120, is the most ribonuclease-resistant part of the native 5S RNA of Escherichia coli, at 0 degrees C. A smaller fragment of nucleotide sequence 69-87 and 90-110 is ribonuclease-resistant at 25 degrees....... Degradation of the L25-5S RNA complex with ribonuclease A or T2 yielded RNA fragments similar to those of the free 5S RNA at 0 degrees C and 25 degrees C; moreover L25 remained strongly bound to both RNA fragments and also produced some opening of the RNA structure in at least two positions. Protein L18...... initially protected most of the 5S RNA against ribonuclease digestion, at 0 degrees C, but was then gradually released prior to the formation of the larger RNA fragment. It cannot be concluded, therefore, as it was earlier (Gray et al., 1973), that this RNA fragment contains the primary binding site of L18....

  9. 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.

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

    Science.gov (United States)

    Chen, Tao; Cui, Peng; Xiong, Liming

    2015-09-30

    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.

  11. RNA-binding protein hnRNPLL regulates mRNA splicing and stability during B-cell to plasma-cell differentiation.

    Science.gov (United States)

    Chang, Xing; Li, Bin; Rao, Anjana

    2015-04-14

    Posttranscriptional regulation is a major mechanism to rewire transcriptomes during differentiation. Heterogeneous nuclear RNA-binding protein LL (hnRNPLL) is specifically induced in terminally differentiated lymphocytes, including effector T cells and plasma cells. To study the molecular functions of hnRNPLL at a genome-wide level, we identified hnRNPLL RNA targets and binding sites in plasma cells through integrated Photoactivatable-Ribonucleoside-Enhanced Cross-Linking and Immunoprecipitation (PAR-CLIP) and RNA sequencing. hnRNPLL preferentially recognizes CA dinucleotide-containing sequences in introns and 3' untranslated regions (UTRs), promotes exon inclusion or exclusion in a context-dependent manner, and stabilizes mRNA when associated with 3' UTRs. During differentiation of primary B cells to plasma cells, hnRNPLL mediates a genome-wide switch of RNA processing, resulting in loss of B-cell lymphoma 6 (Bcl6) expression and increased Ig production--both hallmarks of plasma-cell maturation. Our data identify previously unknown functions of hnRNPLL in B-cell to plasma-cell differentiation and demonstrate that the RNA-binding protein hnRNPLL has a critical role in tuning transcriptomes of terminally differentiating B lymphocytes.

  12. Biophysical analysis of Arabidopsis protein-only RNase P alone and in complex with tRNA provides a refined model of tRNA binding.

    Science.gov (United States)

    Pinker, Franziska; Schelcher, Cédric; Fernandez-Millan, Pablo; Gobert, Anthony; Birck, Catherine; Thureau, Aurélien; Roblin, Pierre; Giegé, Philippe; Sauter, Claude

    2017-08-25

    RNase P is a universal enzyme that removes 5' leader sequences from tRNA precursors. The enzyme is therefore essential for maturation of functional tRNAs and mRNA translation. RNase P represents a unique example of an enzyme that can occur either as ribonucleoprotein or as protein alone. The latter form of the enzyme, called protein-only RNase P (PRORP), is widespread in eukaryotes in which it can provide organellar or nuclear RNase P activities. Here, we have focused on Arabidopsis nuclear PRORP2 and its interaction with tRNA substrates. Affinity measurements helped assess the respective importance of individual pentatricopeptide repeat motifs in PRORP2 for RNA binding. We characterized the PRORP2 structure by X-ray crystallography and by small-angle X-ray scattering in solution as well as that of its complex with a tRNA precursor by small-angle X-ray scattering. Of note, our study reports the first structural data of a PRORP-tRNA complex. Combined with complementary biochemical and biophysical analyses, our structural data suggest that PRORP2 undergoes conformational changes to accommodate its substrate. In particular, the catalytic domain and the RNA-binding domain can move around a central hinge. Altogether, this work provides a refined model of the PRORP-tRNA complex that illustrates how protein-only RNase P enzymes specifically bind tRNA and highlights the contribution of protein dynamics to achieve this specific interaction. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Impaired embryonic development in mice overexpressing the RNA-binding protein TIAR.

    Directory of Open Access Journals (Sweden)

    Yacine Kharraz

    Full Text Available BACKGROUND: TIA-1-related (TIAR protein is a shuttling RNA-binding protein involved in several steps of RNA metabolism. While in the nucleus TIAR participates to alternative splicing events, in the cytoplasm TIAR acts as a translational repressor on specific transcripts such as those containing AU-Rich Elements (AREs. Due to its ability to assemble abortive pre-initiation complexes coalescing into cytoplasmic granules called stress granules, TIAR is also involved in the general translational arrest observed in cells exposed to environmental stress. However, the in vivo role of this protein has not been studied so far mainly due to severe embryonic lethality upon tiar invalidation. METHODOLOGY/PRINCIPAL FINDINGS: To examine potential TIAR tissue-specificity in various cellular contexts, either embryonic or adult, we constructed a TIAR transgenic allele (loxPGFPloxPTIAR allowing the conditional expression of TIAR protein upon Cre recombinase activity. Here, we report the role of TIAR during mouse embryogenesis. We observed that early TIAR overexpression led to low transgene transmission associated with embryonic lethality starting at early post-implantation stages. Interestingly, while pre-implantation steps evolved correctly in utero, in vitro cultured embryos were very sensitive to culture medium. Control and transgenic embryos developed equally well in the G2 medium, whereas culture in M16 medium led to the phosphorylation of eIF2alpha that accumulated in cytoplasmic granules precluding transgenic blastocyst hatching. Our results thus reveal a differential TIAR-mediated embryonic response following artificial or natural growth environment. CONCLUSIONS/SIGNIFICANCE: This study reports the importance of the tightly balanced expression of the RNA-binding protein TIAR for normal embryonic development, thereby emphasizing the role of post-transcriptional regulations in early embryonic programming.

  14. The Arabidopsis RNA-Binding Protein AtRGGA Regulates Tolerance to Salt and Drought Stress

    KAUST Repository

    Ambrosone, Alfredo

    2015-03-17

    Salt and drought stress severely reduce plant growth and crop productivity worldwide. The identification of genes underlying stress response and tolerance is the subject of intense research in plant biology. Through microarray analyses, we previously identified in potato (Solanum tuberosum) StRGGA, coding for an Arginine Glycine Glycine (RGG) box-containing RNA-binding protein, whose expression was specifically induced in potato cell cultures gradually exposed to osmotic stress. Here, we show that the Arabidopsis (Arabidopsis thaliana) ortholog, AtRGGA, is a functional RNA-binding protein required for a proper response to osmotic stress. AtRGGA gene expression was up-regulated in seedlings after long-term exposure to abscisic acid (ABA) and polyethylene glycol, while treatments with NaCl resulted in AtRGGA down-regulation. AtRGGA promoter analysis showed activity in several tissues, including stomata, the organs controlling transpiration. Fusion of AtRGGA with yellow fluorescent protein indicated that AtRGGA is localized in the cytoplasm and the cytoplasmic perinuclear region. In addition, the rgga knockout mutant was hypersensitive to ABA in root growth and survival tests and to salt stress during germination and at the vegetative stage. AtRGGA-overexpressing plants showed higher tolerance to ABA and salt stress on plates and in soil, accumulating lower levels of proline when exposed to drought stress. Finally, a global analysis of gene expression revealed extensive alterations in the transcriptome under salt stress, including several genes such as ASCORBATE PEROXIDASE2, GLUTATHIONE S-TRANSFERASE TAU9, and several SMALL AUXIN UPREGULATED RNA-like genes showing opposite expression behavior in transgenic and knockout plants. Taken together, our results reveal an important role of AtRGGA in the mechanisms of plant response and adaptation to stress.

  15. Impaired Embryonic Development in Mice Overexpressing the RNA-Binding Protein TIAR

    Science.gov (United States)

    Kharraz, Yacine; Salmand, Pierre-Adrien; Camus, Anne; Auriol, Jacques; Gueydan, Cyril; Kruys, Véronique; Morello, Dominique

    2010-01-01

    Background TIA-1-related (TIAR) protein is a shuttling RNA-binding protein involved in several steps of RNA metabolism. While in the nucleus TIAR participates to alternative splicing events, in the cytoplasm TIAR acts as a translational repressor on specific transcripts such as those containing AU-Rich Elements (AREs). Due to its ability to assemble abortive pre-initiation complexes coalescing into cytoplasmic granules called stress granules, TIAR is also involved in the general translational arrest observed in cells exposed to environmental stress. However, the in vivo role of this protein has not been studied so far mainly due to severe embryonic lethality upon tiar invalidation. Methodology/Principal Findings To examine potential TIAR tissue-specificity in various cellular contexts, either embryonic or adult, we constructed a TIAR transgenic allele (loxPGFPloxPTIAR) allowing the conditional expression of TIAR protein upon Cre recombinase activity. Here, we report the role of TIAR during mouse embryogenesis. We observed that early TIAR overexpression led to low transgene transmission associated with embryonic lethality starting at early post-implantation stages. Interestingly, while pre-implantation steps evolved correctly in utero, in vitro cultured embryos were very sensitive to culture medium. Control and transgenic embryos developed equally well in the G2 medium, whereas culture in M16 medium led to the phosphorylation of eIF2α that accumulated in cytoplasmic granules precluding transgenic blastocyst hatching. Our results thus reveal a differential TIAR-mediated embryonic response following artificial or natural growth environment. Conclusions/Significance This study reports the importance of the tightly balanced expression of the RNA-binding protein TIAR for normal embryonic development, thereby emphasizing the role of post-transcriptional regulations in early embryonic programming. PMID:20596534

  16. Salicylic acid-dependent and -independent impact of an RNA-binding protein on plant immunity.

    Science.gov (United States)

    Hackmann, Christian; Korneli, Christin; Kutyniok, Magdalene; Köster, Tino; Wiedenlübbert, Matthias; Müller, Caroline; Staiger, Dorothee

    2014-03-01

    Plants overexpressing the RNA-binding protein AtGRP7 (AtGRP7-ox plants) constitutively express the PR-1 (PATHOGENESIS-RELATED-1), PR-2 and PR-5 transcripts associated with salicylic acid (SA)-mediated immunity and show enhanced resistance against Pseudomonas syringae pv. tomato (Pto) DC3000. Here, we investigated whether the function of AtGRP7 in plant immunity depends on SA. Endogenous SA was elevated fivefold in AtGRP7-ox plants. The elevated PR-1, PR-2 and PR-5 levels were eliminated upon expression of the salicylate hydroxylase nahG in AtGRP7-ox plants and elevated PR-1 levels were suppressed by sid (salicylic acid deficient) 2-1 that is impaired in SA biosynthesis. RNA immunoprecipitation showed that AtGRP7 does not bind the PR-1 transcript in vivo, whereas it binds PDF1.2. Constitutive or inducible AtGRP7 overexpression increases PR-1 promoter activity, indicating that AtGRP7 affects PR-1 transcription. In line with this, the effect of AtGRP7 on PR-1 is suppressed by npr (non-expressor of PR genes) 1. Whereas AtGRP7-ox plants restricted growth of Pto DC3000 compared with wild type (wt), sid2-1 AtGRP7-ox plants allowed more growth than AtGRP7-ox plants. Furthermore, we show an enhanced hypersensitive response triggered by avirulent Pto DC3000 (AvrRpt2) in AtGRP7-ox compared with wt. In sid2-1 AtGRP7-ox, an intermediate phenotype was observed. Thus, AtGRP7 has both SA-dependent and SA-independent effects on plant immunity.

  17. Linking Spermatid Ribonucleic Acid (RNA) Binding Protein and Retrogene Diversity to Reproductive Success*

    Science.gov (United States)

    Chapman, Karen M.; Powell, Heather M.; Chaudhary, Jaideep; Shelton, John M.; Richardson, James A.; Richardson, Timothy E.; Hamra, F. Kent

    2013-01-01

    Spermiogenesis is a postmeiotic process that drives development of round spermatids into fully elongated spermatozoa. Spermatid elongation is largely controlled post-transcriptionally after global silencing of mRNA synthesis from the haploid genome. Here, rats that differentially express EGFP from a lentiviral transgene during early and late steps of spermiogenesis were used to flow sort fractions of round and elongating spermatids. Mass-spectral analysis of 2D gel protein spots enriched >3-fold in each fraction revealed a heterogeneous RNA binding proteome (hnRNPA2/b1, hnRNPA3, hnRPDL, hnRNPK, hnRNPL, hnRNPM, PABPC1, PABPC4, PCBP1, PCBP3, PTBP2, PSIP1, RGSL1, RUVBL2, SARNP2, TDRD6, TDRD7) abundantly expressed in round spermatids prior to their elongation. Notably, each protein within this ontology cluster regulates alternative splicing, sub-cellular transport, degradation and/or translational repression of mRNAs. In contrast, elongating spermatid fractions were enriched with glycolytic enzymes, redox enzymes and protein synthesis factors. Retrogene-encoded proteins were over-represented among the most abundant elongating spermatid factors identified. Consistent with these biochemical activities, plus corresponding histological profiles, the identified RNA processing factors are predicted to collectively drive post-transcriptional expression of an alternative exome that fuels finishing steps of sperm maturation and fitness. PMID:23938467

  18. AtMBD6, a methyl CpG binding domain protein, maintains gene silencing in Arabidopsis by interacting with RNA binding proteins

    Indian Academy of Sciences (India)

    ADWAITA PRASAD PARIDA; AMRAPALI SHARMA; ARUN KUMAR SHARMA

    2017-03-01

    DNA methylation, mediated by double-stranded RNA, is a conserved epigenetic phenomenon that protects a genome fromtransposons, silences unwanted genes and has a paramount function in plant or animal development. Methyl CpG bindingdomain proteins are members of a class of proteins that bind tomethylated DNA. The Arabidopsis thaliana genome encodes13 methyl CpG binding domain (MBD) proteins, but themolecular/biological functions of most of these proteins are still notclear. In the present study, we identified four proteins that interact with AtMBD6. Interestingly, three of them contain RNAbinding domains and are co-localized with AtMBD6 in the nucleus. The interacting partners includes AtRPS2C (a 40Sribosomal protein), AtNTF2 (nuclear transport factor 2) and AtAGO4 (Argonoute 4). The fourth protein that physicallyinteracts with AtMBD6 is a histone-modifying enzyme, histone deacetylase 6 (AtHDA6), which is a known component ofthe RNA-mediated gene silencing system. Analysis of genomic DNA methylation in the atmbd6, atrps2c and atntf2mutants, using methylation-sensitive PCR detected decreased DNA methylation at miRNA/siRNA producing loci,pseudogenes and other targets of RNA-directed DNA methylation. Our results indicate that AtMBD6 is involved inRNA-mediated gene silencing and it binds to RNA binding proteins like AtRPS2C, AtAGO4 and AtNTF2. AtMBD6 alsointeracts with histone deacetylase AtHDA6 that might have a role in chromatin condensation at the targets of RdDM.

  19. The binding specificity of Translocated in LipoSarcoma/FUsed in Sarcoma with lncRNA transcribed from the promoter region of cyclin D1

    OpenAIRE

    Yoneda, Ryoma; Suzuki, Shiho; Mashima, Tsukasa; Kondo, Keiko; Nagata, Takashi; Katahira, Masato; Kurokawa, Riki

    2016-01-01

    Background Translocated in LipoSarcoma (TLS, also known as FUsed in Sarcoma) is an RNA/DNA binding protein whose mutation cause amyotrophic lateral sclerosis. In previous study, we demonstrated that TLS binds to long noncoding RNA, promoter-associated ncRNA-D (pncRNA-D), transcribed from the 5? upstream region of cyclin D1 (CCND1), and inhibits the expression of CCND1. Results In order to elucidate the binding specificity between TLS and pncRNA-D, we divided pncRNA-D into seven fragments and ...

  20. Molecular mechanisms for the regulation of histone mRNA stem-loop-binding protein by phosphorylation

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Jun; Tan, Dazhi; DeRose, Eugene F.; Perera, Lalith; Dominski, Zbigniew; Marzluff, William F.; Tong, Liang; Tanaka Hall, Traci M. [NIH; (UNC); (Columbia)

    2014-08-06

    Replication-dependent histone mRNAs end with a conserved stem loop that is recognized by stem-loop–binding protein (SLBP). The minimal RNA-processing domain of SLBP is phosphorylated at an internal threonine, and Drosophila SLBP (dSLBP) also is phosphorylated at four serines in its 18-aa C-terminal tail. We show that phosphorylation of dSLBP increases RNA-binding affinity dramatically, and we use structural and biophysical analyses of dSLBP and a crystal structure of human SLBP phosphorylated on the internal threonine to understand the striking improvement in RNA binding. Together these results suggest that, although the C-terminal tail of dSLBP does not contact the RNA, phosphorylation of the tail promotes SLBP conformations competent for RNA binding and thereby appears to reduce the entropic penalty for the association. Increased negative charge in this C-terminal tail balances positively charged residues, allowing a more compact ensemble of structures in the absence of RNA.

  1. Exploiting structural and topological information to improve prediction of RNA-protein binding sites

    Directory of Open Access Journals (Sweden)

    Yuan Zheng

    2009-10-01

    Full Text Available Abstract Background RNA-protein interactions are important for a wide range of biological processes. Current computational methods to predict interacting residues in RNA-protein interfaces predominately rely on sequence data. It is, however, known that interface residue propensity is closely correlated with structural properties. In this paper we systematically study information obtained from sequences and structures and compare their contributions in this prediction problem. Particularly, different geometrical and network topological properties of protein structures are evaluated to improve interface residue prediction accuracy. Results We have quantified the impact of structural information on the prediction accuracy in comparison to the purely sequence based approach using two machine learning techniques: Naïve Bayes classifiers and Support Vector Machines. The highest AUC of 0.83 was achieved by a Support Vector Machine, exploiting PSI-BLAST profile, accessible surface area, betweenness-centrality and retention coefficient as input features. Taking into account that our results are based on a larger non-redundant data set, the prediction accuracy is considerably higher than reported in previous, comparable studies. A protein-RNA interface predictor (PRIP and the data set have been made available at http://www.qfab.org/PRIP. Conclusion Graph-theoretic properties of residue contact maps derived from protein structures such as betweenness-centrality can supplement sequence or structure features to improve the prediction accuracy for binding residues in RNA-protein interactions. While Support Vector Machines perform better on this task, Naïve Bayes classifiers also have been found to achieve good prediction accuracies but require much less training time and are an attractive choice for large scale predictions.

  2. An RNA-binding complex involved in ribosome biogenesis contains a protein with homology to tRNA CCA-adding enzyme.

    Directory of Open Access Journals (Sweden)

    Jinzhong Lin

    2013-10-01

    Full Text Available A multitude of proteins and small nucleolar RNAs transiently associate with eukaryotic ribosomal RNAs to direct their modification and processing and the assembly of ribosomal proteins. Utp22 and Rrp7, two interacting proteins with no recognizable domain, are components of the 90S preribosome or the small subunit processome that conducts early processing of 18S rRNA. Here, we determine the cocrystal structure of Utp22 and Rrp7 complex at 1.97 Å resolution and the NMR structure of a C-terminal fragment of Rrp7, which is not visible in the crystal structure. The structure reveals that Utp22 surprisingly resembles a dimeric class I tRNA CCA-adding enzyme yet with degenerate active sites, raising an interesting evolutionary connection between tRNA and rRNA processing machineries. Rrp7 binds extensively to Utp22 using a deviant RNA recognition motif and an extended linker. Functional sites on the two proteins were identified by structure-based mutagenesis in yeast. We show that Rrp7 contains a flexible RNA-binding C-terminal tail that is essential for association with preribosomes. RNA-protein crosslinking shows that Rrp7 binds at the central domain of 18S rRNA and shares a neighborhood with two processing H/ACA snoRNAs snR30 and snR10. Depletion of snR30 prevents the stable assembly of Rrp7 into preribosomes. Our results provide insight into the evolutionary origin and functional context of Utp22 and Rrp7.

  3. Exploratory Study on the RNA-Binding Structural Motifs by Library Screening Targeting pre-miRNA-29 a.

    Science.gov (United States)

    Fukuzumi, Takeo; Murata, Asako; Aikawa, Haruo; Harada, Yasue; Nakatani, Kazuhiko

    2015-11-16

    The metabolic stream of microRNA (miRNA) production, the so-called maturation process of miRNAs, became one of important metabolic paths for drug-targeting to modulate the expression of genes related to a number of diseases. We carried out discovery studies on small molecules binding to the precursor of miR-29a (pre-miR-29a) from a chemical library containing 41,119 compounds (AQ library) by the fluorescent indicator displacement (FID) assay using the xanthone derivative X2SdiMe as a fluorescent indicator. The FID assay provided 1075 compounds, which showed an increase of fluorescence. These compounds were subsequently submitted to a binding analysis in a surface plasmon resonance (SPR) assay on a pre-miR-29a immobilized surface. 21 hit compounds were identified with a good reproducibility in the binding. These compounds have not been reported to bind to RNA until now and can be classified into two groups on the basis of the kinetics in the binding. To gain more information on the motif structures that could be necessary for the binding to pre-miR-29a, 19 substructures were selected from the hit compounds. The substructure library (SS library) which consisted of 362 compounds was prepared from the AQ library. An SPR assay of the SS library on pre-miR-29a-immobilized surface suggested that five substructures could potentially be important structural motifs to bind to pre-miR-29a. These studies demonstrate that the combination of FID-based screening of chemical library and subsequent SPR assay would be one way for obtaining practical solutions for the discovery of molecules which bind to the target pre-miRNAs.

  4. Binding of tRNA nucleotidyltransferase to Affi-Gel Blue: rapid purification of the enzyme and binding studies.

    Science.gov (United States)

    Deutscher, M P; Masiakowski, P

    1978-06-01

    Rabbit liver tRNA nucleotidyldransferase bound to columns of Affi-Gel Blue and could be specifically eluted with tRNA. This observation led to development of a rapid purification procedure for the enzyme. The adsorbent was also used to assess interaction of tRNA nucleotidyltransferase with various polynucleotides and substrates. The enzyme could be efficiently desorbed from Affi-Gel Blue by low concentrations of tRNA-C-C, less well by tRNA-C-C-A, and not at all by poly(A), poly(C), ATP or CTP.

  5. Short peptide nucleic acids bind strongly to homopurine tract of double helical RNA at pH 5.5.

    Science.gov (United States)

    Li, Ming; Zengeya, Thomas; Rozners, Eriks

    2010-06-30

    The important role that noncoding RNA plays in cell biology makes it an attractive target for molecular recognition. However, the discovery of small molecules that bind double helical RNA selectively and may serve as biochemical probes and potential drug leads has been relatively slow. Herein, we show that peptide nucleic acids, as short as six nucleobases, bind very strongly (K(a) > 10(7)) and sequence selectively to a homopurine tract of double helical RNA at pH 5.5. The isothermal titration calorimetry and circular dichroism experiments suggest that the binding mode may be a sequence selective triple helix formation. Our results have implications for development of biochemical probes to study function of noncoding RNAs and design of compounds with potential antibacterial and antiviral activity.

  6. Distinct states of methionyl-tRNA synthetase indicate inhibitor binding by conformational selection.

    Science.gov (United States)

    Koh, Cho Yeow; Kim, Jessica E; Shibata, Sayaka; Ranade, Ranae M; Yu, Mingyan; Liu, Jiyun; Gillespie, J Robert; Buckner, Frederick S; Verlinde, Christophe L M J; Fan, Erkang; Hol, Wim G J

    2012-10-10

    To guide development of new drugs targeting methionyl-tRNA synthetase (MetRS) for treatment of human African trypanosomiasis, crystal structure determinations of Trypanosoma brucei MetRS in complex with its substrate methionine and its intermediate product methionyl-adenylate were followed by those of the enzyme in complex with high-affinity aminoquinolone inhibitors via soaking experiments. Drastic changes in conformation of one of the two enzymes in the asymmetric unit allowed these inhibitors to occupy an enlarged methionine pocket and a new so-called auxiliary pocket. Interestingly, a small low-affinity compound caused the same conformational changes, removed the methionine without occupying the methionine pocket, and occupied the previously not existing auxiliary pocket. Analysis of these structures indicates that the binding of the inhibitors is the result of conformational selection, not induced fit. Copyright © 2012 Elsevier Ltd. All rights reserved.

  7. Molecular recognition of single-stranded RNA: neomycin binding to poly(A).

    Science.gov (United States)

    Xi, Hongjuan; Gray, David; Kumar, Sunil; Arya, Dev P

    2009-07-01

    Poly(A) is a relevant sequence in cell biology due to its importance in mRNA stability and translation initiation. Neomycin is an aminoglycoside antibiotic that is well known for its ability to target various nucleic acid structures. Here it is reported that neomycin is capable of binding tightly to a single-stranded oligonucleotide (A(30)) with a K(d) in the micromolar range. CD melting experiments support complex formation and indicate a melting temperature of 47 degrees C. The poly(A) duplex, which melts at 44 degrees C (pH 5.5), was observed to melt at 61 degrees C in the presence of neomycin, suggesting a strong stabilization of the duplex by the neomycin.

  8. Identification and characterization of RNA-binding activity in the ORF1-encoded replicase protein of Pelargonium flower break virus.

    Science.gov (United States)

    Martínez-Turiño, Sandra; Hernández, Carmen

    2010-12-01

    Pelargonium flower break virus (PFBV) belongs to the genus Carmovirus (family Tombusviridae) and, as with the remaining members of the group, possesses a monopartite genome of single-stranded, positive-sense RNA that contains five ORFs. The two 5'-proximal ORFs (ORFs 1 and 2) encode two polypeptides of 27 and 86 kDa (p27 and p86), respectively, that show homology with replication proteins. The p27 does not present any motif to explain its presumed involvement in replication, while p86 has the motifs conserved in RNA-dependent RNA polymerases. In this work, we have confirmed the necessity of p27 and p86 for PFBV replication. To gain insights into the function(s) of p27, we have expressed and purified the protein from Escherichia coli and tested its ability to bind RNA in vitro. The results have shown that p27 is able to bind ssRNA with high affinity and in a cooperative fashion and that it is also capable of binding other types of nucleic acids, though to a lesser extent. Additionally, competition experiments suggest that p27 has a preference for PFBV-derived ssRNAs. Using truncated forms of p27, it can be concluded that several regions of the protein contribute to its RNA-binding properties and that this contribution is additive. This study is the first to show nucleic acid-binding ability of the ORF1 product of a carmovirus and the data obtained suggest that this product plays an essential role in selection and recruitment of viral RNA replication templates.

  9. The RNA-binding protein CsrA plays a central role in positively regulating virulence factors in Erwinia amylovora

    OpenAIRE

    Veronica Ancona; Jae Hoon Lee; Youfu Zhao

    2016-01-01

    The GacS/GacA two-component system (also called GrrS/GrrA) is a global regulatory system which is highly conserved among gamma-proteobacteria. This system positively regulates non-coding small regulatory RNA csrB, which in turn binds to the RNA-binding protein CsrA. However, how GacS/GacA-Csr system regulates virulence traits in E. amylovora remains unknown. Results from mutant characterization showed that the csrB mutant was hypermotile, produced higher amount of exopolysaccharide amylovoran...

  10. Proteolytic footprinting of transcription factor TFIIIA reveals different tightly binding sites for 5S RNA and 5S DNA.

    OpenAIRE

    Bogenhagen, D F

    1993-01-01

    Transcription factor IIIA (TFIIIA) employs an array of nine N-terminal zinc fingers to bind specifically to both 5S RNA and 5S DNA. The binding of TFIIIA to 5S RNA and 5S DNA was studied by using a protease footprinting technique. Brief treatment of free or bound TFIIA with trypsin or chymotrypsin generated fragments which were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Fragments retaining the N terminus of TFIIA were identified by immunoblotting with an antibody ...

  11. Mapping the ribosomal protein S7 regulatory binding site on mRNA of the E. coli streptomycin operon.

    Science.gov (United States)

    Surdina, A V; Rassokhin, T I; Golovin, A V; Spiridonova, V A; Kopylov, A M

    2010-07-01

    In this work it is shown by deletion analysis that an intercistronic region (ICR) approximately 80 nucleotides in length is necessary for interaction with recombinant E. coli S7 protein (r6hEcoS7). A model is proposed for the interaction of S7 with two ICR sites-region of hairpin bifurcations and Shine-Dalgarno sequence of cistron S7. A de novo RNA binding site for heterologous S7 protein of Thermus thermophilus (r6hTthS7) was constructed by selection of a combinatorial RNA library based on E. coli ICR: it has only a single supposed protein recognition site in the region of bifurcation. The SERW technique was used for selection of two intercistronic RNA libraries in which five nucleotides of a double-stranded region, adjacent to the bifurcation, had the randomized sequence. One library contained an authentic AG (-82/-20) pair, while in the other this pair was replaced by AU. A serwamer capable of specific binding to r6hTthS7 was selected; it appeared to be the RNA68 mutant with eight nucleotide mutations. The serwamer binds to r6hTthS7 with the same affinity as homologous authentic ICR of str mRNA binds to r6hEcoS7; apparent dissociation constants are 89 +/- 43 and 50 +/- 24 nM, respectively.

  12. Elucidation of a C-Rich Signature Motif in Target mRNAs of RNA-Binding Protein TIAR▿ †

    OpenAIRE

    Kim, Henry S.; Kuwano, Yuki; Zhan, Ming; Pullmann, Rudolf; Mazan-Mamczarz, Krystyna; Li, Huai; Kedersha, Nancy; Anderson, Paul; Wilce, Matthew C J; Gorospe, Myriam; Wilce, Jacqueline A.

    2007-01-01

    The RNA-binding protein TIAR (related to TIA-1 [T-cell-restricted intracellular antigen 1]) was shown to associate with subsets of mRNAs bearing U-rich sequences in their 3′ untranslated regions. TIAR can function as a translational repressor, particularly in response to cytotoxic agents. Using unstressed colon cancer cells, collections of mRNAs associated with TIAR were isolated by immunoprecipitation (IP) of (TIAR-RNA) ribonucleoprotein (RNP) complexes, identified by microarray analysis, an...

  13. Nuclear Factor 90, a cellular dsRNA binding protein inhibits the HIV Rev-export function

    Directory of Open Access Journals (Sweden)

    St-Laurent Georges

    2006-11-01

    Full Text Available Abstract Background The HIV Rev protein is known to facilitate export of incompletely spliced and unspliced viral transcripts to the cytoplasm, a necessary step in virus life cycle. The Rev-mediated nucleo-cytoplasmic transport of nascent viral transcripts, dependents on interaction of Rev with the RRE RNA structural element present in the target RNAs. The C-terminal variant of dsRNA-binding nuclear protein 90 (NF90ctv has been shown to markedly attenuate viral replication in stably transduced HIV-1 target cell line. Here we examined a mechanism of interference of viral life cycle involving Rev-NF90ctv interaction. Results Since Rev:RRE complex formations depend on protein:RNA and protein:protein interactions, we investigated whether the expression of NF90ctv might interfere with Rev-mediated export of RRE-containing transcripts. When HeLa cells expressed both NF90ctv and Rev protein, we observed that NF90ctv inhibited the Rev-mediated RNA transport. In particular, three regions of NF90ctv protein are involved in blocking Rev function. Moreover, interaction of NF90ctv with the RRE RNA resulted in the expression of a reporter protein coding sequences linked to the RRE structure. Moreover, Rev influenced the subcellular localization of NF90ctv, and this process is leptomycin B sensitive. Conclusion The dsRNA binding protein, NF90ctv competes with HIV Rev function at two levels, by competitive protein:protein interaction involving Rev binding to specific domains of NF90ctv, as well as by its binding to the RRE-RNA structure. Our results are consistent with a model of Rev-mediated HIV-1 RNA export that envisions Rev-multimerization, a process interrupted by NF90ctv.

  14. Resveratrol post-transcriptionally regulates pro-inflammatory gene expression via regulation of KSRP RNA binding activity

    Science.gov (United States)

    Bollmann, Franziska; Art, Julia; Henke, Jenny; Schrick, Katharina; Besche, Verena; Bros, Matthias; Li, Huige; Siuda, Daniel; Handler, Norbert; Bauer, Florian; Erker, Thomas; Behnke, Felix; Mönch, Bettina; Härdle, Lorena; Hoffmann, Markus; Chen, Ching-Yi; Förstermann, Ulrich; Dirsch, Verena M.; Werz, Oliver; Kleinert, Hartmut; Pautz, Andrea

    2014-01-01

    Resveratrol shows beneficial effects in inflammation-based diseases like cancer, cardiovascular and chronic inflammatory diseases. Therefore, the molecular mechanisms of the anti-inflammatory resveratrol effects deserve more attention. In human epithelial DLD-1 and monocytic Mono Mac 6 cells resveratrol decreased the expression of iNOS, IL-8 and TNF-α by reducing mRNA stability without inhibition of the promoter activity. Shown by pharmacological and siRNA-mediated inhibition, the observed effects are SIRT1-independent. Target-fishing and drug responsive target stability experiments showed selective binding of resveratrol to the RNA-binding protein KSRP, a central post-transcriptional regulator of pro-inflammatory gene expression. Knockdown of KSRP expression prevented resveratrol-induced mRNA destabilization in human and murine cells. Resveratrol did not change KSRP expression, but immunoprecipitation experiments indicated that resveratrol reduces the p38 MAPK-related inhibitory KSRP threonine phosphorylation, without blocking p38 MAPK activation or activity. Mutation of the p38 MAPK target site in KSRP blocked the resveratrol effect on pro-inflammatory gene expression. In addition, resveratrol incubation enhanced KSRP-exosome interaction, which is important for mRNA degradation. Finally, resveratrol incubation enhanced its intra-cellular binding to the IL-8, iNOS and TNF-α mRNA. Therefore, modulation of KSRP mRNA binding activity and, thereby, enhancement of mRNA degradation seems to be the common denominator of many anti-inflammatory effects of resveratrol. PMID:25352548

  15. Expression cloning and characterization of a novel gene that encodes the RNA-binding protein FAU-1 from Pyrococcus furiosus.

    Science.gov (United States)

    Kanai, Akio; Oida, Hanako; Matsuura, Nana; Doi, Hirofumi

    2003-05-15

    We systematically screened a genomic DNA library to identify proteins of the hyperthermophilic archaeon Pyrococcus furiosus using an expression cloning method. One gene product, which we named FAU-1 (P. furiosus AU-binding), demonstrated the strongest binding activity of all the genomic library-derived proteins tested against an AU-rich RNA sequence. The protein was purified to near homogeneity as a 54 kDa single polypeptide, and the gene locus corresponding to this FAU-1 activity was also sequenced. The FAU-1 gene encoded a 472-amino-acid protein that was characterized by highly charged domains consisting of both acidic and basic amino acids. The N-terminal half of the gene had a degree of similarity (25%) with RNase E from Escherichia coli. Five rounds of RNA-binding-site selection and footprinting analysis showed that the FAU-1 protein binds specifically to the AU-rich sequence in a loop region of a possible RNA ligand. Moreover, we demonstrated that the FAU-1 protein acts as an oligomer, and mainly as a trimer. These results showed that the FAU-1 protein is a novel heat-stable protein with an RNA loop-binding characteristic.

  16. Mutagen-specific mutation signature determines global microRNA binding.

    Directory of Open Access Journals (Sweden)

    Eyal Greenberg

    Full Text Available Micro-RNAs (miRNAs are small non-coding RNAs that regulate gene products at the post-transcriptional level. It is thought that loss of cell regulation by miRNAs supports cancer development. Based on whole genome sequencing of a melanoma tumor, we predict, using three different computational algorithms, that the melanoma somatic mutations globally reduce binding of miRNAs to the mutated 3'UTRs. This phenomenon reflects the nature of the characteristic UV-induced mutation, C-to-T. Furthermore, we show that seed regions are enriched with Guanine, thus rendering miRNAs prone to reduced binding to UV-mutated 3'UTRs. Accordingly, mutation patterns in non UV-induced malignancies e.g. lung cancer and leukemia do not yield similar predictions. It is suggested that UV-induced disruption of miRNA-mediated gene regulation plays a carcinogenic role. Remarkably, dark-skinned populations have significantly higher GC content in 3'UTR SNPs than light-skinned populations, which implies on evolutionary pressure to preserve regulation by trans-acting oligonucleotides under conditions with excess UV radiation.

  17. Targeted protein footprinting: where different transcription factors bind to RNA polymerase.

    Science.gov (United States)

    Traviglia, S L; Datwyler, S A; Yan, D; Ishihama, A; Meares, C F

    1999-11-30

    Gene transcription is regulated through the interactions of RNA polymerase (RNAP) with transcription factors, such as the bacterial sigma proteins. We have devised a new strategy that relies on targeted protein footprinting to make an extensive survey of proximity to the protein surface. This involves attaching cutting reagents randomly to lysine residues on the surface of a protein such as sigma. The lysine-labeled sigma protein is then used to cleave the polypeptide backbones of the RNAP proteins at exposed residues adjacent to the sigma binding site. We used targeted protein footprinting to compare the areas near which sigma(70), sigma(54), sigma(38), sigma(E), NusA, GreA, and omega bind to the protein subunits of Escherichia coli RNAP. The sigma proteins and NusA cut sites in similar regions of the two large RNAP subunits, beta and beta', outlining a common surface. GreA cuts a larger set of sites, whereas omega shows no overlap with the others, cutting only the beta' subunit at a unique location.

  18. The pleuromutilin drugs tiamulin and valnemulin bind to the RNA at the peptidyl transferase centre on the ribosome

    DEFF Research Database (Denmark)

    Poulsen, S M; Karlsson, M; Johansson, L B

    2001-01-01

    The pleuromutilin antibiotic derivatives, tiamulin and valnemulin, inhibit protein synthesis by binding to the 50S ribosomal subunit of bacteria. The action and binding site of tiamulin and valnemulin was further characterized on Escherichia coli ribosomes. It was revealed that these drugs...... results that tiamulin and valnemulin interact with the rRNA in the peptidyl transferase slot on the ribosomes in which they prevent the correct positioning of the CCA-ends of tRNAs for peptide transfer....

  19. RNA- binding protein Stau2 is important for spindle integrity and meiosis progression in mouse oocytes.

    Science.gov (United States)

    Cao, Yan; Du, Juan; Chen, Dandan; Wang, Qian; Zhang, Nana; Liu, Xiaoyun; Liu, Xiaoyu; Weng, Jing; Liang, Yuanjing; Ma, Wei

    2016-10-01

    Staufen2 (Stau2) is a double-stranded RNA-binding protein involved in cell fate decision by regulating mRNA transport, mRNA stability, translation, and ribonucleoprotein assembly. Little is known about Stau2 expression and function in mammalian oocytes during meiosis. Herein we report the sub-cellular distribution and function of Stau2 in mouse oocyte meiosis. Western blot analysis revealed high and stable expression of Stau2 in oocytes from germinal vesicle (GV) to metaphase II (MII). Immunofluorescence showed that Stau2 was evenly distributed in oocytes at GV stage, and assembled as filaments after germinal vesicle breakdown (GVBD), particularly, colocalized with spindle at MI and MII. Stau2 was disassembled when microtubules were disrupted with nocodazole, on the other hand, when MTs were stabilized with taxol, Stau2 was not colocalized with the stabilized microtubules, but aggregated around the chromosomes array, indicating Stau2 assembly and colocalization with microtubules require both microtubule integrity and its normal dynamics. During interphase and mitosis of BHK and MEF cells, Stau2 was not distributed on microtubules, but colocalized with cis-Golgi marker GM130, implying its association with Golgi complex but not the spindle in fully differentiated somatic cells. Specific morpholino oligo-mediated Stau2 knockdown disrupted spindle formation, chromosome alignment and microtubule-kinetochore attachment in oocytes. The majority oocytes were arrested at MI stage, with bright MAD1 at kinetochores, indicating activation of spindle assembly checkpoint (SAC). Some oocytes were stranded at telophase I (TI), implying suppressed first polar body extrution. Together these data demonstrate that Stau2 is required for spindle formation and timely meiotic progression in mouse oocytes.

  20. Ablation of the Sam68 RNA binding protein protects mice from age-related bone loss.

    Directory of Open Access Journals (Sweden)

    Stéphane Richard

    2005-12-01

    Full Text Available The Src substrate associated in mitosis of 68 kDa (Sam68 is a KH-type RNA binding protein that has been shown to regulate several aspects of RNA metabolism; however, its physiologic role has remained elusive. Herein we report the generation of Sam68-null mice by homologous recombination. Aged Sam68-/- mice preserved their bone mass, in sharp contrast with 12-month-old wild-type littermates in which bone mass was decreased up to approximately 75%. In fact, the bone volume of the 12-month-old Sam68-/- mice was virtually indistinguishable from that of 4-month-old wild-type or Sam68-/- mice. Sam68-/- bone marrow stromal cells had a differentiation advantage for the osteogenic pathway. Moreover, the knockdown of Sam68 using short hairpin RNA in the embryonic mesenchymal multipotential progenitor C3H10T1/2 cells resulted in more pronounced expression of the mature osteoblast marker osteocalcin when differentiation was induced with bone morphogenetic protein-2. Cultures of mouse embryo fibroblasts generated from Sam68+/+ and Sam68-/- littermates were induced to differentiate into adipocytes with culture medium containing pioglitazone and the Sam68-/- mouse embryo fibroblasts shown to have impaired adipocyte differentiation. Furthermore, in vivo it was shown that sections of bone from 12-month-old Sam68-/- mice had few marrow adipocytes compared with their age-matched wild-type littermate controls, which exhibited fatty bone marrow. Our findings identify endogenous Sam68 as a positive regulator of adipocyte differentiation and a negative regulator of osteoblast differentiation, which is consistent with Sam68 being a modulator of bone marrow mesenchymal cell differentiation, and hence bone metabolism, in aged mice.

  1. Characterization of RNA binding and chaperoning activities of HIV-1 Vif protein. Importance of the C-terminal unstructured tail.

    Science.gov (United States)

    Sleiman, Dona; Bernacchi, Serena; Xavier Guerrero, Santiago; Brachet, Franck; Larue, Valéry; Paillart, Jean-Christophe; Tisne, Carine

    2014-01-01

    The viral infectivity factor (Vif) is essential for the productive infection and dissemination of HIV-1 in non-permissive cells, containing the cellular anti-HIV defense cytosine deaminases APOBEC3 (A3G and A3F). Vif neutralizes the antiviral activities of the APOBEC3G/F by diverse mechanisms including their degradation through the ubiquitin/proteasome pathway and their translational inhibition. In addition, Vif appears to be an active partner of the late steps of viral replication by interacting with Pr55(Gag), reverse transcriptase and genomic RNA. Here, we expressed and purified full-length and truncated Vif proteins, and analyzed their RNA binding and chaperone properties. First, we showed by CD and NMR spectroscopies that the N-terminal domain of Vif is highly structured in solution, whereas the C-terminal domain remains mainly unfolded. Both domains exhibited substantial RNA binding capacities with dissociation constants in the nanomolar range, whereas the basic unfolded C-terminal domain of Vif was responsible in part for its RNA chaperone activity. Second, we showed by NMR chemical shift mapping that Vif and NCp7 share the same binding sites on tRNA(Lys) 3, the primer of HIV-1 reverse transcriptase. Finally, our results indicate that Vif has potent RNA chaperone activity and provide direct evidence for an important role of the unstructured C-terminal domain of Vif in this capacity.

  2. Dissecting the expression relationships between RNA-binding proteins and their cognate targets in eukaryotic post-transcriptional regulatory networks

    Science.gov (United States)

    Nishtala, Sneha; Neelamraju, Yaseswini; Janga, Sarath Chandra

    2016-05-01

    RNA-binding proteins (RBPs) are pivotal in orchestrating several steps in the metabolism of RNA in eukaryotes thereby controlling an extensive network of RBP-RNA interactions. Here, we employed CLIP (cross-linking immunoprecipitation)-seq datasets for 60 human RBPs and RIP-ChIP (RNP immunoprecipitation-microarray) data for 69 yeast RBPs to construct a network of genome-wide RBP- target RNA interactions for each RBP. We show in humans that majority (~78%) of the RBPs are strongly associated with their target transcripts at transcript level while ~95% of the studied RBPs were also found to be strongly associated with expression levels of target transcripts when protein expression levels of RBPs were employed. At transcript level, RBP - RNA interaction data for the yeast genome, exhibited a strong association for 63% of the RBPs, confirming the association to be conserved across large phylogenetic distances. Analysis to uncover the features contributing to these associations revealed the number of target transcripts and length of the selected protein-coding transcript of an RBP at the transcript level while intensity of the CLIP signal, number of RNA-Binding domains, location of the binding site on the transcript, to be significant at the protein level. Our analysis will contribute to improved modelling and prediction of post-transcriptional networks.

  3. A viral nuclear noncoding RNA binds re-localized poly(A binding protein and is required for late KSHV gene expression.

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    Sumit Borah

    2011-10-01

    Full Text Available During the lytic phase of infection, the gamma herpesvirus Kaposi's Sarcoma-Associated Herpesvirus (KSHV expresses a highly abundant, 1.1 kb nuclear noncoding RNA of unknown function. We observe that this polyadenylated nuclear (PAN RNA avidly binds host poly(A-binding protein C1 (PABPC1, which normally functions in the cytoplasm to bind the poly(A tails of mRNAs, regulating mRNA stability and translation efficiency. During the lytic phase of KSHV infection, PABPC1 is re-localized to the nucleus as a consequence of expression of the viral shutoff exonuclease (SOX protein; SOX also mediates the host shutoff effect in which host mRNAs are downregulated while viral mRNAs are selectively expressed. We show that whereas PAN RNA is not required for the host shutoff effect or for PABPC1 re-localization, SOX strongly upregulates the levels of PAN RNA in transient transfection experiments. This upregulation is destroyed by the same SOX mutation that ablates the host shutoff effect and PABPC1 nuclear re-localization or by removal of the poly(A tail of PAN. In cells induced into the KSHV lytic phase, depletion of PAN RNA using RNase H-targeting antisense oligonucleotides reveals that it is necessary for the production of late viral proteins from mRNAs that are themselves polyadenylated. Our results add to the repertoire of functions ascribed to long noncoding RNAs and suggest a mechanism of action for nuclear noncoding RNAs in gamma herpesvirus infection.

  4. RNA targeting by DNA binding drugs: structural, conformational and energetic aspects of the binding of quinacrine and DAPI to A-form and H(L)-form of poly(rC).poly(rG).

    Science.gov (United States)

    Sinha, Rangana; Hossain, Maidul; Kumar, Gopinatha Suresh

    2007-12-01

    A key step in the rational design of new RNA binding small molecules necessitates a complete elucidation of the molecular aspects of the binding of existing molecules to RNA structures. This work focuses towards the understanding of the interaction of a DNA intercalator, quinacrine and a minor groove binder 4',6-diamidino-2-phenylindole (DAPI) with the right handed Watson-Crick base paired A-form and the left-handed Hoogsteen base paired H(L)-form of poly(rC).poly(rG) evaluated by multifaceted spectroscopic and viscometric techniques. The energetics of their interaction has also been elucidated by isothermal titration calorimetry. Results of this study converge to suggest that (i) quinacrine intercalates to both A-form and H(L)-form of poly(rC).poly(rG); (ii) DAPI shows both intercalative and groove-binding modes to the A-form of the RNA but binds by intercalative mode to the H(L)-form. Isothermal calorimetric patterns of quinacrine binding to both the forms of RNA and of DAPI binding to the H(L)-form are indicative of single binding while the binding of DAPI to the A-form reveals two kinds of binding. The binding of both the drugs to both conformations of RNA is exothermic; while the binding of quinacrine to both conformations and DAPI to the A-form (first site) is entropy driven, the binding of DAPI to the second site of A-form and H(L)-conformation is enthalpy driven. Temperature dependence of the binding enthalpy revealed that the RNA-ligand interaction reactions are accompanied by small heat capacity changes that are nonetheless significant. We conclude that the binding affinity characteristics and energetics of interaction of these DNA binding molecules to the RNA conformations are significantly different and may serve as data for the development of effective structure selective RNA-based antiviral drugs.

  5. Structure, tissue distribution and genomic organization of the murine RRM-type RNA binding proteins TIA-1 and TIAR.

    OpenAIRE

    Beck, A.R.; Medley, Q G; O'Brien, S; Anderson, P; Streuli, M

    1996-01-01

    TIA-1 and TIAR are RNA binding proteins of the RNA recognition motif (RRM)/ribonucleoprotein (RNP) family that have been implicated as effectors of apoptotic cell death. We report the structures of murine TIA-1 and TIAR (mTIA-1 and mTIAR) deduced from cDNA cloning, the mRNA and protein tissue distribution of mTIA-1 and mTIAR, and the exon-intron structures of the mTIA-1 and mTIAR genes. Both mTIA-1 and mTIAR are comprised of three approximately 100 amino acid N-terminal RRM domains and a appr...

  6. Design and Synthesis of Paromomycin Related Heterocyclic Aminogly coside Mimetics Based on the Mass Spectroscopy RNA Binding Assay

    Institute of Scientific and Technical Information of China (English)

    DING Yi-Li

    2003-01-01

    @@ Aminoglycoside antibiotics are known to interact with a variety of RNA molecules, including ribosomal RNA, and the hammerhead ribozyme. These low molecular weight antibiotics have also been found to block the binding of HIV-1 Rev protein to its viral RNA recognition site, thereby inhibiting the production of the virus. But due to their toxicity, poor bioavailability, cellular penetration, and instability, they can not be used as an inhibitory drug direct ly. Therefore, it is highly desirable to synthesize aminoglycoside mimetics, which may be less toxic and more active than original aminoglycosides.

  7. RNA-binding Domain of the Key Structural Protein P7 for the Rice dwarf virus Particle Assembly

    Institute of Scientific and Technical Information of China (English)

    Bo-Xiong ZHONG; Yan-Wei SHEN; Toshihiro OMURA

    2005-01-01

    The Rice dwarf virus (RDV) P7 structural protein is the key protein in the RDV particle assembly. The P7 protein was digested partially or completely by Staphylococcus aureus V8 protease and/or Pseudomonas fragi Asp-N protease. The molecular mass and the N-terminal amino acid sequence of the polypeptide fragments of the P7 protein were determined by SDS-PAGE and the Edman degradation method,respectively. Then the polypeptides were located in the deduced amino acid sequence of the RDV P7 protein based on the nucleotide sequence information, with the knowledge of the specific cleavage sites of the Staphylococcus aureus V8 and Pseudomonasfragi Asp-N protease, and the two RNA-binding domains in the P7 protein were identified. Domain 1 was located in the residue 128-249 containing 122 amino acids and domain 2 was located in the residue 325-355 containing 31 amino acids. Thus, these two domains may play an important role in the virus particle assembly by contributing to the packaging of viral dsRNAs inside the particles. The two domains may be novel RNA-binding domains, because no amino acid sequences highly similar to the conservative sequences of known dsRNA-binding domains reported so far. The similarity between the motif of domain 1 and the motif of the DNA-binding protein suggests that the DNA-binding activity of the RDV P7 protein may be due to this sequence. The similarity between the motif of domain 1 and the motif of the RNA polymerase domain suggests that the P7 protein may also play a role in RNA synthesis,besides its function in the assembly and subsequent packaging of viral dsRNA into core particles.

  8. A microRNA-7 binding site polymorphism in HOXB5 leads to differential gene expression in bladder cancer.

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    Junhua Luo

    Full Text Available PURPOSE: To investigate the biological function of HOXB5 in human bladder cancer and explore whether the HOXB5 3'-UTR SNP (1010A/G, which is located within the microRNA-7 binding site, was correlated with clinical features of bladder cancer. METHODS: Expression of HOXB5 in 35 human bladder cancer tissues and 8 cell lines were examined using real-time PCR and immunohistochemistry. Next, we explored the biological function of HOXB5 in vitro using cell proliferation, migration and colony formation assays. Using bioinformatics, a SNP (1010A/G was found located within the microRNA-7 binding site in the 3'-UTR of HOXB5. Real-time PCR was used to test HOXB5 expression affected by different alleles. Finally, multivariate logistic regression analysis was used to determine the relationship between SNP (1010A/G frequency and clinical features in 391 cases. RESULTS: HOXB5 was frequently over-expressed both in bladder cancer tissues and cell lines. Inhibition of HOXB5 suppressed the oncogenic function of cancer cells. Next, we demonstrated that a SNP (1010A/G, located within the microRNA-7 binding site in the 3'-UTR of HOXB5, could affect HOXB5 expression in bladder cancer mainly by differential binding activity of microRNA-7 and SNP-related mRNA stability. Finally, we also showed the frequency of 1010G genotype was higher in cancer group compared to normal controls and correlated with the risk of high grade and high stage. CONCLUSION: HOXB5 is overexpressed in bladder cancer. A miRNA-binding SNP (1010A/G located within 3'-UTR of HOXB5 is associated with gene expression and may be a promising prognostic factor for bladder cancer.

  9. Widespread regulation of miRNA biogenesis at the Dicer step by the cold-inducible RNA-binding protein, RBM3.

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    Julie Pilotte

    Full Text Available MicroRNAs (miRNAs play critical roles in diverse cellular events through their effects on translation. Emerging data suggest that modulation of miRNA biogenesis at post-transcriptional steps by RNA-binding proteins is a key point of regulatory control over the expression of some miRNAs and the cellular processes they influence. However, the extent and conditions under which the miRNA pathway is amenable to regulation at posttranscriptional steps are poorly understood. Here we show that RBM3, a cold-inducible, developmentally regulated RNA-binding protein and putative protooncogene, is an essential regulator of miRNA biogenesis. Utilizing miRNA array, Northern blot, and PCR methods, we observed that over 60% of miRNAs detectable in a neuronal cell line were significantly downregulated by knockdown of RBM3. Conversely, for select miRNAs assayed by Northern blot, induction of RBM3 by overexpression or mild hypothermia increased their levels. Changes in miRNA expression were accompanied by changes in the levels of their ~70 nt precursors, whereas primary transcript levels were unaffected. Mechanistic studies revealed that knockdown of RBM3 does not reduce Dicer activity or impede transport of pre-miRNAs into the cytoplasm. Rather, we find that RBM3 binds directly to ~70 nt pre-miRNA intermediates and promotes / de-represses their ability as larger ribonucleoproteins (pre-miRNPs to associate with active Dicer complexes. Our findings suggest that the processing of a majority of pre-miRNPs by Dicer is subject to an intrinsic inhibitory influence that is overcome by RBM3 expression. RBM3 may thus orchestrate changes in miRNA expression during hypothermia and other cellular stresses, and in the euthermic contexts of early development, differentiation, and oncogenesis where RBM3 expression is highly elevated. Additionally, our data suggest that temperature-dependent changes in miRNA expression mediated by RBM3 may contribute to the therapeutic effects of

  10. Long Non-Coding RNA HOTAIR Promotes Cell Migration and Invasion via Down-Regulation of RNA Binding Motif Protein 38 in Hepatocellular Carcinoma Cells

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    Chaofeng Ding

    2014-03-01

    Full Text Available Long non-coding RNA HOTAIR exerts regulatory functions in various biological processes in cancer cells, such as proliferation, apoptosis, mobility, and invasion. We previously found that HOX transcript antisense RNA (HOTAIR is a negative prognostic factor and exhibits oncogenic activity in hepatocellular carcinoma (HCC. In this study, we aimed to investigate the role and molecular mechanism of HOTAIR in promoting HCC cell migration and invasion. Firstly, we profiled its gene expression pattern by microarray analysis of HOTAIR loss in Bel-7402 HCC cell line. The results showed that 129 genes were significantly down-regulated, while 167 genes were significantly up-regulated (fold change >2, p < 0.05. Bioinformatics analysis indicated that RNA binding proteins were involved in this biological process. HOTAIR suppression using RNAi strategy with HepG2 and Bel-7402 cells increased the mRNA and protein expression levels of RNA binding motif protein 38 (RBM38. Moreover, the expression levels of RBM38 in HCC specimens were significantly lower than paired adjacent noncancerous tissues. In addition, knockdown of HOTAIR resulted in a decrease of cell migration and invasion, which could be specifically rescued by down-regulation of RBM38. Taken together, HOTAIR could promote migration and invasion of HCC cells by inhibiting RBM38, which indicated critical roles of HOTAIR and RBM38 in HCC progression.

  11. Differential expression of the RNA-binding motif protein 3 in human astrocytoma

    Institute of Scientific and Technical Information of China (English)

    ZHANG Hai-tao; ZHANG Zhi-wen; XUE Jing-hui; KONG Hai-bo; LIU Ai-jun; LI Shou-chun; LIU Yu-xiao

    2013-01-01

    Background The RNA-binding motif protein 3 (RBM3),which is transcriptionally induced by low temperature and hypoxia,has recently been found to be upregulated in human tumors.However,its expression status in human astrocytoma is not well defined.This article focuses on the differential expression of RBM3 in human astrocytomas of different grades and normal brain tissues.Methods RBM3 was detected in astrocytomas and normal brain tissues by quantitative real-time PCR,immunohistochemistry,and Western blotting.Analysis of variance was performed on the data from quantitative real-time PCR.The Fisher's exact test was used to analyze the immunohistochemistry results.A P-value of less than 0.05 indicates a statistically significant difference.Results On one hand,the mRNA expression levels of three X-chromosome-related RBM genes (RBMX,RBM3,and RBM10) were detected by quantitative real-time PCR.The results showed that there were no significant differences in RBMX and RBM10 mRNA expression levels in human astrocytomas of different grades and normal brain tissues.However,RBM3 mRNA expression levels were elevated in high-grade (World Health Organization (WHO) Grade Ⅲ-Ⅳ) astrocytomas versus low-grade (WHO Grade Ⅰ-Ⅱ) astrocytomas (5.06±0.66 vs.1.60±0.58; P <0.05) or normal controls (5.06±0.66 vs.1.03±0.22; P <0.05) as determined by quantitative real-time PCR analysis.On the other hand,immunohistochemistry showed an increased RBM3 labeling index in astrocytomas of different grades and normal brain tissues (positive staining rate:astrocytoma Grade Ⅳ,92.9%; astrocytoma Grade Ⅲ,81.8%; astrocytoma Grade Ⅰ-Ⅱ,50%;normal brain tissues,37.5%; high-grade astrocytoma versus normal brain tissues,P <0.05; high-grade astrocytoma versus low-grade astrocytoma,P <0.05).The higher protein levels of RBM3 were also validated in high-grade astrocytomas and low-grade astrocytomas compared with normal brain tissues by Western blotting.Conclusions These

  12. Functional Significance of the Interaction between the mRNA-binding Protein, Nab2, and the Nuclear Pore-associated Protein, Mlp1, in mRNA Export* S⃞

    OpenAIRE

    Fasken, Milo B.; Stewart, Murray; Corbett, Anita H.

    2008-01-01

    Nuclear export of mRNA requires several key mRNA-binding proteins that recognize and remodel the mRNA and target it for export via interactions with the nuclear pore complex. In Saccharomyces cerevisiae, the shuttling heterogeneous nuclear ribonucleoprotein, Nab2, which is essential for mRNA export, specifically recognizes poly(A) RNA and binds to the nuclear pore-associated protein, myosin-like protein 1 (Mlp1), which functions in mRNA export and quality control. Specifically, the N-terminal...

  13. The magic spot: a ppGpp binding site on E. coli RNA polymerase responsible for regulation of transcription initiation.

    Science.gov (United States)

    Ross, Wilma; Vrentas, Catherine E; Sanchez-Vazquez, Patricia; Gaal, Tamas; Gourse, Richard L

    2013-05-01

    The global regulatory nucleotide ppGpp ("magic spot") regulates transcription from a large subset of Escherichia coli promoters, illustrating how small molecules can control gene expression promoter-specifically by interacting with RNA polymerase (RNAP) without binding to DNA. However, ppGpp's target site on RNAP, and therefore its mechanism of action, has remained unclear. We report here a binding site for ppGpp on E. coli RNAP, identified by crosslinking, protease mapping, and analysis of mutant RNAPs that fail to respond to ppGpp. A strain with a mutant ppGpp binding site displays properties characteristic of cells defective for ppGpp synthesis. The binding site is at an interface of two RNAP subunits, ω and β', and its position suggests an allosteric mechanism of action involving restriction of motion between two mobile RNAP modules. Identification of the binding site allows prediction of bacterial species in which ppGpp exerts its effects by targeting RNAP.

  14. Phosphate-binding pocket in Dicer-2 PAZ domain for high-fidelity siRNA production.

    Science.gov (United States)

    Kandasamy, Suresh K; Fukunaga, Ryuya

    2016-12-06

    The enzyme Dicer produces small silencing RNAs such as micro-RNAs (miRNAs) and small interfering RNAs (siRNAs). In Drosophila, Dicer-1 produces ∼22-24-nt miRNAs from pre-miRNAs, whereas Dicer-2 makes 21-nt siRNAs from long double-stranded RNAs (dsRNAs). How Dicer-2 precisely makes 21-nt siRNAs with a remarkably high fidelity is unknown. Here we report that recognition of the 5'-monophosphate of a long dsRNA substrate by a phosphate-binding pocket in the Dicer-2 PAZ (Piwi, Argonaute, and Zwille/Pinhead) domain is crucial for the length fidelity, but not the efficiency, in 21-nt siRNA production. Loss of the length fidelity, meaning increased length heterogeneity of siRNAs, caused by point mutations in the phosphate-binding pocket of the Dicer-2 PAZ domain decreased RNA silencing activity in vivo, showing the importance of the high fidelity to make 21-nt siRNAs. We propose that the 5'-monophosphate of a long dsRNA substrate is anchored by the phosphate-binding pocket in the Dicer-2 PAZ domain and the distance between the pocket and the RNA cleavage active site in the RNaseIII domain corresponds to the 21-nt pitch in the A-form duplex of a long dsRNA substrate, resulting in high-fidelity 21-nt siRNA production. This study sheds light on the molecular mechanism by which Dicer-2 produces 21-nt siRNAs with a remarkably high fidelity for efficient RNA silencing.

  15. Does unpaired adenosine-66 from helix II of Escherichia coli 5S RNA bind to protein L18?

    DEFF Research Database (Denmark)

    Christiansen, J; Douthwaite, S R; Christensen, A

    1985-01-01

    plasmid derived from pKK3535. Binding studies with protein L18 revealed that the protein bound much more weakly to the mutated 5S RNA. We consider the most likely explanation of this result is that L18 interacts with adenosine-66, and we present a tentative model for an interaction between the unpaired...

  16. A petunia ocs element binding factor, PhOBF1, plays an important role in antiviral RNA silencing

    Science.gov (United States)

    Virus-induced gene silencing (VIGS) is a common strategy of reverse genetics for characterizing function of genes in plant. The detailed mechanism governing RNA silencing efficiency triggered by virus is largely unclear. Here, we revealed that a petunia (Petunia hybrida) ocs element binding factor, ...

  17. The magic spot: identification of the binding site for ppGpp on E. coli RNA polymerase

    Science.gov (United States)

    Despite more than 40 years of study of the global regulatory nucleotide ppGpp ("magic spot") in Escherichia coli, its target site on RNA polymerase (RNAP), and therefore its mechanism of action, is unknown. We report here a binding site for ppGpp on E. coli RNAP, identified by crosslinking, protease...

  18. Prediction of altered 3'- UTR miRNA-binding sites from RNA-Seq data: the swine leukocyte antigen complex (SLA as a model region.

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    Marie-Laure Endale Ahanda

    Full Text Available THE SLA (swine leukocyte antigen, MHC: SLA genes are the most important determinants of immune, infectious disease and vaccine response in pigs; several genetic associations with immunity and swine production traits have been reported. However, most of the current knowledge on SLA is limited to gene coding regions. MicroRNAs (miRNAs are small molecules that post-transcriptionally regulate the expression of a large number of protein-coding genes in metazoans, and are suggested to play important roles in fine-tuning immune mechanisms and disease responses. Polymorphisms in either miRNAs or their gene targets may have a significant impact on gene expression by abolishing, weakening or creating miRNA target sites, possibly leading to phenotypic variation. We explored the impact of variants in the 3'-UTR miRNA target sites of genes within the whole SLA region. The combined predictions by TargetScan, PACMIT and TargetSpy, based on different biological parameters, empowered the identification of miRNA target sites and the discovery of polymorphic miRNA target sites (poly-miRTSs. Predictions for three SLA genes characterized by a different range of sequence variation provided proof of principle for the analysis of poly-miRTSs from a total of 144 M RNA-Seq reads collected from different porcine tissues. Twenty-four novel SNPs were predicted to affect miRNA-binding sites in 19 genes of the SLA region. Seven of these genes (SLA-1, SLA-6, SLA-DQA, SLA-DQB1, SLA-DOA, SLA-DOB and TAP1 are linked to antigen processing and presentation functions, which is reminiscent of associations with disease traits reported for altered miRNA binding to MHC genes in humans. An inverse correlation in expression levels was demonstrated between miRNAs and co-expressed SLA targets by exploiting a published dataset (RNA-Seq and small RNA-Seq of three porcine tissues. Our results support the resource value of RNA-Seq collections to identify SNPs that may lead to altered miRNA

  19. Prediction of altered 3'- UTR miRNA-binding sites from RNA-Seq data: the swine leukocyte antigen complex (SLA) as a model region.

    Science.gov (United States)

    Endale Ahanda, Marie-Laure; Fritz, Eric R; Estellé, Jordi; Hu, Zhi-Liang; Madsen, Ole; Groenen, Martien A M; Beraldi, Dario; Kapetanovic, Ronan; Hume, David A; Rowland, Robert R R; Lunney, Joan K; Rogel-Gaillard, Claire; Reecy, James M; Giuffra, Elisabetta

    2012-01-01

    THE SLA (swine leukocyte antigen, MHC: SLA) genes are the most important determinants of immune, infectious disease and vaccine response in pigs; several genetic associations with immunity and swine production traits have been reported. However, most of the current knowledge on SLA is limited to gene coding regions. MicroRNAs (miRNAs) are small molecules that post-transcriptionally regulate the expression of a large number of protein-coding genes in metazoans, and are suggested to play important roles in fine-tuning immune mechanisms and disease responses. Polymorphisms in either miRNAs or their gene targets may have a significant impact on gene expression by abolishing, weakening or creating miRNA target sites, possibly leading to phenotypic variation. We explored the impact of variants in the 3'-UTR miRNA target sites of genes within the whole SLA region. The combined predictions by TargetScan, PACMIT and TargetSpy, based on different biological parameters, empowered the identification of miRNA target sites and the discovery of polymorphic miRNA target sites (poly-miRTSs). Predictions for three SLA genes characterized by a different range of sequence variation provided proof of principle for the analysis of poly-miRTSs from a total of 144 M RNA-Seq reads collected from different porcine tissues. Twenty-four novel SNPs were predicted to affect miRNA-binding sites in 19 genes of the SLA region. Seven of these genes (SLA-1, SLA-6, SLA-DQA, SLA-DQB1, SLA-DOA, SLA-DOB and TAP1) are linked to antigen processing and presentation functions, which is reminiscent of associations with disease traits reported for altered miRNA binding to MHC genes in humans. An inverse correlation in expression levels was demonstrated between miRNAs and co-expressed SLA targets by exploiting a published dataset (RNA-Seq and small RNA-Seq) of three porcine tissues. Our results support the resource value of RNA-Seq collections to identify SNPs that may lead to altered miRNA regulation patterns.

  20. Prediction of Altered 3′- UTR miRNA-Binding Sites from RNA-Seq Data: The Swine Leukocyte Antigen Complex (SLA) as a Model Region

    Science.gov (United States)

    Endale Ahanda, Marie-Laure; Fritz, Eric R.; Estellé, Jordi; Hu, Zhi-Liang; Madsen, Ole; Groenen, Martien A. M.; Beraldi, Dario; Kapetanovic, Ronan; Hume, David A.; Rowland, Robert R. R.; Lunney, Joan K.; Rogel-Gaillard, Claire; Reecy, James M.; Giuffra, Elisabetta

    2012-01-01

    The SLA (swine leukocyte antigen, MHC: SLA) genes are the most important determinants of immune, infectious disease and vaccine response in pigs; several genetic associations with immunity and swine production traits have been reported. However, most of the current knowledge on SLA is limited to gene coding regions. MicroRNAs (miRNAs) are small molecules that post-transcriptionally regulate the expression of a large number of protein-coding genes in metazoans, and are suggested to play important roles in fine-tuning immune mechanisms and disease responses. Polymorphisms in either miRNAs or their gene targets may have a significant impact on gene expression by abolishing, weakening or creating miRNA target sites, possibly leading to phenotypic variation. We explored the impact of variants in the 3′-UTR miRNA target sites of genes within the whole SLA region. The combined predictions by TargetScan, PACMIT and TargetSpy, based on different biological parameters, empowered the identification of miRNA target sites and the discovery of polymorphic miRNA target sites (poly-miRTSs). Predictions for three SLA genes characterized by a different range of sequence variation provided proof of principle for the analysis of poly-miRTSs from a total of 144 M RNA-Seq reads collected from different porcine tissues. Twenty-four novel SNPs were predicted to affect miRNA-binding sites in 19 genes of the SLA region. Seven of these genes (SLA-1, SLA-6, SLA-DQA, SLA-DQB1, SLA-DOA, SLA-DOB and TAP1) are linked to antigen processing and presentation functions, which is reminiscent of associations with disease traits reported for altered miRNA binding to MHC genes in humans. An inverse correlation in expression levels was demonstrated between miRNAs and co-expressed SLA targets by exploiting a published dataset (RNA-Seq and small RNA-Seq) of three porcine tissues. Our results support the resource value of RNA-Seq collections to identify SNPs that may lead to altered miRNA regulation

  1. Binding of a candidate splice regulator to a calcitonin-specific splice enhancer regulates calcitonin/CGRP pre-mRNA splicing.

    Science.gov (United States)

    Coleman, Timothy P; Tran, Quincy; Roesser, James R

    2003-01-27

    The calcitonin/calcitonin gene-related peptide (CGRP) pre-mRNA is alternatively processed in a tissue-specific manner leading to the production of calcitonin mRNA in thyroid C cells and CGRP mRNA in neurons. A candidate calcitonin/CGRP splice regulator (CSR) isolated from rat brain was shown to inhibit calcitonin-specific splicing in vitro. CSR specifically binds to two regions in the calcitonin-specific exon 4 RNA previously demonstrated to function as a bipartate exonic splice enhancer (ESE). The two regions, A and B element, are necessary for inclusion of exon 4 into calcitonin mRNA. A novel RNA footprinting method based on the UV cross-linking assay was used to define the site of interaction between CSR and B element RNA. Base changes at the CSR binding site prevented CSR binding to B element RNA and CSR was unable to inhibit in vitro splicing of pre-mRNAs containing the mutated CSR binding site. When expressed in cells that normally produce predominantly CGRP mRNA, a calcitonin/CGRP gene containing the mutated CSR binding site expressed predominantly calcitonin mRNA. These observations demonstrate that CSR binding to the calcitonin-specific ESE regulates calcitonin/CGRP pre-mRNA splicing.

  2. Effects of single-base substitutions within the acanthamoeba castellanii rRNA promoter on transcription and on binding of transcription initiation factor and RNA polymerase I

    Energy Technology Data Exchange (ETDEWEB)

    Kownin, P.; Bateman, E.; Paule, M.R.

    1988-02-01

    Single-point mutations were introduced into the promoter region of the Acanthamoeba castellanii rRNA gene by chemical mutagen treatment of a single-stranded clone in vitro, followed by reverse transcription and cloning of the altered fragment. The promoter mutants were tested for transcription initiation factor (TIF) binding by a template commitment assay plus DNase I footprinting and for transcription by an in vitro runoff assay. Point mutations within the previously identified TIF interaction region (between -20 and -47, motifs A and B) indicated that TIF interacts most strongly with a sequence centered at -29 and less tightly with sequences upstream and downstream. Some alterations of the base sequence closer to the transcription start site (and outside the TIF-protected site) also significantly decrease specific RNA synthesis in vitro. These were within the region which is protected from DNAse I digestion by polymerase I, but these mutations did not detectably affect the binding of polymerase to the promoter.

  3. MD Simulations of tRNA and Aminoacyl-tRNA Synthetases: Dynamics, Folding, Binding, and Allostery

    Directory of Open Access Journals (Sweden)

    Rongzhong Li

    2015-07-01

    Full Text Available While tRNA and aminoacyl-tRNA synthetases are classes of biomolecules that have been extensively studied for decades, the finer details of how they carry out their fundamental biological functions in protein synthesis remain a challenge. Recent molecular dynamics (MD simulations are verifying experimental observations and providing new insight that cannot be addressed from experiments alone. Throughout the review, we briefly discuss important historical events to provide a context for how far the field has progressed over the past few decades. We then review the background of tRNA molecules, aminoacyl-tRNA synthetases, and current state of the art MD simulation techniques for those who may be unfamiliar with any of those fields. Recent MD simulations of tRNA dynamics and folding and of aminoacyl-tRNA synthetase dynamics and mechanistic characterizations are discussed. We highlight the recent successes and discuss how important questions can be addressed using current MD simulations techniques. We also outline several natural next steps for computational studies of AARS:tRNA complexes.

  4. IGF2 mRNA-binding protein 2: biological function and putative role in type 2 diabetes

    DEFF Research Database (Denmark)

    Christiansen, J.; Kolte, A.M.; Hansen, T.O.;

    2009-01-01

    Recent genome-wide association (GWA) studies of type 2 diabetes (T2D) have implicated IGF2 mRNA-binding protein 2 (IMP2/IGF2BP2) as one of the several factors in the etiology of late onset diabetes. IMP2 belongs to a family of oncofetal mRNA-binding proteins implicated in RNA localization......, stability, and translation that are essential for normal embryonic growth and development. This review provides a background to the IMP protein family with an emphasis on human IMP2, followed by a closer look at the GWA studies to evaluate the significance, if any, of the proposed correlation between IMP2...... and T2D Udgivelsesdato: 2009/11...

  5. Genetic variation and RNA binding proteins: tools and techniques to detect functional polymorphisms.

    Science.gov (United States)

    Soemedi, Rachel; Vega, Hugo; Belmont, Judson M; Ramachandran, Sohini; Fairbrother, William G

    2014-01-01

    At its most fundamental level the goal of genetics is to connect genotype to phenotype. This question is asked at a basic level evaluating the role of genes and pathways in genetic model organism. Increasingly, this question is being asked in the clinic. Genomes of individuals and populations are being sequenced and compared. The challenge often comes at the stage of analysis. The variant positions are analyzed with the hope of understanding human disease. However after a genome or exome has been sequenced, the researcher is often deluged with hundreds of potentially relevant variations. Traditionally, amino-acid changing mutations were considered the tractable class of disease-causing mutations; however, mutations that disrupt noncoding elements are the subject of growing interest. These noncoding changes are a major avenue of disease (e.g., one in three hereditary disease alleles are predicted to affect splicing). Here, we review some current practices of medical genetics, the basic theory behind biochemical binding and functional assays, and then explore technical advances in how variations that alter RNA protein recognition events are detected and studied. These advances are advances in scale-high-throughput implementations of traditional biochemical assays that are feasible to perform in any molecular biology laboratory. This chapter utilizes a case study approach to illustrate some methods for analyzing polymorphisms. The first characterizes a functional intronic SNP that deletes a high affinity PTB site using traditional low-throughput biochemical and functional assays. From here we demonstrate the utility of high-throughput splicing and spliceosome assembly assays for screening large sets of SNPs and disease alleles for allelic differences in gene expression. Finally we perform three pilot drug screens with small molecules (G418, tetracycline, and valproic acid) that illustrate how compounds that rescue specific instances of differential pre-mRNA processing

  6. Cold-inducible RNA-binding protein inhibits neuron apoptosis through the suppression of mitochondrial apoptosis.

    Science.gov (United States)

    Zhang, Hai-Tao; Xue, Jing-Hui; Zhang, Zhi-Wen; Kong, Hai-Bo; Liu, Ai-Jun; Li, Shou-Chun; Xu, Dong-Gang

    2015-10-01

    Cold-inducible RNA-binding protein (CIRP) is induced by mild hypothermia in several mammals, but the precise mechanism by which CIRP mediates hypothermia-induced neuroprotection remains unknown. We aimed to investigate the molecular mechanisms by which CIRP protects the nervous system during mild hypothermia. Rat cortical neurons were isolated and cultured in vitro under mild hypothermia (32°C). Apoptosis was measured by annexin V and propidium iodide staining, visualized by flow cytometry. Neuron ultrastructure was visualized by transmission electron microscopy. CIRP overexpression and knockdown were achieved via infection with pL/IRES/GFP-CIRP and pL/shRNA/F-CIRP-A lentivirus. RT(2) Profiler PCR Array Pathway Analysis and western blotting were used to evaluate the effects of CIRP overexpresion/knockdown on the neurons׳ transcriptome. Neuron late apoptosis was significantly reduced at day 7 of culture by 12h hypothermia, but neuron ultrastructure remained relatively intact. RT(2) Profiler PCR Array Pathway Analysis of 84 apoptosis pathway-associated factors revealed that mild hypothermia and CIRP overexpression induce similar gene expression profiles, specifically alterations of genes implicated in the mitochondrial apoptosis pathway. Mild hypothermia-treated neurons up-regulated 12 and down-regulated 38 apoptosis pathway-associated genes. CIRP-overexpressing neurons up-regulated 15 and down-regulated 46 genes. CIRP-knocked-down hypothermia-treated cells up-regulated 9 and down-regulated 40 genes. Similar results were obtained at the protein level. In conclusion, CIRP may inhibit neuron apoptosis through the suppression of the mitochondria apoptosis pathway during mild hypothermia.

  7. Extensive association of functionally and cytotopically related mRNAs with Puf family RNA-binding proteins in yeast.

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    André P Gerber

    2004-03-01

    Full Text Available Genes encoding RNA-binding proteins are diverse and abundant in eukaryotic genomes. Although some have been shown to have roles in post-transcriptional regulation of the expression of specific genes, few of these proteins have been studied systematically. We have used an affinity tag to isolate each of the five members of the Puf family of RNA-binding proteins in Saccharomyces cerevisiae and DNA microarrays to comprehensively identify the associated mRNAs. Distinct groups of 40-220 different mRNAs with striking common themes in the functions and subcellular localization of the proteins they encode are associated with each of the five Puf proteins: Puf3p binds nearly exclusively to cytoplasmic mRNAs that encode mitochondrial proteins; Puf1p and Puf2p interact preferentially with mRNAs encoding membrane-associated proteins; Puf4p preferentially binds mRNAs encoding nucleolar ribosomal RNA-processing factors; and Puf5p is associated with mRNAs encoding chromatin modifiers and components of the spindle pole body. We identified distinct sequence motifs in the 3'-untranslated regions of the mRNAs bound by Puf3p, Puf4p, and Puf5p. Three-hybrid assays confirmed the role of these motifs in specific RNA-protein interactions in vivo. The results suggest that combinatorial tagging of transcripts by specific RNA-binding proteins may be a general mechanism for coordinated control of the localization, translation, and decay of mRNAs and thus an integral part of the global gene expression program.

  8. Interactions among rsmX ncRNAs and Rsm RNA-binding proteins in the plant pathogen Pseudomonas syringae DC3000

    Science.gov (United States)

    In response to changing environmental stimuli, many bacterial species utilize the Csr/Rsm system of posttranscriptional gene expression regulation to control metabolism, motility, biofilm formation, and quorum sensing. Most Csr/Rsm RNA binding proteins are thought to bind near the 5’ end of mRNA tra...

  9. Problem-Solving Test: Nucleocytoplasmic Shuttling of Pre-mRNA Binding Proteins

    Science.gov (United States)

    Szeberenyi, Jozsef

    2012-01-01

    Terms to be familiar with before you start to solve the test: transcription, pre-mRNA, RNA processing, RNA transport, RNA polymerase II, direct and indirect immunofluorescence staining, cell fractionation by centrifugation, oligo(dT)-cellulose chromatography, washing and elution of the column, ribonuclease, SDS-polyacrylamide gel electrophoresis,…

  10. RNA-binding protein HuR sequesters microRNA-21 to prevent translation repression of proinflammatory tumor suppressor gene programmed cell death 4.

    Science.gov (United States)

    Poria, D K; Guha, A; Nandi, I; Ray, P S

    2016-03-31

    Translation control of proinflammatory genes has a crucial role in regulating the inflammatory response and preventing chronic inflammation, including a transition to cancer. The proinflammatory tumor suppressor protein programmed cell death 4 (PDCD4) is important for maintaining the balance between inflammation and tumorigenesis. PDCD4 messenger RNA translation is inhibited by the oncogenic microRNA, miR-21. AU-rich element-binding protein HuR was found to interact with the PDCD4 3'-untranslated region (UTR) and prevent miR-21-mediated repression of PDCD4 translation. Cells stably expressing miR-21 showed higher proliferation and reduced apoptosis, which was reversed by HuR expression. Inflammatory stimulus caused nuclear-cytoplasmic relocalization of HuR, reversing the translation repression of PDCD4. Unprecedentedly, HuR was also found to bind to miR-21 directly, preventing its interaction with the PDCD4 3'-UTR, thereby preventing the translation repression of PDCD4. This suggests that HuR might act as a 'miRNA sponge' to regulate miRNA-mediated translation regulation under conditions of stress-induced nuclear-cytoplasmic translocation of HuR, which would allow fine-tuned gene expression in complex regulatory environments.

  11. Comparative sequence analysis of double stranded RNA binding protein encoding gene of parapoxviruses from Indian camels

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    G. Nagarajan

    2014-03-01

    Full Text Available The dsRNA binding protein (RBP encoding gene of parapoxviruses (PPVs from the Dromedary camels, inhabitating different geographical region of Rajasthan, India were amplified by polymerase chain reaction using the primers of pseudocowpoxvirus (PCPV from Finnish reindeer and cloned into pGEM-T for sequence analysis. Analysis of RBP encoding gene revealed that PPV DNA from Bikaner shared 98.3% and 76.6% sequence identity at the amino acid level, with Pali and Udaipur PPV DNA, respectively. Reference strains of Bovine papular stomatitis virus (BPSV and PCPV (reindeer PCPV and human PCPV shared 52.8% and 86.9% amino acid identity with RBP gene of camel PPVs from Bikaner, respectively. But different strains of orf virus (ORFV from different geographical areas of the world shared 69.5–71.7% amino acid identity with RBP gene of camel PPVs from Bikaner. These findings indicate that the camel PPVs described are closely related to bovine PPV (PCPV in comparison to caprine and ovine PPV (ORFV.

  12. RNA-binding protein LIN28 is a sensitive marker of ovarian primitive germ cell tumours.

    Science.gov (United States)

    Xue, Debin; Peng, Yan; Wang, Fenghua; Allan, Robert W; Cao, Dengfeng

    2011-09-01

    LIN28 is an RNA-binding protein that has been detected in testicular germ cell tumours (GCTs), but its status in ovarian GCTs is unknown. The aim was to determine the immunohistochemical profile of LIN28 in ovarian GCTs. Immunohistochemistry of LIN28 was performed in 110 primary and 11 metastatic ovarian GCTs. The percentage of tumour cells stained was scored as 0, 1+ (1-30% cells), 2+ (31-60%), 3+ (61-90%), and 4+ (>90%). To determine its specificity, we stained LIN28 in 119 non-GCTs, including 37 clear cell carcinomas. Strong 4+ LIN28 staining was seen in 4/4 (100%) gonadoblastomas, 7/7 (100%) embryonal carcinomas (ECs), and 41/41 (100%) yolk sac tumours (YSTs). Among 39 dysgerminomas, 4+ staining was seen in 37 and 3+ staining in two (strong in 37; mixed weak and strong in two). Twelve of 14 immature teratomas showed variable LIN28 staining (1+ to 4+) in the immature neuroepithelium (weak to strong staining), whereas mature teratomas, carcinoids, struma ovarii and strumal carcinoids were negative. Only 5/117 non-GCTs (1/37 clear cell carcinomas) showed weak to moderate 1-2+ staining. LIN28 is a sensitive marker for gonadoblastomas, dysgerminomas, ECs, and YSTs. LIN28 can be used to distinguish them from non-GCTs. © 2011 Blackwell Publishing Limited.

  13. An RNA Aptamer Targets the PDZ-Binding Motif of the HPV16 E6 Oncoprotein

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    Belyaeva, Tamara A.; Nicol, Clare; Cesur, Özlem [School of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT (United Kingdom); Travé, Gilles [UMR 7242 CNRS-Université de Strasbourg, Ecole Supérieure de Biotechnologie, Boulevard Sébastien Brant, Illkirch 67412 (France); Blair, George Eric; Stonehouse, Nicola J., E-mail: n.j.stonehouse@leeds.ac.uk [School of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT (United Kingdom)

    2014-07-24

    Human papillomavirus 16 (HPV16) is a high-risk DNA tumour virus which is the primary causative agent of cervical cancer. Cell transformation arises from deregulated expression of the E6 and E7 oncogenes. E6 has been shown to bind a number of cellular proteins, including p53 and proteins containing a PDZ domain. This study reports the first RNA aptamers to E6. These have been employed as molecular tools to further investigate E6-p53 and E6-PDZ interactions. This study is focussed on two aptamers (termed F2 and F4) which induced apoptosis in cells derived from an HPV16-transformed cervical carcinoma. The molecules were able to inhibit the interaction between E6 and PDZ1 from Magi1, with F2 being the most effective inhibitor. Neither of the aptamers inhibited E6-p53 interaction or p53 degradation. This study shows the specificity of this approach and highlights the potential benefits of the E6 aptamers as potential therapeutic or diagnostic agents in the future.

  14. An RNA Aptamer Targets the PDZ-Binding Motif of the HPV16 E6 Oncoprotein

    Directory of Open Access Journals (Sweden)

    Tamara A. Belyaeva

    2014-07-01

    Full Text Available Human papillomavirus 16 (HPV16 is a high-risk DNA tumour virus which is the primary causative agent of cervical cancer. Cell transformation arises from deregulated expression of the E6 and E7 oncogenes. E6 has been shown to bind a number of cellular proteins, including p53 and proteins containing a PDZ domain. This study reports the first RNA aptamers to E6. These have been employed as molecular tools to further investigate E6-p53 and E6-PDZ interactions. This study is focussed on two aptamers (termed F2 and F4 which induced apoptosis in cells derived from an HPV16-transformed cervical carcinoma. The molecules were able to inhibit the interaction between E6 and PDZ1 from Magi1, with F2 being the most effective inhibitor. Neither of the aptamers inhibited E6-p53 interaction or p53 degradation. This study shows the specificity of this approach and highlights the potential benefits of the E6 aptamers as potential therapeutic or diagnostic agents in the future.

  15. RNA binding proteins in spermatogenesis: an in depth focus on the Musashi family

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    Jessie M Sutherland

    2015-01-01

    Full Text Available Controlled gene regulation during gamete development is vital for maintaining reproductive potential. During the complex process of mammalian spermatogenesis, male germ cells experience extended periods of the inactive transcription despite heavy translational requirements for continued growth and differentiation. Hence, spermatogenesis is highly reliant on mechanisms of posttranscriptional regulation of gene expression, facilitated by RNA binding proteins (RBPs, which remain abundantly expressed throughout this process. One such group of proteins is the Musashi family, previously identified as critical regulators of testis germ cell development and meiosis in Drosophila, and also shown to be vital to sperm development and reproductive potential in the mouse. This review describes the role and function of RBPs within the scope of male germ cell development, focusing on our recent knowledge of the Musashi proteins in spermatogenesis. The functional mechanisms utilized by RBPs within the cell are outlined in depth, and the significance of sub-cellular localization and stage-specific expression in relation to the mode and impact of posttranscriptional regulation is also highlighted. We emphasize the historical role of the Musashi family of RBPs in stem cell function and cell fate determination, as originally characterized in Drosophila and Xenopus, and conclude with our current understanding of the differential roles and functions of the mammalian Musashi proteins, Musashi-1 and Musashi-2, with a primary focus on our findings in spermatogenesis. This review highlights both the essential contribution of RBPs to posttranscriptional regulation and the importance of the Musashi family as master regulators of male gamete development.

  16. TDP-43-Mediated Neuron Loss In Vivo Requires RNA-Binding Activity

    Science.gov (United States)

    Kaur, Kavita; Müller, Daniel; Karsten, Peter; Weber, Stephanie S.; Kahle, Philipp J.; Marquardt, Till; Schulz, Jörg B.

    2010-01-01

    Alteration and/or mutations of the ribonucleoprotein TDP-43 have been firmly linked to human neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). The relative impacts of TDP-43 alteration, mutation, or inherent protein function on neural integrity, however, remain less clear—a situation confounded by conflicting reports based on transient and/or random-insertion transgenic expression. We therefore performed a stringent comparative investigation of impacts of these TDP-43 modifications on neural integrity in vivo. To achieve this, we systematically screened ALS/FTLD-associated and synthetic TDP-43 isoforms via same-site gene insertion and neural expression in Drosophila; followed by transposon-based motor neuron-specific transgenesis in a chick vertebrate system. Using this bi-systemic approach we uncovered a requirement of inherent TDP-43 RNA-binding function—but not ALS/FTLD-linked mutation, mislocalization, or truncation—for TDP-43-mediated neurotoxicity in vivo. PMID:20806063

  17. A new generation of proto-oncogenes: cold-inducible RNA binding proteins.

    Science.gov (United States)

    Lleonart, M E

    2010-01-01

    This review focuses on the roles of two major cold-inducible RNA binding proteins known in human cells: CIRP and RBM3. Both proteins were discovered when they were shown to be induced after exposure to a moderate cold-shock and other cellular stresses such as UV radiation and hypoxia. Initially, it was suggested that these proteins have a suppressive rather stimulatory effect on proliferation; however, proliferative and/or proto-oncogenic functions have recently been assigned to CIRP and RBM3. In a high throughput genetic screen, we recently identified CIRP as an immortalized gene in murine primary cells. On the other hand, the role of RBM3 in transformation has already been demonstrated. Interestingly, both CIRP and RBM3 have been found to be up-regulated in human tumors. This article highlights the roles of CIRP and RBM3 in tumorigenesis, and proposes a model by which CIRP might contribute to senescence bypass by counteracting the deleterious effects of oxidative damage.

  18. Novel RNA-binding properties of Pop3p support a role for eukaryotic RNase P protein subunits in substrate recognition.

    Science.gov (United States)

    Brusca, E M; True, H L; Celander, D W

    2001-11-09

    Ribonuclease P (RNase P) catalyzes the 5'-end maturation of transfer RNA molecules. Recent evidence suggests that the eukaryotic protein subunits may provide substrate-binding functions (True, H. L., and Celander, D. W. (1998) J. Biol. Chem. 273, 7193-7196). We now report that Pop3p, an essential protein subunit of the holoenzyme in Saccharomyces cerevisiae, displays novel RNA-binding properties. A recombinant form of Pop3p (H6Pop3p) displays a 3-fold greater affinity for binding pre-tRNA substrates relative to tRNA products. The recognition sequence for the H6Pop3p-substrate interaction in vitro was mapped to a 39-nucleotide long sequence that extends from position -21 to +18 surrounding the natural processing site in pre-tRNA substrates. H6Pop3p binds a variety of RNA molecules with high affinity (K(d) = 16-25 nm) and displays a preference for single-stranded RNAs. Removal or modification of basic C-terminal residues attenuates the RNA-binding properties displayed by the protein specifically for a pre-tRNA substrate. These studies support the model that eukaryotic RNase P proteins bind simultaneously to the RNA subunit and RNA substrate.

  19. The Human dsRNA binding protein PACT is unable to functionally substitute for the Drosophila dsRNA binding protein R2D2 [v1; ref status: indexed, http://f1000r.es/201

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    Benjamin K Dickerman

    2013-10-01

    Full Text Available The primary function of the dsRNA binding protein (dsRBP PACT/RAX is to activate the dsRNA dependent protein kinase PKR in response to stress signals.  Additionally, it has been identified as a component of the small RNA processing pathway.  A role for PACT/RAX in this pathway represents an important interplay between two modes of post-transcriptional gene regulation.  The function of PACT/RAX in this context is poorly understood.  Thus, additional models are required to clarify the mechanism by which PACT/RAX functions.  In this study, Drosophila melanogaster was employed to identify functionally orthologous dsRNA-binding proteins.  Transgenic Drosophila expressing human PACT were generated to determine whether PACT is capable of functionally substituting for the Drosophila dsRBP R2D2, which has a well-defined role in small RNA biogenesis.  Results presented here indicate that PACT is unable to substitute for R2D2 at the whole organism level.

  20. Structural arrangement of tRNA binding sites on Escherichia coli ribosomes, as revealed from data on affinity labelling with photoactivatable tRNA derivatives.

    Science.gov (United States)

    Graifer, D M; Babkina, G T; Matasova, N B; Vladimirov, S N; Karpova, G G; Vlassov, V V

    1989-07-01

    A systematic study of protein environment of tRNA in ribosomes in model complexes representing different translation steps was carried out using the affinity labelling of the ribosomes with tRNA derivatives bearing aryl azide groups scattered statistically over tRNA guanine residues. Analysis of the proteins crosslinked to tRNA derivatives showed that the location of the derivatives in the aminoacyl (A) site led to the labelling of the proteins S5 and S7 in all complexes studied, whereas the labelling of the proteins S2, S8, S9, S11, S14, S16, S17, S18, S19, S21 as well as L9, L11, L14, L15, L21, L23, L24, L29 depended on the state of tRNA in A site. Similarly, the location of tRNA derivatives in the peptidyl (P) site resulted in the labelling of the proteins L27, S11, S13 and S19 in all states, whereas the labelling of the proteins S5, S7, S9, S12, S14, S20, S21 as well as L2, L13, L14, L17, L24, L27, L31, L32, L33 depended on the type of complex. The derivatives of tRNA(fMet) were found to crosslink to S1, S3, S5, S7, S9, S14 and L1, L2, L7/L12, L27. Based on the data obtained, a general principle of the dynamic functioning of ribosomes has been proposed: (i) the formation of each type of ribosomal complex is accompanied by changes in mutual arrangement of proteins - 'conformational adjustment' of the ribosome - and (ii) a ribosome can dynamically change its internal structure at each step of initiation and elongation; on the 70 S ribosome there are no rigidly fixed structures forming tRNA-binding sites (primarily A and P sites).

  1. The RNA Binding Protein IMP2 Preserves Glioblastoma Stem Cells by Preventing let-7 Target Gene Silencing

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    Nils Degrauwe

    2016-05-01

    Full Text Available Cancer stem cells (CSCs can drive tumor growth, and their maintenance may rely on post-transcriptional regulation of gene expression, including that mediated by microRNAs (miRNAs. The let-7 miRNA family has been shown to induce differentiation by silencing stem cell programs. Let-7-mediated target gene suppression is prevented by LIN28A/B, which reduce let-7 biogenesis in normal embryonic and some cancer stem cells and ensure maintenance of stemness. Here, we find that glioblastoma stem cells (GSCs lack LIN28 and express both let-7 and their target genes, suggesting LIN28-independent protection from let-7 silencing. Using photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP, we show that insulin-like growth factor 2 mRNA-binding protein 2 (IMP2 binds to let-7 miRNA recognition elements (MREs and prevents let-7 target gene silencing. Our observations define the RNA-binding repertoire of IMP2 and identify a mechanism whereby it supports GSC and neural stem cell specification.

  2. Sequence-Based Prediction of RNA-Binding Proteins Using Random Forest with Minimum Redundancy Maximum Relevance Feature Selection

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    Xin Ma

    2015-01-01

    Full Text Available The prediction of RNA-binding proteins is one of the most challenging problems in computation biology. Although some studies have investigated this problem, the accuracy of prediction is still not sufficient. In this study, a highly accurate method was developed to predict RNA-binding proteins from amino acid sequences using random forests with the minimum redundancy maximum relevance (mRMR method, followed by incremental feature selection (IFS. We incorporated features of conjoint triad features and three novel features: binding propensity (BP, nonbinding propensity (NBP, and evolutionary information combined with physicochemical properties (EIPP. The results showed that these novel features have important roles in improving the performance of the predictor. Using the mRMR-IFS method, our predictor achieved the best performance (86.62% accuracy and 0.737 Matthews correlation coefficient. High prediction accuracy and successful prediction performance suggested that our method can be a useful approach to identify RNA-binding proteins from sequence information.

  3. Regulation of the mRNA-binding protein HuR by posttranslational modification: spotlight on phosphorylation.

    Science.gov (United States)

    Eberhardt, Wolfgang; Doller, Anke; Pfeilschifter, Josef

    2012-06-01

    The ubiquitous mRNA-binding protein human antigen R (HuR) and its neuronal relatives (HuB, HuC, HuD) participate in the post-transcriptional regulation of many AU-rich element-bearing mRNAs. In addition to its originally described role in controlling mRNA decay, the binding of HuR to target mRNAs can affect many aspects of mRNA processing including splicing, polyadenylation, intracellular trafficking, translation and modulation of mRNA repression by miRNAs. In accordance to the growing list of signalling events which are involved in regulating these different HuR functions, recent data implicate that posttranslational modification, namely protein kinase-triggered phosphorylation of HuR plays a crucial role in connecting extracellular signal inputs to a specific post-transcriptional program by HuR. Notably, in addition to directly targeting HuR functions, posttranslational modifications of HuR have a major impact on the sequestration and binding to various HuR ligand proteins as has been demonstrated e.g. for the 14-3-3 chaperones. However, the detailed mechanisms of how a specific modification of HuR coordinates different aspects in HuR regulation are currently poorly understood. Due to the fact that most of the described HuR activities are closely related to its subcellular localization and the binding to cargo mRNA, this review will focus on these aspects of HuR functions and their control by posttranslational modification, particularly by HuR phosphorylations by different protein kinases.

  4. The Runt domain of AML1 (RUNX1) binds a sequence-conserved RNA motif that mimics a DNA element

    Science.gov (United States)

    Fukunaga, Junichi; Nomura, Yusuke; Tanaka, Yoichiro; Amano, Ryo; Tanaka, Taku; Nakamura, Yoshikazu; Kawai, Gota; Sakamoto, Taiichi; Kozu, Tomoko

    2013-01-01

    AML1 (RUNX1) is a key transcription factor for hematopoiesis that binds to the Runt-binding double-stranded DNA element (RDE) of target genes through its N-terminal Runt domain. Aberrations in the AML1 gene are frequently found in human leukemia. To better understand AML1 and its potential utility for diagnosis and therapy, we obtained RNA aptamers that bind specifically to the AML1 Runt domain. Enzymatic probing and NMR analyses revealed that Apt1-S, which is a truncated variant of one of the aptamers, has a CACG tetraloop and two stem regions separated by an internal loop. All the isolated aptamers were found to contain the conserved sequence motif 5′-NNCCAC-3′ and 5′-GCGMGN′N′-3′ (M:A or C; N and N′ form Watson–Crick base pairs). The motif contains one AC mismatch and one base bulged out. Mutational analysis of Apt1-S showed that three guanines of the motif are important for Runt binding as are the three guanines of RDE, which are directly recognized by three arginine residues of the Runt domain. Mutational analyses of the Runt domain revealed that the amino acid residues used for Apt1-S binding were similar to those used for RDE binding. Furthermore, the aptamer competed with RDE for binding to the Runt domain in vitro. These results demonstrated that the Runt domain of the AML1 protein binds to the motif of the aptamer that mimics DNA. Our findings should provide new insights into RNA function and utility in both basic and applied sciences. PMID:23709277

  5. 14-3-3 protein binds to the low molecular weight neurofilament (NFL) mRNA 3' UTR.

    Science.gov (United States)

    Ge, Wei-Wen; Volkening, Kathryn; Leystra-Lantz, Cheryl; Jaffe, Howard; Strong, Michael J

    2007-01-01

    We have previously reported that altered stability of low molecular weight neurofilament (NFL) mRNA in lumbar spinal cord homogenates in amyotrophic lateral sclerosis (ALS) is associated with altered expression of trans-acting 3' UTR mRNA binding proteins. We have identified two hexanucleotide motifs as the main cis elements and, using LC/MS/MS of peptide digests of NFL 3' UTR interacting proteins from human spinal cord, observed that 14-3-3 proteins interact with these motifs. 14-3-3 beta, zeta, tau, gamma, and eta isoforms were found to be expressed in human spinal cord. Each isoform was expressed in vitro and shown to interact with NFL 3' UTR mRNA. Mutation of one or both motifs resulted in decreased 14-3-3 interaction, changes in predicted mRNA structure or alteration in stability of the mRNA. These data show a novel interaction for 14-3-3 with NFL mRNA, and suggests that 14-3-3 may play a role in regulating NFL mRNA stability.

  6. [Study of the photoaffinity modification of Escherichia coli ribosomes near the donor tRNA-binding center].

    Science.gov (United States)

    Bausk, E V; Graĭfer, D M; Karpova, G G

    1985-01-01

    Affinity labelling of E. coli ribosomes near the donor tRNA-binding (P) site was studied with the use of photoreactive derivatives of tRNAPhe bearing arylazidogroups on N7 atoms of guanine residues (azido-tRNA). UV-irradiation of complexes 70S ribosome.poly(U).azido- tRNA(P-site) and 70S ribosome.poly(U).azido-tRNA(P-site).Phe- tRNAPhe(A-site) resulted in covalent attachment of azido-tRNA to ribosomes, both subunits being labelled. In both cases modification extent of 30S subunit was two-fold than that of the 50S one. It was shown that when the A-site was free the azido-tRNA located in P-site labelled proteins S9, S11, S12, S13, S21 and L14, L27, L31. Azido-tRNA located in P-site when the A-site was occupied with Phe-tRNAPhe labelled proteins S11, S12, S13, S14, S19, L32/L33 and possibly L23, L25. From the comparison of the sets of proteins labelled when A-site was free or occupied a conclusion was drawn that aminoacyl-tRNA located in ribosomal A-site affects the arrangement of deacylated tRNA in P-site. Data obtained allow to propose that proteins S5, S19, S20 and L24, L33 interact with guanine residues important for the tRNA tertiary structure formation.

  7. Symportin 1 chaperones 5S RNP assembly during ribosome biogenesis by occupying an essential rRNA-binding site.

    Science.gov (United States)

    Calviño, Fabiola R; Kharde, Satyavati; Ori, Alessandro; Hendricks, Astrid; Wild, Klemens; Kressler, Dieter; Bange, Gert; Hurt, Ed; Beck, Martin; Sinning, Irmgard

    2015-04-07

    During 60S biogenesis, mature 5S RNP consisting of 5S RNA, RpL5 and RpL11, assembles into a pre-60S particle, where docking relies on RpL11 interacting with helix 84 (H84) of the 25S RNA. How 5S RNP is assembled for recruitment into the pre-60S is not known. Here we report the crystal structure of a ternary symportin Syo1-RpL5-N-RpL11 complex and provide biochemical and structural insights into 5S RNP assembly. Syo1 guards the 25S RNA-binding surface on RpL11 and competes with H84 for binding. Pull-down experiments show that H84 releases RpL11 from the ternary complex, but not in the presence of 5S RNA. Crosslinking mass spectrometry visualizes structural rearrangements on incorporation of 5S RNA into the Syo1-RpL5-RpL11 complex supporting the formation of a pre-5S RNP. Our data underline the dual role of Syo1 in ribosomal protein transport and as an assembly platform for 5S RNP.

  8. Symportin 1 chaperones 5S RNP assembly during ribosome biogenesis by occupying an essential rRNA-binding site

    Science.gov (United States)

    Calviño, Fabiola R.; Kharde, Satyavati; Ori, Alessandro; Hendricks, Astrid; Wild, Klemens; Kressler, Dieter; Bange, Gert; Hurt, Ed; Beck, Martin; Sinning, Irmgard

    2015-04-01

    During 60S biogenesis, mature 5S RNP consisting of 5S RNA, RpL5 and RpL11, assembles into a pre-60S particle, where docking relies on RpL11 interacting with helix 84 (H84) of the 25S RNA. How 5S RNP is assembled for recruitment into the pre-60S is not known. Here we report the crystal structure of a ternary symportin Syo1-RpL5-N-RpL11 complex and provide biochemical and structural insights into 5S RNP assembly. Syo1 guards the 25S RNA-binding surface on RpL11 and competes with H84 for binding. Pull-down experiments show that H84 releases RpL11 from the ternary complex, but not in the presence of 5S RNA. Crosslinking mass spectrometry visualizes structural rearrangements on incorporation of 5S RNA into the Syo1-RpL5-RpL11 complex supporting the formation of a pre-5S RNP. Our data underline the dual role of Syo1 in ribosomal protein transport and as an assembly platform for 5S RNP.

  9. The rotaviral NSP3 protein stimulates translation of polyadenylated target mRNAs independently of its RNA-binding domain

    Energy Technology Data Exchange (ETDEWEB)

    Keryer-Bibens, Cecile, E-mail: cecile.keryer-bibens@univ-rennes1.fr [Universite de Rennes 1, IFR 140, Institut de Genetique et Developpement de Rennes, 35000 Rennes (France); CNRS, UMR 6061, equipe Expression Genetique et Developpement, 35000 Rennes (France); Universite Europeenne de Bretagne, 35000 Rennes (France); Legagneux, Vincent; Namanda-Vanderbeken, Allen [Universite de Rennes 1, IFR 140, Institut de Genetique et Developpement de Rennes, 35000 Rennes (France); CNRS, UMR 6061, equipe Expression Genetique et Developpement, 35000 Rennes (France); Universite Europeenne de Bretagne, 35000 Rennes (France); Cosson, Bertrand [UPMC Universite de Paris 06, UMR 7150, Equipe Traduction Cycle Cellulaire et Developpement, Station Biologique de Roscoff, 29682 Roscoff (France); CNRS, UMR 7150, Station Biologique de Roscoff, 29682 Roscoff (France); Universite Europeenne de Bretagne, 35000 Rennes (France); Paillard, Luc [Universite de Rennes 1, IFR 140, Institut de Genetique et Developpement de Rennes, 35000 Rennes (France); CNRS, UMR 6061, equipe Expression Genetique et Developpement, 35000 Rennes (France); Universite Europeenne de Bretagne, 35000 Rennes (France); Poncet, Didier [Virologie Moleculaire et Structurale, UMR CNRS, 2472, INRA, 1157, 91198 Gif sur Yvette (France); Osborne, H. Beverley, E-mail: beverley.osborne@univ-rennes1.fr [Universite de Rennes 1, IFR 140, Institut de Genetique et Developpement de Rennes, 35000 Rennes (France); CNRS, UMR 6061, equipe Expression Genetique et Developpement, 35000 Rennes (France); Universite Europeenne de Bretagne, 35000 Rennes (France)

    2009-12-11

    The non-structural protein 3 (NSP3) of rotaviruses is an RNA-binding protein that specifically recognises a 4 nucleotide sequence at the 3' extremity of the non-polyadenylated viral mRNAs. NSP3 also has a high affinity for eIF4G. These two functions are clearly delimited in separate domains the structures of which have been determined. They are joined by a central domain implicated in the dimerisation of the full length protein. The bridging function of NSP3 between the 3' end of the viral mRNA and eIF4G has been proposed to enhance the synthesis of viral proteins. However, this role has been questioned as knock-down of NSP3 did not impair viral protein synthesis. We show here using a MS2/MS2-CP tethering assay that a C-terminal fragment of NSP3 containing the eIF4G binding domain and the dimerisation domain can increase the expression of a protein encoded by a target reporter mRNA in HEK 293 cells. The amount of reporter mRNA in the cells is not significantly affected by the presence of the NSP3 derived fusion protein showing that the enhanced protein expression is due to increased translation. These results show that NSP3 can act as a translational enhancer even on a polyadenylated mRNA that should be a substrate for PABP1.

  10. Inhibition of 3T3-L1 adipocyte differentiation by expression of acyl-CoA-binding protein antisense RNA

    DEFF Research Database (Denmark)

    Mandrup, S; Sorensen, R V; Helledie, T

    1998-01-01

    adipocyte differentiation. In this report we describe the effects of expression of high levels of ACBP antisense RNA on the differentiation of 3T3-L1 cells. Pools of 3T3-L1 cells transfected with vectors expressing ACBP antisense RNA showed significantly less lipid accumulation as compared with cells...... of the adipogenic transcription factors peroxisome proliferator-activated receptor gamma and CCAAT/enhancer-binding protein alpha as well as several adipocyte-specific genes was significantly delayed and reduced. The adipogenic potential of antisense-expressing cells was partially restored by transfection...

  11. RNA-binding protein LIN28 is a marker for testicular germ cell tumors.

    Science.gov (United States)

    Cao, Dengfeng; Allan, Robert W; Cheng, Liang; Peng, Yan; Guo, Charles C; Dahiya, Neha; Akhi, Shirin; Li, Jianping

    2011-05-01

    LIN28 is an RNA-binding protein involved in maintaining the pluripotency of embryonic stem cells. Using formalin-fixed, paraffin-embedded tissue blocks, we performed immunohistochemical staining of LIN28 in 103 primary and 81 metastatic testicular germ cell tumors (54 intratubular germ cell neoplasias, unclassified type; 49 primary and 20 metastatic classic seminomas; 35 primary and 24 metastatic embryonal carcinomas; 35 primary and 15 metastatic yolk sac tumors; 23 primary and 12 metastatic teratomas; 6 primary and 10 metastatic choriocarcinomas; and 5 spermatocytic seminomas). The percentage of tumor cell stained was scored as 0 (0%), 1+ (≤30%), 2+ (31%-60%), 3+ (61%-90%), and 4+ (>90%). We stained LIN28 in 327 non-germ cell tumors to determine its specificity. We also compared LIN28 with SALL4 (Sal-like 4) and OCT4 (octamer-binding transcription factor 4) in all germ cell tumors. The staining was cytoplasmic for LIN28 and nuclear for SALL4 and OCT4. Strong 4+ LIN28 staining was seen in all 54 intratubular germ cell neoplasias, 59 embryonal carcinomas, and 50 yolk sac tumors. Positive LIN28 staining was seen in all 69 classic seminomas (1+ in 3, 3+ in 3, and 4+ in 63) (63, strong). Variable staining of LIN28 was seen in 10 of 35 teratomas (1+ to 3+, weak to strong intensity), 12 of 16 choriocarcinomas (1+ to 4+, weak to strong intensity), and 1 of 5 spermatocytic seminomas (2+, weak). Only 10 of 327 non-germ cell tumors showed 1+ weak LIN28 staining. Therefore, LIN28 is a highly sensitive marker for testicular intratubular germ cell neoplasias, classic seminomas, embryonal carcinomas, and yolk sac tumors with relatively high specificity. LIN28 can be used as a diagnostic marker for these tumors and has demonstrated a similar level of diagnostic utility as SALL4 (except for a few classic seminomas), although it does not show an advantage over SALL4. The major advantage of LIN28 over OCT4 is in diagnosing yolk sac tumors (yolk sac tumors negative for OCT4

  12. A family of insulin-like growth factor II mRNA-binding proteins represses translation in late development

    DEFF Research Database (Denmark)

    Nielsen, J; Christiansen, J; Lykke-Andersen, J;

    1999-01-01

    Insulin-like growth factor II (IGF-II) is a major fetal growth factor. The IGF-II gene generates multiple mRNAs with different 5' untranslated regions (5' UTRs) that are translated in a differential manner during development. We have identified a human family of three IGF-II mRNA-binding proteins.......5 followed by a decline towards birth, and, similar to IGF-II, IMPs are especially expressed in developing epithelia, muscle, and placenta in both mouse and human embryos. The results imply that cytoplasmic 5' UTR-binding proteins control IGF-II biosynthesis during late mammalian development....

  13. Rbms3, an RNA-binding protein, mediates the expression of Ptf1a by binding to its 3'UTR during mouse pancreas development.

    Science.gov (United States)

    Lu, Chung-Kuang; Lai, Yi-Chyi; Chen, Hau-Ren; Chiang, Ming-Ko

    2012-07-01

    The development of the pancreas is a complicated process that is regulated on several levels. Pancreas transcription factor 1, alpha subunit (Ptf1a), also known as p48, is a pancreas-specific basic helix-loop-helix transcription factor that is critical for both exocrine pancreas development and maintenance of acinar cell differentiation. Based on a differential screening assay, we identified Rbms3, a gene encoding a glycine-rich RNA-binding protein, to be specifically expressed in the neural tube and the pancreatic rudiment of e10.5 embryos. The presence of Rbms3 in the early developing pancreas suggests that specific post-transcriptional regulation mechanisms play an important role in controlling pancreas development. In this study, we show that Rbms3 binds to the 3'UTR of Ptf1a mRNA, but not the 3'UTR of Pdx1, which is another pancreatic transcription factor. The ectopic expression of Rbms3 stimulates the translation of a reporter gene carrying the Ptf1a 3'UTR. In addition, when Rbms3 expression is suppressed in the AR42J-B13 pancreatic exocrine cell line, the expression of Ptf1a is also down-regulated. These results suggest that binding of Rbms3 to the 3'UTR of Ptf1a regulates the production of the Ptf1a protein and, thereby, indirectly regulates the expression of the Ptf1a downstream target genes.

  14. Evolutionary history of double-stranded RNA binding proteins in plants: identification of new cofactors involved in easiRNA biogenesis.

    Science.gov (United States)

    Clavel, Marion; Pélissier, Thierry; Montavon, Thomas; Tschopp, Marie-Aude; Pouch-Pélissier, Marie-Noëlle; Descombin, Julie; Jean, Viviane; Dunoyer, Patrice; Bousquet-Antonelli, Cécile; Deragon, Jean-Marc

    2016-05-01

    In this work, we retrace the evolutionary history of plant double-stranded RNA binding proteins (DRBs), a group of non-catalytic factors containing one or more double-stranded RNA binding motif (dsRBM) that play important roles in small RNA biogenesis and functions. Using a phylogenetic approach, we show that multiple dsRBM DRBs are systematically composed of two different types of dsRBMs evolving under different constraints and likely fulfilling complementary functions. In vascular plants, four distinct clades of multiple dsRBM DRBs are always present with the exception of Brassicaceae species, that do not possess member of the newly identified clade we named DRB6. We also identified a second new and highly conserved DRB family (we named DRB7) whose members possess a single dsRBM that shows concerted evolution with the most C-terminal dsRBM domain of the Dicer-like 4 (DCL4) proteins. Using a BiFC approach, we observed that Arabidopsis thaliana DRB7.2 (AtDRB7.2) can directly interact with AtDRB4 but not with AtDCL4 and we provide evidence that both AtDRB7.2 and AtDRB4 participate in the epigenetically activated siRNAs pathway.

  15. Rice MEL2, the RNA recognition motif (RRM) protein, binds in vitro to meiosis-expressed genes containing U-rich RNA consensus sequences in the 3'-UTR.

    Science.gov (United States)

    Miyazaki, Saori; Sato, Yutaka; Asano, Tomoya; Nagamura, Yoshiaki; Nonomura, Ken-Ichi

    2015-10-01

    Post-transcriptional gene regulation by RNA recognition motif (RRM) proteins through binding to cis-elements in the 3'-untranslated region (3'-UTR) is widely used in eukaryotes to complete various biological processes. Rice MEIOSIS ARRESTED AT LEPTOTENE2 (MEL2) is the RRM protein that functions in the transition to meiosis in proper timing. The MEL2 RRM preferentially associated with the U-rich RNA consensus, UUAGUU[U/A][U/G][A/U/G]U, dependently on sequences and proportionally to MEL2 protein amounts in vitro. The consensus sequences were located in the putative looped structures of the RNA ligand. A genome-wide survey revealed a tendency of MEL2-binding consensus appearing in 3'-UTR of rice genes. Of 249 genes that conserved the consensus in their 3'-UTR, 13 genes spatiotemporally co-expressed with MEL2 in meiotic flowers, and included several genes whose function was supposed in meiosis; such as Replication protein A and OsMADS3. The proteome analysis revealed that the amounts of small ubiquitin-related modifier-like protein and eukaryotic translation initiation factor3-like protein were dramatically altered in mel2 mutant anthers. Taken together with transcriptome and gene ontology results, we propose that the rice MEL2 is involved in the translational regulation of key meiotic genes on 3'-UTRs to achieve the faithful transition of germ cells to meiosis.

  16. RNA-binding protein LIN28 is a sensitive marker of pediatric yolk sac tumors.

    Science.gov (United States)

    Feng, Shaoguang; Huang, Songsong; Tong, Yulong; Chen, Zhongliang; Shen, Delei; Wu, Dazhou; Lai, Xin-He; Chen, Xiaoming

    2016-08-01

    RNA-binding protein LIN28 is involved in maintaining the pluripotency of embryonic stem cells. It has been detected in different types of testicular and ovarian germ cell tumors (GCTs), but its status in pediatric YSTs (yolk sac tumors) is still unknown. The aim of this study was to determine the immunohistochemical profile of LIN28 in pediatric YSTs. Immunohistochemistry detection of LIN28 was performed in 22 cases of pediatric YSTs and 10 mature teratomas. The percentage of tumor cells stained was scored as 0, 1+ (1-30 % cells), 2+ (31-60 %), 3+ (61-90 %), and 4+ (>90 %). To compare its sensitive and specificity with alpha-fetoprotein (AFP), we also stained AFP in 22 cases of pediatric YSTs and 10 mature teratomas in children. LIN28 staining was high in all 22 pediatric yolk sac tumor (2+ in 1, 3+ in 1, and 4+ in 20), and weak staining of LIN28 was seen in 1 of 10 mature teratomas (1+), 9 of 10 mature teratomas were negative expression. However, the expression of AFP in pediatric YST was lower compared with Lin28 (- in 1, 1+ in 8, 2+ in 12, and 3+ in 1), and weak expression of AFP was seen in 2 of 10 mature teratomas (1+), 8 of 10 mature teratomas were negative. LIN28 had higher intensity expression than AFP in pediatric YSTs (P LIN28 is a sensitive marker for pediatric YSTs and it can be used to distinguish them from mature teratomas. LIN28 is likely to become a new and valuable biomarker for diagnosing of pediatric YST.

  17. Selection of functional tRNA primers and primer binding site sequences from a retroviral combinatorial library: identification of new functional tRNA primers in murine leukemia virus replication

    DEFF Research Database (Denmark)

    Lund, Anders Henrik; Duch, M; Pedersen, F S

    2000-01-01

    . While most of the selected primer binding sites are complementary to the 3'-end of tRNA((Pro)), we also retrieved PBS sequences matching four other tRNA molecules and demonstrate that Akv murine leukemia virus vectors may efficiently replicate using tRNA(Arg(CCU)), tRNA(Phe(GAA))and a hitherto unknown......Retroviral reverse transcription is initiated from a cellular tRNA molecule and all known exogenous isolates of murine leukemia virus utilise a tRNA(Pro)molecule. While several studies suggest flexibility in murine leukemia virus primer utilisation, studies on human immunodeficiency virus and avian...... retro-viruses have revealed evidence of molecular adapt-ation towards the specific tRNA isoacceptor used as replication primer. In this study, murine leukemia virus tRNA utilisation is investigated by in vivo screening of a retroviral vector combinatorial library with randomised primer binding sites...

  18. Effect of Hfq on RprA-rpoS mRNA pairing: Hfq-RNA binding and the influence of the 5' rpoS mRNA leader region.

    Science.gov (United States)

    Updegrove, Taylor; Wilf, Nabil; Sun, Xueguang; Wartell, Roger M

    2008-10-28

    The rpoS mRNA encodes a stress response transcription factor in Escherichia coli. It is one of a growing number of mRNAs found to be regulated by small RNAs (sRNA). Translation initiation of rpoS mRNA is enhanced by two sRNAs, DsrA and RprA, that pair to the same site near the rpoS start codon in the presence of the Hfq protein. In this work, we examine the interaction of E. coli Hfq with RprA and two portions of the rpoS mRNA leader region. One rpoS RNA, rpoS-L, contained the entire 565-nucleotide leader region, while the other, rpoS-S, contained the 199-nucleotide sequence surrounding the start codon. An RNase H assay indicated both rpoS RNAs have similar secondary structures in the translation initiation region. Hfq formed two complexes with RprA in a gel mobility assay with binding parameters similar to values previously determined for DsrA. Unlike DsrA, Hfq binding to RprA was inhibited by poly(A) and influenced by Hfq mutations on both the distal and proximal surfaces. Hfq increased the level of RprA binding to both rpoS RNAs but showed a much larger enhancement when rpoS-L, the entire leader region, was examined. The lower affinity of RprA for rpoS-L versus rpoS-S in the absence of Hfq suggests that Hfq overcomes an inhibitory structure within rpoS-L in stimulating RprA binding. Similar results were obtained with DsrA. The results indicate that the full upstream leader sequence of rpoS mRNA influences Hfq-facilitated annealing of RprA and DsrA and is likely to be involved in its regulation.

  19. Small Molecule Binding, Docking, and Characterization of the Interaction between Pth1 and Peptidyl-tRNA

    Directory of Open Access Journals (Sweden)

    Mary C. Hames

    2013-11-01

    Full Text Available Bacterial Pth1 is essential for viability. Pth1 cleaves the ester bond between the peptide and nucleotide of peptidyl-tRNA generated from aborted translation, expression of mini-genes, and short ORFs. We have determined the shape of the Pth1:peptidyl-tRNA complex using small angle neutron scattering. Binding of piperonylpiperazine, a small molecule constituent of a combinatorial synthetic library common to most compounds with inhibitory activity, was mapped to Pth1 via NMR spectroscopy. We also report computational docking results, modeling piperonylpiperazine binding based on chemical shift perturbation mapping. Overall these studies promote Pth1 as a novel antibiotic target, contribute to understanding how Pth1 interacts with its substrate, advance the current model for cleavage, and demonstrate feasibility of small molecule inhibition.

  20. Structure of the RNA polymerase core-binding domain of sigma(54) reveals a likely conformational fracture point.

    Science.gov (United States)

    Hong, Eunmi; Doucleff, Michaeleen; Wemmer, David E

    2009-07-03

    Transcription initiation by bacterial sigma(54)-RNA polymerase requires a conformational change of the holopolymerase-DNA complex, driven by an enhancer-binding protein. Although structures of the core polymerase and the more common sigma(70) factor have been determined, little is known about the structure of the sigma(54) variant. We report here the structure of an Aquifex aeolicus sigma(54) domain (residues 69-198), which binds core RNA polymerase. The structure is composed of two distinct subdomains held together by a small, conserved hydrophobic interface that appears to act as a fracture point in the structure. The N-terminal, four-helical subdomain has a negative surface and conserved residues that likely contact the core polymerase, while the C-terminal, three-helical bundle has a strongly positive patch that could contact DNA. Sequence conservation indicates that these structural features are conserved and are important for the role of sigma(54) in the polymerase complex.

  1. Structure and associated DNA-helicase activity of a general transcription initiation factor that binds to RNA polymerase II.

    Science.gov (United States)

    Sopta, M; Burton, Z F; Greenblatt, J

    1989-10-05

    RAP30/74 is a heteromeric general transcription initiation factor which binds to RNA polymerase II. Here we report that preparations of RAP30/74 contain an ATP-dependent DNA helicase whose probable function is to melt the DNA at transcriptional start sites. The sequence of the RAP30 subunit of RAP30/74 indicates that RAP30 may be distantly related to bacterial sigma factors.

  2. Cytoplasmic polyadenylation-element-binding protein (CPEB)1 and 2 bind to the HIF-1alpha mRNA 3'-UTR and modulate HIF-1alpha protein expression.

    Science.gov (United States)

    Hägele, Sonja; Kühn, Uwe; Böning, Melanie; Katschinski, Dörthe M

    2009-01-01

    The heterodimeric HIF (hypoxia-inducible factor)-1 is a transcriptional master regulator of several genes involved in mammalian oxygen homoeostasis. Besides the well described regulation of the HIF-1alpha subunit via hydroxylation-mediated protein stability in hypoxia, there are several indications of an additional translational control of the HIF-1alpha mRNA, especially after growth factor stimulation. We identified an interaction of CPEB (cytoplasmic polyadenylation-element-binding protein) 1 and CPEB2 with the 3'-UTR (untranslated region) of HIF-1alpha mRNA. Overexpression of CPEB1 and CPEB2 affected HIF-1alpha protein levels mediated by the 3'-UTR of HIF-1alpha mRNA. Stimulation of neuroblastoma SK-N-MC cells with insulin and thus activation of endogenous CPEBs increased the expression of a luciferase reporter gene fused to the 3'-UTR of HIF-1alpha as well as endogenous HIF-1alpha protein levels. This could be abrogated by treating the cells with CPEB1 or CPEB2 siRNAs (short interfering RNAs). Injection of HIF-1alpha cRNA into Xenopus oocytes verified the elongation of the poly(A)+ (polyadenylated) tail by cytoplasmic polyadenylation. Thus CPEB1 and CPEB2 are involved in the regulation of HIF-1alpha following insulin stimulation.

  3. The Drosophila hnRNP F/H Homolog Glorund Uses Two Distinct RNA-Binding Modes to Diversify Target Recognition

    Energy Technology Data Exchange (ETDEWEB)

    Tamayo, Joel V.; Teramoto, Takamasa; Chatterjee, Seema; Hall, Traci M. Tanaka; Gavis, Elizabeth R.

    2017-04-01

    The Drosophila hnRNP F/H homolog, Glorund (Glo), regulates nanos mRNA translation by interacting with a structured UA-rich motif in the nanos 3' untranslated region. Glo regulates additional RNAs, however, and mammalian homologs bind G-tract sequences to regulate alternative splicing, suggesting that Glo also recognizes G-tract RNA. To gain insight into how Glo recognizes both structured UA-rich and G-tract RNAs, we used mutational analysis guided by crystal structures of Glo’s RNA-binding domains and identified two discrete RNA-binding surfaces that allow Glo to recognize both RNA motifs. By engineering Glo variants that favor a single RNA-binding mode, we show that a subset of Glo’s functions in vivo is mediated solely by the G-tract binding mode, whereas regulation of nanos requires both recognition modes. Our findings suggest a molecular mechanism for the evolution of dual RNA motif recognition in Glo that may be applied to understanding the functional diversity of other RNA-binding proteins.

  4. Cyclophilin A binds to the viral RNA and replication proteins, resulting in inhibition of tombusviral replicase assembly.

    Science.gov (United States)

    Kovalev, Nikolay; Nagy, Peter D

    2013-12-01

    Replication of plus-stranded RNA viruses is greatly affected by numerous host-encoded proteins that act as restriction factors. Cyclophilins, which are a large family of cellular prolyl isomerases, have been found to inhibit Tomato bushy stunt tombusvirus (TBSV) replication in a Saccharomyces cerevisiae model based on genome-wide screens and global proteomics approaches. In this report, we further characterize single-domain cyclophilins, including the mammalian cyclophilin A and plant Roc1 and Roc2, which are orthologs of the yeast Cpr1p cyclophilin, a known inhibitor of TBSV replication in yeast. We found that recombinant CypA, Roc1, and Roc2 strongly inhibited TBSV replication in a cell-free replication assay. Additional in vitro studies revealed that CypA, Roc1, and Roc2 cyclophilins bound to the viral replication proteins, and CypA and Roc1 also bound to the viral RNA. These interactions led to inhibition of viral RNA recruitment, the assembly of the viral replicase complex, and viral RNA synthesis. A catalytically inactive mutant of CypA was also able to inhibit TBSV replication in vitro due to binding to the replication proteins and the viral RNA. Overexpression of CypA and its mutant in yeast or plant leaves led to inhibition of tombusvirus replication, confirming that CypA is a restriction factor for TBSV. Overall, the current work has revealed a regulatory role for the cytosolic single-domain Cpr1-like cyclophilins in RNA virus replication.

  5. The RNA-binding protein CsrA plays a central role in positively regulating virulence factors in Erwinia amylovora.

    Science.gov (United States)

    Ancona, Veronica; Lee, Jae Hoon; Zhao, Youfu

    2016-11-15

    The GacS/GacA two-component system (also called GrrS/GrrA) is a global regulatory system which is highly conserved among gamma-proteobacteria. This system positively regulates non-coding small regulatory RNA csrB, which in turn binds to the RNA-binding protein CsrA. However, how GacS/GacA-Csr system regulates virulence traits in E. amylovora remains unknown. Results from mutant characterization showed that the csrB mutant was hypermotile, produced higher amount of exopolysaccharide amylovoran, and had increased expression of type III secretion (T3SS) genes in vitro. In contrast, the csrA mutant exhibited complete opposite phenotypes, including non-motile, reduced amylovoran production and expression of T3SS genes. Furthermore, the csrA mutant did not induce hypersensitive response on tobacco or cause disease on immature pear fruits, indicating that CsrA is a positive regulator of virulence factors. These findings demonstrated that CsrA plays a critical role in E. amylovora virulence and suggested that negative regulation of virulence by GacS/GacA acts through csrB sRNA, which binds to CsrA and neutralizes its positive effect on T3SS gene expression, flagellar formation and amylovoran production. Future research will be focused on determining the molecular mechanism underlying the positive regulation of virulence traits by CsrA.

  6. Fragile X mental retardation protein recognition of G quadruplex structure per se is sufficient for high affinity binding to RNA.

    Science.gov (United States)

    Bole, Medhavi; Menon, Lakshmi; Mihailescu, Mihaela-Rita

    2008-12-01

    Fragile X syndrome, the most common form of inherited mental retardation is caused by the expansion of a CGG trinucleotide repeat in the fragile X mental retardation 1 (fmr1) gene. The abnormal expansion of the CGG repeat causes hypermethylation and subsequent silencing of the fmr1 gene, resulting in the loss of the fragile X mental retardation protein (FMRP). FMRP has been shown to use its arginine-glycine-glycine rich region (RGG box) to bind to messenger RNAs that form G quadruplex structures. Several studies reported that the G quadruplex RNA recognition alone is not sufficient for FMRP RGG box binding and that an additional stem and/or a G quadruplex-stem junction region may also be important in recognition. In this study we have used biophysical methods such as fluorescence, UV, CD and NMR spectroscopy to demonstrate that the recognition of the RNA G quadruplex structure per se, in the absence of a stem region, is sufficient for the FMRP high affinity and specific binding. These findings indicate that the presence of a stem structure in some of the FMRP G quadruplex forming mRNAs is not a requirement for protein recognition as previously believed, but rather for the proper formation of the correct RNA G quadruplex structure recognized by FMRP.

  7. The RNA-binding protein CsrA plays a central role in positively regulating virulence factors in Erwinia amylovora

    Science.gov (United States)

    Ancona, Veronica; Lee, Jae Hoon; Zhao, Youfu

    2016-01-01

    The GacS/GacA two-component system (also called GrrS/GrrA) is a global regulatory system which is highly conserved among gamma-proteobacteria. This system positively regulates non-coding small regulatory RNA csrB, which in turn binds to the RNA-binding protein CsrA. However, how GacS/GacA-Csr system regulates virulence traits in E. amylovora remains unknown. Results from mutant characterization showed that the csrB mutant was hypermotile, produced higher amount of exopolysaccharide amylovoran, and had increased expression of type III secretion (T3SS) genes in vitro. In contrast, the csrA mutant exhibited complete opposite phenotypes, including non-motile, reduced amylovoran production and expression of T3SS genes. Furthermore, the csrA mutant did not induce hypersensitive response on tobacco or cause disease on immature pear fruits, indicating that CsrA is a positive regulator of virulence factors. These findings demonstrated that CsrA plays a critical role in E. amylovora virulence and suggested that negative regulation of virulence by GacS/GacA acts through csrB sRNA, which binds to CsrA and neutralizes its positive effect on T3SS gene expression, flagellar formation and amylovoran production. Future research will be focused on determining the molecular mechanism underlying the positive regulation of virulence traits by CsrA. PMID:27845410

  8. Binding of helix-threading peptides to E. coli 16S ribosomal RNA and inhibition of the S15-16S complex.

    Science.gov (United States)

    Gooch, Barry D; Krishnamurthy, Malathy; Shadid, Mohammad; Beal, Peter A

    2005-12-01

    Helix-threading peptides (HTPs) constitute a new class of small molecules that bind selectively to duplex RNA structures adjacent to helix defects and project peptide functionality into the dissimilar duplex grooves. To further explore and develop the capabilities of the HTP design for binding RNA selectively, we identified helix 22 of the prokaryotic ribosomal RNA 16S as a target. This helix is a component of the binding site for the ribosomal protein S15. In addition, the S15-16S RNA interaction is important for the ordered assembly of the bacterial ribosome. Here we present the synthesis and characterization of helix-threading peptides that bind selectively to helix 22 of E. coli 16S RNA. These compounds bind helix 22 by threading intercalation placing the N termini in the minor groove and the C termini in the major groove. Binding is dependent on the presence of a highly conserved purine-rich internal loop in the RNA, whereas removal of the loop minimally affects binding of the classical intercalators ethidium bromide and methidiumpropyl-EDTAFe (MPEFe). Moreover, binding selectivity translates into selective inhibition of formation of the S15-16S complex.

  9. Acute reduction of neuronal RNA binding Elavl2 protein and Gap43 mRNA in mouse hippocampus after kainic acid treatment.

    Science.gov (United States)

    Ohtsuka, Takafumi; Yano, Masato; Okano, Hideyuki

    2015-10-09

    Activity-dependent gene regulation in neurons has been hypothesized to be under transcriptional control and to include dramatic increases in immediate early genes (IEGs) after neuronal activity. In addition, several reports have focused on post-transcriptional regulation, which could be mediated by neuronal post-transcriptional regulators, including RNA binding proteins (RNABPs). One such protein family is the neuronal Elavls (nElavls; Elavl2, Elavl3, and Elavl4), whose members are widely expressed in peripheral and central nervous system. Previous reports showed that Elavl3 and 4 are up-regulated following repeated stimulation such as during cocaine administration, a seizure, or a spatial discrimination task. In this study, we focused on Elavl2, a candidate gene for schizophrenia and studied its role in neuronal activity. First we found that Elavl2 has a cell-type specific expression pattern that is highly expressed in hippocampal CA3 pyramidal neurons and hilar interneurons using Elavl2 specific antibody. Second, unexpectedly, we discovered that the Elavl2 protein level in the hippocampus was acutely down-regulated for 3 h after a kainic acid (KA)-induced seizure in the hippocampal CA3 region. In addition, level of Gap43 mRNA, a target mRNA of Elavl2 is decreased 12 h after KA treatment, thus suggesting the involvement of Elavl2 in activity-dependent RNA regulation.

  10. Predicting protein-binding RNA nucleotides using the feature-based removal of data redundancy and the interaction propensity of nucleotide triplets.

    Science.gov (United States)

    Choi, Sungwook; Han, Kyungsook

    2013-11-01

    Several learning approaches have been used to predict RNA-binding amino acids in a protein sequence, but there has been little attempt to predict protein-binding nucleotides in an RNA sequence. One of the reasons is that the differences between nucleotides in their interaction propensity are much smaller than those between amino acids. Another reason is that RNA exhibits less diverse sequence patterns than protein. Therefore, predicting protein-binding RNA nucleotides is much harder than predicting RNA-binding amino acids. We developed a new method that removes data redundancy in a training set of sequences based on their features. The new method constructs a larger and more informative training set than the standard redundancy removal method based on sequence similarity, and the constructed dataset is guaranteed to be redundancy-free. We computed the interaction propensity (IP) of nucleotide triplets by applying a new definition of IP to an extensive dataset of protein-RNA complexes, and developed a support vector machine (SVM) model to predict protein binding sites in RNA sequences. In a 5-fold cross-validation with 812 RNA sequences, the SVM model predicted protein-binding nucleotides with an accuracy of 86.4%, an F-measure of 84.8%, and a Matthews correlation coefficient of 0.66. With an independent dataset of 56 RNA sequences that were not used in training, the resulting accuracy was 68.1% with an F-measure of 71.7% and a Matthews correlation coefficient of 0.35. To the best of our knowledge, this is the first attempt to predict protein-binding RNA nucleotides in a given RNA sequence from the sequence data alone. The SVM model and datasets are freely available for academics at http://bclab.inha.ac.kr/primer.

  11. Study of the Binding Energies between Unnatural Amino Acids and Engineered Orthogonal Tyrosyl-tRNA Synthetases

    Science.gov (United States)

    Ren, Wei; Truong, Tan M.; Ai, Hui-wang

    2015-01-01

    We utilized several computational approaches to evaluate the binding energies of tyrosine (Tyr) and several unnatural Tyr analogs, to several orthogonal aaRSes derived from Methanocaldococcus jannaschii and Escherichia coli tyrosyl-tRNA synthetases. The present study reveals the following: (1) AutoDock Vina and ROSETTA were able to distinguish binding energy differences for individual pairs of favorable and unfavorable aaRS-amino acid complexes, but were unable to cluster together all experimentally verified favorable complexes from unfavorable aaRS-Tyr complexes; (2) MD-MM/PBSA provided the best prediction accuracy in terms of clustering favorable and unfavorable enzyme-substrate complexes, but also required the highest computational cost; and (3) MM/PBSA based on single energy-minimized structures has a significantly lower computational cost compared to MD-MM/PBSA, but still produced sufficiently accurate predictions to cluster aaRS-amino acid interactions. Although amino acid-aaRS binding is just the first step in a complex series of processes to acylate a tRNA with its corresponding amino acid, the difference in binding energy, as shown by MD-MM/PBSA, is important for a mutant orthogonal aaRS to distinguish between a favorable unnatural amino acid (unAA) substrate from unfavorable natural amino acid substrates. Our computational study should assist further designing and engineering of orthogonal aaRSes for the genetic encoding of novel unAAs. PMID:26220470

  12. Dimerization and heme binding are conserved in amphibian and starfish homologues of the microRNA processing protein DGCR8.

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    Rachel Senturia

    Full Text Available Human DiGeorge Critical Region 8 (DGCR8 is an essential microRNA (miRNA processing factor that is activated via direct interaction with Fe(III heme. In order for DGCR8 to bind heme, it must dimerize using a dimerization domain embedded within its heme-binding domain (HBD. We previously reported a crystal structure of the dimerization domain from human DGCR8, which demonstrated how dimerization results in the formation of a surface important for association with heme. Here, in an attempt to crystallize the HBD, we search for DGCR8 homologues and show that DGCR8 from Patiria miniata (bat star also binds heme. The extinction coefficients (ε of DGCR8-heme complexes are determined; these values are useful for biochemical analyses and allow us to estimate the heme occupancy of DGCR8 proteins. Additionally, we present the crystal structure of the Xenopus laevis dimerization domain. The structure is very similar to that of human DGCR8. Our results indicate that dimerization and heme binding are evolutionarily conserved properties of DGCR8 homologues not only in vertebrates, but also in at least some invertebrates.

  13. RNA-binding proteins regulate cell respiration and coenzyme Q biosynthesis by post-transcriptional regulation of COQ7.

    Science.gov (United States)

    Cascajo, María V; Abdelmohsen, Kotb; Noh, Ji Heon; Fernández-Ayala, Daniel J M; Willers, Imke M; Brea, Gloria; López-Lluch, Guillermo; Valenzuela-Villatoro, Marina; Cuezva, José M; Gorospe, Myriam; Siendones, Emilio; Navas, Plácido

    2016-07-01

    Coenzyme Q (CoQ) is a key component of the mitochondrial respiratory chain carrying electrons from complexes I and II to complex III and it is an intrinsic component of the respirasome. CoQ concentration is highly regulated in cells in order to adapt the metabolism of the cell to challenges of nutrient availability and stress stimuli. At least 10 proteins have been shown to be required for CoQ biosynthesis in a multi-peptide complex and COQ7 is a central regulatory factor of this pathway. We found that the first 765 bp of the 3'-untranslated region (UTR) of COQ7 mRNA contains cis-acting elements of interaction with RNA-binding proteins (RBPs) HuR and hnRNP C1/C2. Binding of hnRNP C1/C2 to COQ7 mRNA was found to require the presence of HuR, and hnRNP C1/C2 silencing appeared to stabilize COQ7 mRNA modestly. By contrast, lowering HuR levels by silencing or depriving cells of serum destabilized and reduced the half-life of COQ7 mRNA, thereby reducing COQ7 protein and CoQ biosynthesis rate. Accordingly, HuR knockdown decreased oxygen consumption rate and mitochondrial production of ATP, and increased lactate levels. Taken together, our results indicate that a reduction in COQ7 mRNA levels by HuR depletion causes mitochondrial dysfunction and a switch toward an enhanced aerobic glycolysis, the characteristic phenotype exhibited by primary deficiency of CoQ10. Thus HuR contributes to efficient oxidative phosphorylation by regulating of CoQ10 biosynthesis.

  14. The importance of CELF control: molecular and biological roles of the CUG-BP, Elav-like family of RNA-binding proteins.

    Science.gov (United States)

    Dasgupta, Twishasri; Ladd, Andrea N

    2012-01-01

    RNA processing is important for generating protein diversity and modulating levels of protein expression. The CUG-BP, Elav-like family (CELF) of RNA-binding proteins regulate several steps of RNA processing in the nucleus and cytoplasm, including pre-mRNA alternative splicing, C to U RNA editing, deadenylation, mRNA decay, and translation. In vivo, CELF proteins have been shown to play roles in gametogenesis and early embryonic development, heart and skeletal muscle function, and neurosynaptic transmission. Dysregulation of CELF-mediated programs has been implicated in the pathogenesis of human diseases affecting the heart, skeletal muscles, and nervous system.

  15. The Puf family of RNA-binding proteins in plants: phylogeny, structural modeling, activity and subcellular localization

    Directory of Open Access Journals (Sweden)

    Tam Michael WC

    2010-03-01

    Full Text Available Abstract Background Puf proteins have important roles in controlling gene expression at the post-transcriptional level by promoting RNA decay and repressing translation. The Pumilio homology domain (PUM-HD is a conserved region within Puf proteins that binds to RNA with sequence specificity. Although Puf proteins have been well characterized in animal and fungal systems, little is known about the structural and functional characteristics of Puf-like proteins in plants. Results The Arabidopsis and rice genomes code for 26 and 19 Puf-like proteins, respectively, each possessing eight or fewer Puf repeats in their PUM-HD. Key amino acids in the PUM-HD of several of these proteins are conserved with those of animal and fungal homologs, whereas other plant Puf proteins demonstrate extensive variability in these amino acids. Three-dimensional modeling revealed that the predicted structure of this domain in plant Puf proteins provides a suitable surface for binding RNA. Electrophoretic gel mobility shift experiments showed that the Arabidopsis AtPum2 PUM-HD binds with high affinity to BoxB of the Drosophila Nanos Response Element I (NRE1 RNA, whereas a point mutation in the core of the NRE1 resulted in a significant reduction in binding affinity. Transient expression of several of the Arabidopsis Puf proteins as fluorescent protein fusions revealed a dynamic, punctate cytoplasmic pattern of localization for most of these proteins. The presence of predicted nuclear export signals and accumulation of AtPuf proteins in the nucleus after treatment of cells with leptomycin B demonstrated that shuttling of these proteins between the cytosol and nucleus is common among these proteins. In addition to the cytoplasmically enriched AtPum proteins, two AtPum proteins showed nuclear targeting with enrichment in the nucleolus. Conclusions The Puf family of RNA-binding proteins in plants consists of a greater number of members than any other model species studied to

  16. Importance of Basic Residues in Binding of Rous Sarcoma Virus Nucleocapsid to the RNA Packaging Signal

    OpenAIRE

    Lee, Eun-Gyung; Alidina, Annie; May, Cynthia; Linial, Maxine L.

    2003-01-01

    In the context of the Rous sarcoma virus Gag polyprotein, only the nucleocapsid (NC) domain is required to mediate the specificity of genomic RNA packaging. We have previously showed that the Saccharomyces cerevisiae three-hybrid system provides a rapid genetic assay to analyze the RNA and protein components of the avian retroviral RNA-Gag interactions necessary for specific encapsidation. In this study, using both site-directed mutagenesis and in vivo random screening in the yeast three-hybr...

  17. Comparison of specific binding sites for Escherichia coli RNA polymerase with naturally occurring hairpin regions in single-stranded DNA of coliphage M13. [Aspergillus oryzae

    Energy Technology Data Exchange (ETDEWEB)

    Niyogi, S.K.; Mitra, S.

    1978-08-25

    Escherichia coli RNA polymerase binds specifically to the single-stranded circular DNA of coliphage M13 in the presence of a saturating concentration of the bacterial DNA binding protein presumably as an essential step in the synthesis of the RNA primer required for synthesizing the complementary DNA strand in parental replicative-form DNA. The RNA polymerase-protected DNA regions were isolated after extensive digestion with pancreatic DNase, S1 endonuclease of Aspergillus oryzae, and exonuclease I of E. coli. The physicochemical properties of the RNA polymerase-protected segments (called PI and PII) were compared with those of the naturally occurring hairpin regions.

  18. RNA-protein binding motifs mining with a new hybrid deep learning based cross-domain knowledge integration approach.

    Science.gov (United States)

    Pan, Xiaoyong; Shen, Hong-Bin

    2017-02-28

    RNAs play key roles in cells through the interactions with proteins known as the RNA-binding proteins (RBP) and their binding motifs enable crucial understanding of the post-transcriptional regulation of RNAs. How the RBPs correctly recognize the target RNAs and why they bind specific positions is still far from clear. Machine learning-based algorithms are widely acknowledged to be capable of speeding up this process. Although many automatic tools have been developed to predict the RNA-protein binding sites from the rapidly growing multi-resource data, e.g. sequence, structure, their domain specific features and formats have posed significant computational challenges. One of current difficulties is that the cross-source shared common knowledge is at a higher abstraction level beyond the observed data, resulting in a low efficiency of direct integration of observed data across domains. The other difficulty is how to interpret the prediction results. Existing approaches tend to terminate after outputting the potential discrete binding sites on the sequences, but how to assemble them into the meaningful binding motifs is a topic worth of further investigation. In viewing of these challenges, we propose a deep learning-based framework (iDeep) by using a novel hybrid convolutional neural network and deep belief network to predict the RBP interaction sites and motifs on RNAs. This new protocol is featured by transforming the original observed data into a high-level abstraction feature space using multiple layers of learning blocks, where the shared representations across different domains are integrated. To validate our iDeep method, we performed experiments on 31 large-scale CLIP-seq datasets, and our results show that by integrating multiple sources of data, the average AUC can be improved by 8% compared to the best single-source-based predictor; and through cross-domain knowledge integration at an abstraction level, it outperforms the state-of-the-art predictors by 6

  19. Similarities in transcription factor IIIC subunits that bind to the posterior regions of internal promoters for RNA polymerase III

    Directory of Open Access Journals (Sweden)

    Matsutani Sachiko

    2004-08-01

    Full Text Available Abstract Background In eukaryotes, RNA polymerase III (RNAP III transcribes the genes for small RNAs like tRNAs, 5S rRNA, and several viral RNAs, and short interspersed repetitive elements (SINEs. The genes for these RNAs and SINEs have internal promoters that consist of two regions. These two regions are called the A and B blocks. The multisubunit transcription factor TFIIIC is required for transcription initiation of RNAP III; in transcription of tRNAs, the B-block binding subunit of TFIIIC recognizes a promoter. Although internal promoter sequences are conserved in eukaryotes, no evidence of homology between the B-block binding subunits of vertebrates and yeasts has been reported previously. Results Here, I reported the results of PSI-BLAST searches using the B-block binding subunits of human and Shizosacchromyces pombe as queries, showing that the same Arabidopsis proteins were hit with low E-values in both searches. Comparison of the convergent iterative alignments obtained by these PSI-BLAST searches revealed that the vertebrate, yeast, and Arabidopsis proteins have similarities in their N-terminal one-third regions. In these regions, there were three domains with conserved sequence similarities, one located in the N-terminal end region. The N-terminal end region of the B-block binding subunit of Saccharomyces cerevisiae is tentatively identified as a HMG box, which is the DNA binding motif. Although I compared the alignment of the N-terminal end regions of the B-block binding subunits, and their homologs, with that of the HMG boxes, it is not clear whether they are related. Conclusion Molecular phylogenetic analyses using the small subunit rRNA and ubiquitous proteins like actin and α-tubulin, show that fungi are more closely related to animals than either is to plants. Interestingly, the results obtained in this study show that, with respect to the B-block binding subunits of TFIIICs, animals appear to be evolutionarily closer to plants

  20. Global transcript profiling of transgenic plants constitutively overexpressing the RNA-binding protein AtGRP7

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    Hennig Lars

    2010-10-01

    Full Text Available Abstract Background The clock-controlled RNA-binding protein AtGRP7 influences circadian oscillations of its own transcript at the post-transcriptional level. To identify additional targets that are regulated by AtGRP7, transcript profiles of transgenic plants constitutively overexpressing AtGRP7 (AtGRP7-ox and wild type plants were compared. Results Approximately 1.4% of the transcripts represented on the Affymetrix ATH1 microarray showed changes in steady-state abundance upon AtGRP7 overexpression. One third of the differentially expressed genes are controlled by the circadian clock, and they show a distinct bias of their phase: The up-regulated genes preferentially peak around dawn, roughly opposite to the AtGRP7 peak abundance whereas the down-regulated genes preferentially peak at the end of the day. Further, transcripts responsive to abiotic and biotic stimuli were enriched among AtGRP7 targets. Transcripts encoding the pathogenesis-related PR1 and PR2 proteins were elevated in AtGRP7-ox plants but not in plants overexpressing AtGRP7 with a point mutation in the RNA-binding domain, indicating that the regulation involves RNA binding activity of AtGRP7. Gene set enrichment analysis uncovered components involved in ribosome function and RNA metabolism among groups of genes upregulated in AtGRP7-ox plants, consistent with its role in post-transcriptional regulation. Conclusion Apart from regulating a suite of circadian transcripts in a time-of-day dependent manner AtGRP7, both directly and indirectly, affects other transcripts including transcripts responsive to abiotic and biotic stimuli. This suggests a regulatory role of AtGRP7 in the output of the endogenous clock and a complex network of transcripts responsive to external stimuli downstream of the AtGRP7 autoregulatory circuit.

  1. Double-Stranded RNA-Binding Protein 4 Is Required for Resistance Signaling against Viral and Bacterial Pathogens

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    Shifeng Zhu

    2013-09-01

    Full Text Available Plant viruses often encode suppressors of host RNA silencing machinery, which occasionally function as avirulence factors that are recognized by host resistance (R proteins. For example, the Arabidopsis R protein, hypersensitive response to TCV (HRT, recognizes the turnip crinkle virus (TCV coat protein (CP. HRT-mediated resistance requires the RNA-silencing component double-stranded RNA-binding protein 4 (DRB4 even though it neither is associated with the accumulation of TCV-specific small RNA nor requires the RNA silencing suppressor function of CP. HRT interacts with the cytosolic fraction of DRB4. Interestingly, TCV infection both increases the cytosolic DRB4 pool and inhibits the HRT-DRB4 interaction. The virulent R8A CP derivative, which induces a subset of HRT-derived responses, also disrupts this interaction. The differential localization of DRB4 in the presence of wild-type and R8A CP implies the importance of subcellular compartmentalization of DRB4. The requirement of DRB4 in resistance to bacterial infection suggests a universal role in R-mediated defense signaling.

  2. Mapping Escherichia coli elongation factor Tu residues involved in binding of aminoacyl-tRNA

    DEFF Research Database (Denmark)

    Wiborg, Ove; Andersen, C; Knudsen, Charlotte Rohde

    1996-01-01

    were characterized with respect to thermal and chemical stability, GTPase activity, tRNA affinity, and activity in an in vitro translation assay. Most conspicuously tRNA affinities were reduced for all mutants. The results verify our structural analysis of elongation factor Tu in complex with aminoacyl...

  3. The human cap-binding complex is functionally connected to the nuclear RNA exosome

    DEFF Research Database (Denmark)

    Andersen, Peter Refsing; Domanski, Michal; Kristiansen, Maiken S

    2013-01-01

    of combinatorial depletion of CBCN and exosome components underscore the functional relevance of CBC-exosome bridging at the level of target RNA. Specifically, CBCA suppresses read-through products of several RNA families by promoting their transcriptional termination. We suggest that the RNP 5' cap links...

  4. Mechanism of regulation of bcl-2 mRNA by nucleolin and A+U-rich element-binding factor 1 (AUF1).

    Science.gov (United States)

    Ishimaru, Daniella; Zuraw, Lisa; Ramalingam, Sivakumar; Sengupta, Tapas K; Bandyopadhyay, Sumita; Reuben, Adrian; Fernandes, Daniel J; Spicer, Eleanor K

    2010-08-27

    The antiapoptotic Bcl-2 protein is overexpressed in a variety of cancers, particularly leukemias. In some cell types this is the result of enhanced stability of bcl-2 mRNA, which is controlled by elements in its 3'-untranslated region. Nucleolin is one of the proteins that binds to bcl-2 mRNA, thereby increasing its half-life. Here, we examined the site on the bcl-2 3'-untranslated region that is bound by nucleolin as well as the protein binding domains important for bcl-2 mRNA recognition. RNase footprinting and RNA fragment binding assays demonstrated that nucleolin binds to a 40-nucleotide region at the 5' end of the 136-nucleotide bcl-2 AU-rich element (ARE(bcl-2)). The first two RNA binding domains of nucleolin were sufficient for high affinity binding to ARE(bcl-2). In RNA decay assays, ARE(bcl-2) transcripts were protected from exosomal decay by the addition of nucleolin. AUF1 has been shown to recruit the exosome to mRNAs. When MV-4-11 cell extracts were immunodepleted of AUF1, the rate of decay of ARE(bcl-2) transcripts was reduced, indicating that nucleolin and AUF1 have opposing roles in bcl-2 mRNA turnover. When the function of nucleolin in MV-4-11 cells was impaired by treatment with the nucleolin-targeting aptamer AS1411, association of AUF1 with bcl-2 mRNA was increased. This suggests that the degradation of bcl-2 mRNA induced by AS1411 results from both interference with nucleolin protection of bcl-2 mRNA and recruitment of the exosome by AUF1. Based on our findings, we propose a model that illustrates the opposing roles of nucleolin and AUF1 in regulating bcl-2 mRNA stability.

  5. Transcription of lncRNA prt, clustered prt RNA sites for Mmi1 binding, and RNA polymerase II CTD phospho-sites govern the repression of pho1 gene expression under phosphate-replete conditions in fission yeast.

    Science.gov (United States)

    Chatterjee, Debashree; Sanchez, Ana M; Goldgur, Yehuda; Shuman, Stewart; Schwer, Beate

    2016-07-01

    Expression of fission yeast Pho1 acid phosphatase is repressed during growth in phosphate-rich medium. Repression is mediated by transcription of the prt locus upstream of pho1 to produce a long noncoding (lnc) prt RNA. Repression is also governed by RNA polymerase II CTD phosphorylation status, whereby inability to place a Ser7-PO4 mark (as in S7A) derepresses Pho1 expression, and inability to place a Thr4-PO4 mark (as in T4A) hyper-represses Pho1 in phosphate replete cells. Here we find that basal pho1 expression from the prt-pho1 locus is inversely correlated with the activity of the prt promoter, which resides in a 110-nucleotide DNA segment preceding the prt transcription start site. CTD mutations S7A and T4A had no effect on the activity of the prt promoter or the pho1 promoter, suggesting that S7A and T4A affect post-initiation events in prt lncRNA synthesis that make it less and more repressive of pho1, respectively. prt lncRNA contains clusters of DSR (determinant of selective removal) sequences recognized by the YTH-domain-containing protein Mmi1. Altering the nucleobase sequence of two DSR clusters in the prt lncRNA caused hyper-repression of pho1 in phosphate replete cells, concomitant with increased levels of the prt transcript. The isolated Mmi1 YTH domain binds to RNAs with single or tandem DSR elements, to the latter in a noncooperative fashion. We report the 1.75 Å crystal structure of the Mmi1 YTH domain and provide evidence that Mmi1 recognizes DSR RNA via a binding mode distinct from that of structurally homologous YTH proteins that recognize m(6)A-modified RNA.

  6. RNA targeting by small molecule alkaloids: Studies on the binding of berberine and palmatine to polyribonucleotides and comparison to ethidium

    Science.gov (United States)

    Islam, Md. Maidul; Suresh Kumar, Gopinatha

    2008-03-01

    The binding affinity, energetics and conformational aspects of the interaction of isoquinoline alkaloids berberine and palmatine to four single stranded polyribonucleotides polyguanylic acid [poly(G)], polyinosinic acid [poly(I)], polycytidylic acid [poly(C)] and polyuridylic acid [poly(U)] were studied by absorption, fluorescence, isothermal titration calorimetry and circular dichroism spectroscopy and compared with ethidium. Berberine, palmatine and ethidium binds strongly with poly(G) and poly(I) with affinity in the order 10 5 M -1 while their binding to poly(C) and poly(U) were very weak or practically nil. The same conclusions have also emerged from isothermal titration calorimetric studies. The binding of all the three compounds to poly(C) and poly(I) was exothermic and favored by both negative enthalpy change and positive entropy change. Conformational change in the polymer associated with the binding was observed in poly(I) with all the three molecules and poly(U) with ethidium but not in poly(G) and poly(C) revealing differences in the orientation of the bound molecules in the hitherto different helical organization of these polymers. These fundamental results may be useful and serve as database for the development of futuristic RNA based small molecule therapeutics.

  7. Double-stranded RNA-activated protein kinase PKR of fishes and amphibians: Varying the number of double-stranded RNA binding domains and lineage-specific duplications

    Directory of Open Access Journals (Sweden)

    Dever Thomas E

    2008-03-01

    Full Text Available Abstract Background Double-stranded (ds RNA, generated during viral infection, binds and activates the mammalian anti-viral protein kinase PKR, which phosphorylates the translation initiation factor eIF2α leading to the general inhibition of protein synthesis. Although PKR-like activity has been described in fish cells, the responsible enzymes eluded molecular characterization until the recent discovery of goldfish and zebrafish PKZ, which contain Z-DNA-binding domains instead of dsRNA-binding domains (dsRBDs. Fish and amphibian PKR genes have not been described so far. Results Here we report the cloning and identification of 13 PKR genes from 8 teleost fish and amphibian species, including zebrafish, demonstrating the coexistence of PKR and PKZ in this latter species. Analyses of their genomic organization revealed up to three tandemly arrayed PKR genes, which are arranged in head-to-tail orientation. At least five duplications occurred independently in fish and amphibian lineages. Phylogenetic analyses reveal that the kinase domains of fish PKR genes are more closely related to those of fish PKZ than to the PKR kinase domains of other vertebrate species. The duplication leading to fish PKR and PKZ genes occurred early during teleost fish evolution after the divergence of the tetrapod lineage. While two dsRBDs are found in mammalian and amphibian PKR, one, two or three dsRBDs are present in fish PKR. In zebrafish, both PKR and PKZ were strongly upregulated after immunostimulation with some tissue-specific expression differences. Using genetic and biochemical assays we demonstrate that both zebrafish PKR and PKZ can phosphorylate eIF2α in yeast. Conclusion Considering the important role for PKR in host defense against viruses, the independent duplication and fixation of PKR genes in different lineages probably provided selective advantages by leading to the recognition of an extended spectrum of viral nucleic acid structures, including both dsRNA

  8. Single nucleotide polymorphisms in miRNA binding sites and miRNA genes as breast/ovarian cancer risk modifiers in Jewish high-risk women.

    Science.gov (United States)

    Kontorovich, Tair; Levy, Asaf; Korostishevsky, Michael; Nir, Uri; Friedman, Eitan

    2010-08-01

    We hypothesized that aberrant gene silencing by miRNA may affect mutant BRCA penetrance. To test this notion, frequency of single nucleotide polymorphisms (SNPs; n = 42) within predicted miRNA binding sites or miRNA precursors were determined and compared in 363 BRCA1 mutation carriers: asymptomatic (n = 160), breast cancer (n = 140) and ovarian cancer (n = 63) patients, and in 125 BRCA2 mutation carriers: asymptomatic (n = 48), breast cancer (n = 58) and ovarian cancer (n = 19) patients. Overall, 16 of 42 SNPs were polymorphic, 11 had a minor allele frequency greater than 5% and 9 of them maintained the Hardy-Weinberg Equilibrium. Based on Cox regression and Kaplan-Meier analyses, statistically significant differences were noted in BRCA2 mutation carriers by health status in 3 SNPs: CC homozygosity at rs6505162 increased ovarian cancer risk (RR 2.77; p = 0.028; 95% CI, 1.11-6.9); heterozygote SNP carriers of rs11169571 had an approximately 2 fold increased risk for developing breast/ovarian cancer, whereas heterozygotes of the rs895819 SNP had an approximately 50% reduced risk for developing breast/ovarian cancer. This study provides preliminary evidence for another regulatory level of penetrance of deleterious mutations in cancer predisposition genes.

  9. Localization of vasa protein to the Drosophila pole plasm is independent of its RNA-binding and helicase activities.

    Science.gov (United States)

    Liang, L; Diehl-Jones, W; Lasko, P

    1994-05-01

    The Drosophila gene vasa encodes a DEAD-box protein, which is localized during early oogenesis to the perinuclear region of the nurse cells and later to the pole plasm at the posterior end of the oocyte. Posterior localization of vasa protein depends upon the functions of four genes: capu, spir, osk and stau. We have found that localization of vasa to the perinuclear nuage is abolished in most vas alleles, but is unaffected by mutations in four genes required upstream for its pole plasm localization. Thus localization of vasa to the nuage particles is independent of the pole plasm assembly pathway. Furthermore, electron-dense nuage particles are less abundant in the cytoplasm of nurse cells from vas mutants that fail to exhibit perinuclear localization, suggesting that the formation of the nuage depends upon vas function. Eight of nine vas point mutations cause codon substitutions in a region conserved among DEAD-box genes. The proteins from two mutant alleles that retain the capacity to localize to the posterior pole of the oocyte, vasO14 and vasO11, are both severely reduced in RNA-binding and -unwinding activity as compared to the wild-type protein on a variety of RNA substrates including in vitro synthesized pole plasm RNAs. Initial recruitment of vasa to the pole plasm must consequently depend upon protein-protein interactions but, once localized, vasa must bind to RNA to mediate germ cell formation.

  10. Translational regulation of viral secretory proteins by the 5' coding regions and a viral RNA-binding protein.

    Science.gov (United States)

    Nordholm, Johan; Petitou, Jeanne; Östbye, Henrik; da Silva, Diogo V; Dou, Dan; Wang, Hao; Daniels, Robert

    2017-08-07

    A primary function of 5' regions in many secretory protein mRNAs is to encode an endoplasmic reticulum (ER) targeting sequence. In this study, we show how the regions coding for the ER-targeting sequences of the influenza glycoproteins NA and HA also function as translational regulatory elements that are controlled by the viral RNA-binding protein (RBP) NS1. The translational increase depends on the nucleotide composition and 5' positioning of the ER-targeting sequence coding regions and is facilitated by the RNA-binding domain of NS1, which can associate with ER membranes. Inserting the ER-targeting sequence coding region of NA into different 5' UTRs confirmed that NS1 can promote the translation of secretory protein mRNAs based on the nucleotides within this region rather than the resulting amino acids. By analyzing human protein mRNA sequences, we found evidence that this mechanism of using 5' coding regions and particular RBPs to achieve gene-specific regulation may extend to human-secreted proteins. © 2017 Nordholm et al.

  11. Evaluation the susceptibility of five polymorphisms in microRNA-binding sites to female breast cancer risk in Chinese population.

    Science.gov (United States)

    He, Bang-Shun; Pan, Yu-Qin; Lin, Kang; Ying, Hou-Qun; Wang, Feng; Deng, Qi-Wen; Sun, Hui-Ling; Gao, Tian-Yi; Wang, Shu-Kui

    2015-11-15

    Polymorphisms in microRNA (miRNA) binding site have been widely discussed to be associated with cancer risk; however, the associations were unclear in Chinese population. To investigate the associations of five polymorphisms (rs11097457, rs1434536, rs1970801, rs1044129, rs11169571) in miRNA binding sites with breast cancer risk, a total of 435 female patients with breast cancer and 439 age- and gender-matched tumor-free individuals were enrolled in this case-control study. Sequenom MassARRAY was applied to detect the polymorphisms, and the immunohistochemistry assay was used to measure the expression of estrogen receptor (ER) and progesterone receptor (PR) and CerbB-2. The data showed that these polymorphisms were not associated with breast cancer risk or clinical characters of breast cancer in all participants and sub-group with the exception that, in the sub-group of women with their first menstruation after 14 years old, those who carried rs1970801 T allele (genotype TT/GT) were associated with decreased breast cancer risk. In short, this case-control study provided the evidence that women with their first menstruation after 14 years old and carried rs1970801 T allele (genotype TT/GT) were associated with decreased breast cancer risk.

  12. The Puf-family RNA-binding protein Puf2 controls sporozoite conversion to liver stages in the malaria parasite.

    Directory of Open Access Journals (Sweden)

    Katja Müller

    Full Text Available Malaria is a vector-borne infectious disease caused by unicellular, obligate intracellular parasites of the genus Plasmodium. During host switch the malaria parasite employs specialized latent stages that colonize the new host environment. Previous work has established that gametocytes, sexually differentiated stages that are taken up by the mosquito vector, control expression of genes required for mosquito colonization by translational repression. Sexual parasite development is controlled by a DEAD-box RNA helicase of the DDX6 family, termed DOZI. Latency of sporozoites, the transmission stage injected during an infectious blood meal, is controlled by the eIF2alpha kinase IK2, a general inhibitor of protein synthesis. Whether RNA-binding proteins participate in translational regulation in sporozoites remains to be studied. Here, we investigated the roles of two RNA-binding proteins of the Puf-family, Plasmodium Puf1 and Puf2, during sporozoite stage conversion. Our data reveal that, in the rodent malaria parasite P. berghei, Puf2 participates in the regulation of IK2 and inhibits premature sporozoite transformation. Inside mosquito salivary glands puf2⁻ sporozoites transform over time to round forms resembling early intra-hepatic stages. As a result, mutant parasites display strong defects in initiating a malaria infection. In contrast, Puf1 is dispensable in vivo throughout the entire Plasmodium life cycle. Our findings support the notion of a central role for Puf2 in parasite latency during switch between the insect and mammalian hosts.

  13. Crystallization of the avian reovirus double-stranded RNA-binding and core protein σA

    Energy Technology Data Exchange (ETDEWEB)

    Hermo-Parrado, X. Lois; Guardado-Calvo, Pablo [Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de Santiago de Compostela, Campus Sur, E-15782 Santiago de Compostela (Spain); Llamas-Saiz, Antonio L. [Unidad de Difracción de Rayos X, Laboratorio Integral de Dinámica y Estructura de Biomoléculas José R. Carracido, Edificio CACTUS, Universidad de Santiago de Compostela, Campus Sur, E-15782 Santiago de Compostela (Spain); Fox, Gavin C. [Spanish CRG Beamline BM16, European Synchrotron Radiation Facility (ESRF), 6 Rue Jules Horowitz, BP 220, F-38043 Grenoble (France); Vazquez-Iglesias, Lorena; Martínez-Costas, José; Benavente, Javier [Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de Santiago de Compostela, Campus Sur, E-15782 Santiago de Compostela (Spain); Raaij, Mark J. van, E-mail: vanraaij@usc.es [Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de Santiago de Compostela, Campus Sur, E-15782 Santiago de Compostela (Spain); Unidad de Difracción de Rayos X, Laboratorio Integral de Dinámica y Estructura de Biomoléculas José R. Carracido, Edificio CACTUS, Universidad de Santiago de Compostela, Campus Sur, E-15782 Santiago de Compostela (Spain)

    2007-05-01

    The avian reovirus double-stranded RNA-binding and core protein σA has been crystallized in space group P1, with unit-cell parameters a = 103.2, b = 129.9, c = 144.0 Å, α = 93.8, β = 105.1, γ = 98.2°. A complete data set has been collected to 2.3 Å resolution and analyzed. The avian reovirus protein σA plays a dual role: it is a structural protein forming part of the transcriptionally active core, but it has also been implicated in the resistance of the virus to interferon by strongly binding double-stranded RNA and thus inhibiting the double-stranded RNA-dependent protein kinase. The σA protein has been crystallized from solutions containing ammonium sulfate at pH values around 6. Crystals belonging to space group P1, with unit-cell parameters a = 103.2, b = 129.9, c = 144.0 Å, α = 93.8, β = 105.1, γ = 98.2° were grown and a complete data set has been collected to 2.3 Å resolution. The self-rotation function suggests that σA may form symmetric arrangements in the crystals.

  14. SNPs in microRNA binding sites in 3'-UTRs of RAAS genes influence arterial blood pressure and risk of myocardial infarction

    DEFF Research Database (Denmark)

    Nossent, Anne Yaël; Hansen, Jakob Liebe; Doggen, Carine

    2011-01-01

    We hypothesized that single nucleotide polymorphisms (SNPs) located in microRNA (miR) binding sites in genes of the renin angiotensin aldosterone system (RAAS) can influence blood pressure and risk of myocardial infarction.......We hypothesized that single nucleotide polymorphisms (SNPs) located in microRNA (miR) binding sites in genes of the renin angiotensin aldosterone system (RAAS) can influence blood pressure and risk of myocardial infarction....

  15. The Role of RNA Binding Proteins in Insulin Messenger Stability and Translation

    OpenAIRE

    2010-01-01

    Although the reason for insufficient release of insulin in diabetes mellitus may vary depending on the type and stage of the disease, it is of vital importance that an amplified insulin biosynthesis can meet the increased need during periods of hyperglycemia. The insulin mRNA is highly abundant in beta cells and changes in insulin mRNA levels are, at least in part, controlled by altered rates of mRNA degradation. Since the mechanisms behind the control of insulin messenger stability and trans...

  16. The B-WICH chromatin-remodelling complex regulates RNA polymerase III transcription by promoting Max-dependent c-Myc binding

    Science.gov (United States)

    Sadeghifar, Fatemeh; Böhm, Stefanie; Vintermist, Anna; Östlund Farrants, Ann-Kristin

    2015-01-01

    The chromatin-remodelling complex B-WICH, comprised of William syndrome transcription factor, the ATPase SNF2h and nuclear myosin, specifically activates RNA polymerase III transcription of the 5S rRNA and 7SL genes. However, the underlying mechanism is unknown. Using high-resolution MN walking we demonstrate here that B-WICH changes the chromatin structure in the vicinity of the 5S rRNA and 7SL RNA genes during RNA polymerase III transcription. The action of B-WICH is required for the binding of the RNA polymerase machinery and the regulatory factors c-Myc at the 5S rRNA and 7SL RNA genes. In addition to the c-Myc binding site at the 5S genes, we have revealed a novel c-Myc and Max binding site in the intergenic spacer of the 5S rDNA. This region also contains a region remodelled by B-WICH. We demonstrate that c-Myc binds to both sites in a Max-dependent way, and thereby activate transcription by acetylating histone H3. The novel binding patterns of c-Myc and Max link transcription of 5S rRNA to the Myc/Max/Mxd network. Since B-WICH acts prior to c-Myc and other factors, we propose a model in which the B-WICH complex is required to maintain an open chromatin structure at these RNA polymerase III genes. This is a prerequisite for the binding of additional regulatory factors. PMID:25883140

  17. Interaction between the tRNA-binding and C-terminal domains of Yeast Gcn2 regulates kinase activity in vivo.

    Directory of Open Access Journals (Sweden)

    Sebastien Lageix

    2015-02-01

    Full Text Available The stress-activated protein kinase Gcn2 regulates protein synthesis by phosphorylation of translation initiation factor eIF2α. Gcn2 is activated in amino acid-deprived cells by binding of uncharged tRNA to the regulatory domain related to histidyl-tRNA synthetase, but the molecular mechanism of activation is unclear. We used a genetic approach to identify a key regulatory surface in Gcn2 that is proximal to the predicted active site of the HisRS domain and likely remodeled by tRNA binding. Mutations leading to amino acid substitutions on this surface were identified that activate Gcn2 at low levels of tRNA binding (Gcd- phenotype, while other substitutions block kinase activation (Gcn- phenotype, in some cases without altering tRNA binding by Gcn2 in vitro. Remarkably, the Gcn- substitutions increase affinity of the HisRS domain for the C-terminal domain (CTD, previously implicated as a kinase autoinhibitory segment, in a manner dampened by HisRS domain Gcd- substitutions and by amino acid starvation in vivo. Moreover, tRNA specifically antagonizes HisRS/CTD association in vitro. These findings support a model wherein HisRS-CTD interaction facilitates the autoinhibitory function of the CTD in nonstarvation conditions, with tRNA binding eliciting kinase activation by weakening HisRS-CTD association with attendant disruption of the autoinhibitory KD-CTD interaction.

  18. NF-κB-dependent role for cold-inducible RNA binding protein in regulating interleukin 1β.

    Directory of Open Access Journals (Sweden)

    Christian Brochu

    Full Text Available The cold inducible RNA binding protein (CIRBP responds to a wide array of cellular stresses, including short wavelength ultraviolet light (UVC, at the transcriptional and post-translational level. CIRBP can bind the 3'untranslated region of specific transcripts to stabilize them and facilitate their transport to ribosomes for translation. Here we used RNA interference and oligonucleotide microarrays to identify potential downstream targets of CIRBP induced in response to UVC. Twenty eight transcripts were statistically increased in response to UVC and these exhibited a typical UVC response. Only 5 of the 28 UVC-induced transcripts exhibited a CIRBP-dependent pattern of expression. Surprisingly, 3 of the 5 transcripts (IL1B, IL8 and TNFAIP6 encoded proteins important in inflammation with IL-1β apparently contributing to IL8 and TNFAIP6 expression in an autocrine fashion. UVC-induced IL1B expression could be inhibited by pharmacological inhibition of NFκB suggesting that CIRBP was affecting NF-κB signaling as opposed to IL1B mRNA stability directly. Bacterial lipopolysaccharide (LPS was used as an activator of NF-κB to further study the potential link between CIRBP and NFκB. Transfection of siRNAs against CIRBP reduced the extent of the LPS-induced phosphorylation of IκBα, NF-κB DNA binding activity and IL-1β expression. The present work firmly establishes a novel link between CIRBP and NF-κB signaling in response to agents with diverse modes of action. These results have potential implications for disease states associated with inflammation.

  19. NF-κB-dependent role for cold-inducible RNA binding protein in regulating interleukin 1β.

    Science.gov (United States)

    Brochu, Christian; Cabrita, Miguel A; Melanson, Brian D; Hamill, Jeffrey D; Lau, Rosanna; Pratt, M A Christine; McKay, Bruce C

    2013-01-01

    The cold inducible RNA binding protein (CIRBP) responds to a wide array of cellular stresses, including short wavelength ultraviolet light (UVC), at the transcriptional and post-translational level. CIRBP can bind the 3'untranslated region of specific transcripts to stabilize them and facilitate their transport to ribosomes for translation. Here we used RNA interference and oligonucleotide microarrays to identify potential downstream targets of CIRBP induced in response to UVC. Twenty eight transcripts were statistically increased in response to UVC and these exhibited a typical UVC response. Only 5 of the 28 UVC-induced transcripts exhibited a CIRBP-dependent pattern of expression. Surprisingly, 3 of the 5 transcripts (IL1B, IL8 and TNFAIP6) encoded proteins important in inflammation with IL-1β apparently contributing to IL8 and TNFAIP6 expression in an autocrine fashion. UVC-induced IL1B expression could be inhibited by pharmacological inhibition of NFκB suggesting that CIRBP was affecting NF-κB signaling as opposed to IL1B mRNA stability directly. Bacterial lipopolysaccharide (LPS) was used as an activator of NF-κB to further study the potential link between CIRBP and NFκB. Transfection of siRNAs against CIRBP reduced the extent of the LPS-induced phosphorylation of IκBα, NF-κB DNA binding activity and IL-1β expression. The present work firmly establishes a novel link between CIRBP and NF-κB signaling in response to agents with diverse modes of action. These results have potential implications for disease states associated with inflammation.

  20. Evolutionarily dynamic roles of a PUF RNA-binding protein in the somatic development of Caenorhabditis briggsae.

    Science.gov (United States)

    Liu, Qinwen; Haag, Eric S

    2014-05-01

    Gene duplication and divergence has emerged as an important aspect of developmental evolution. The genomes of Caenorhabditis nematodes encode an ancient family of PUF RNA-binding proteins. Most have been implicated in germline development, and are often redundant with paralogs of the same sub-family. An exception is Cbr-puf-2 (one of three Caenorhabditis briggsae PUF-2 sub-family paralogs), which is required for development past the second larval stage. Here, we provide a detailed functional characterization of Cbr-puf-2. The larval arrest of Cbr-puf-2 mutant animals is caused by inefficient breakdown of bacterial food, which leads to starvation. Cbr-puf-2 is required for the normal grinding cycle of the muscular terminal bulb during early larval stages, and is transiently expressed in this tissue. In addition, rescue of larval arrest reveals that Cbr-puf-2 also promotes normal vulval development. It is expressed in the anchor cell (which induces vulval fate) and vulval muscles, but not in the vulva precursor cells (VPCs) themselves. This contrasts with the VPC-autonomous repression of vulval development described for the Caenorhabditis elegans homologs fbf-1/2. These different roles for PUF proteins occur even as the vulva and pharynx maintain highly conserved anatomies across Caenorhabditis, indicating pervasive developmental system drift (DSD). Because Cbr-PUF-2 shares RNA-binding specificity with its paralogs and with C. elegans FBF, we suggest that functional novelty of RNA-binding proteins evolves through changes in the site of their expression, perhaps in concert with cis-regulatory evolution in target mRNAs.

  1. PTB and TIAR binding to insulin mRNA 3′- and 5′UTRs; implications for insulin biosynthesis and messenger stability

    Directory of Open Access Journals (Sweden)

    Rikard G. Fred

    2016-09-01

    Conclusions: These experiments indicate that alterations in insulin mRNA stability and translation correlate with differential RBP binding. We propose that the balance between PTB on one hand and TIAR on the other participates in the control of insulin mRNA stability and utilization for insulin biosynthesis.

  2. Mutant forms of Escherichia coli protein L25 unable to bind to 5S rRNA are incorporated efficiently into the ribosome in vivo.

    Science.gov (United States)

    Anikaev, A Y; Korepanov, A P; Korobeinikova, A V; Kljashtorny, V G; Piendl, W; Nikonov, S V; Garber, M B; Gongadze, G M

    2014-08-01

    5S rRNA-binding ribosomal proteins of the L25 family are an evolutional acquisition of bacteria. Earlier we showed that (i) single replacements in the RNA-binding module of the protein of this family result in destabilization or complete impossibility to form a complex with 5S rRNA in vitro; (ii) ΔL25 ribosomes of Escherichia coli are less efficient in protein synthesis in vivo than the control ribosomes. In the present work, the efficiency of incorporation of the E. coli protein L25 with mutations in the 5S rRNA-binding region into the ribosome in vivo was studied. It was found that the mutations in L25 that abolish its ability to form the complex with free 5S rRNA do not prevent its correct and efficient incorporation into the ribosome. This is supported by the fact that even the presence of a very weakly retained mutant form of the protein in the ribosome has a positive effect on the activity of the translational machinery in vivo. All this suggests the existence of an alternative incorporation pathway for this protein into the ribosome, excluding the preliminary formation of the complex with 5S rRNA. At the same time, the stable L25-5S rRNA contact is important for the retention of the protein within the ribosome, and the conservative amino acid residues of the RNA-binding module play a key role in this.

  3. Alterations in the expression of DEAD-box and other RNA binding proteins during HIV-1 replication

    Directory of Open Access Journals (Sweden)

    Zeichner Steven L

    2004-12-01

    Full Text Available Abstract Recent results showed that certain DEAD box protein RNA helicases, DDX3 and DDX1, play an important role in the HIV infection cycle by facilitating the export of long, singly spliced or unspliced HIV RNAs from the nucleus via the CRM1-Rev pathway. Close examination of an extensive microarray expression profiling dataset obtained from cells latently infected with HIV induced to undergo lytic viral replication indicated that additional DEAD box proteins, beyond DDX3 and DDX1, exhibit differential expression during lytic HIV replication, and in latently infected cells prior to induction into active replication. This finding provides additional evidence that the involvement of DEAD box proteins and other RNA-binding proteins may play roles in active HIV replication and in the control of viral latency. Agents targeting these functions may offer new approaches to antiretroviral therapy and the therapeutic manipulation of HIV latency.

  4. Four isoforms of the signal-transduction and RNA-binding protein QKI expressed during chicken spermatogenesis.

    Science.gov (United States)

    Mezquita, J; Pau, M; Mezquita, C

    1998-05-01

    Genes expressed during spermatogenesis undergo alternative initiation and alternative splicing and may be under the control of a coordinated mechanism of RNA processing. A family of proteins that combine features of signal-transduction and RNA-binding molecules could be instrumental in this process. We have characterized a cDNA from adult chicken testis that codifies a highly conserved member of the STAR protein family, the orthologue of the mouse quaking gene qki. The predicted chicken protein differs only in four amino acids from the corresponding mouse protein. Messages of 7, 6, and 5 kb are expressed differentially during chicken spermatogenesis. The 5-kb message, the predominant form in adult testis, presents heterogeneity in the coding region, showing insertions of 51 and 75 bp and a deletion of 24 bp, which gives rise to four possible isoforms of the protein.

  5. Neuroprotective effects of cold-inducible RNA-binding protein during mild hypothermia on traumatic brain injur y

    Institute of Scientific and Technical Information of China (English)

    Guan Wang; Jian-ning Zhang; Jia-kui Guo; Ying Cai; Hong-sheng Sun; Kun Dong; Cheng-gang Wu

    2016-01-01

    Cold-inducible RNA-binding protein (CIRP), a key regulatory protein, could be facilitated by mild hypothermia in the brain, heart and liver. This study observed the effects of mild hypothermia at 31 ± 0.5°C on traumatic brain injury in rats. Results demonstrated that mild hypothermia suppressed apoptosis in the cortex, hippocampus and hypothalamus, facilitated CIRP mRNA and protein expression in these regions, especially in the hypothalamus. The anti-apoptotic effect of mild hypothermia disappeared after CIRP silencing. There was no correlation between mitogen-activated extracellular signal-regulated kinase activation and CIRP silencing. CIRP silencing inhibited extracellular signal-regulated kinase-1/2 activation. These indicate that CIRP inhibits apoptosis by affecting extracellular signal-regulated kinase-1/2 activation, and exerts a neuroprotective effect during mild hypothermia for traumatic brain injury.

  6. Accurate microRNA target prediction using detailed binding site accessibility and machine learning on proteomics data

    Directory of Open Access Journals (Sweden)

    Martin eReczko

    2012-01-01

    Full Text Available MicroRNAs (miRNAs are a class of small regulatory genes regulating gene expression by targetingmessenger RNA. Though computational methods for miRNA target prediction are the prevailingmeans to analyze their function, they still miss a large fraction of the targeted genes and additionallypredict a large number of false positives. Here we introduce a novel algorithm called DIANAmicroT-ANN which combines multiple novel target site features through an artificial neural network(ANN and is trained using recently published high-throughput data measuring the change of proteinlevels after miRNA overexpression, providing positive and negative targeting examples. The featurescharacterizing each miRNA recognition element include binding structure, conservation level and aspecific profile of structural accessibility. The ANN is trained to integrate the features of eachrecognition element along the 3’ untranslated region into a targeting score, reproducing the relativerepression fold change of the protein. Tested on two different sets the algorithm outperforms otherwidely used algorithms and also predicts a significant number of unique and reliable targets notpredicted by the other methods. For 542 human miRNAs DIANA-microT-ANN predicts 120,000targets not provided by TargetScan 5.0. The algorithm is freely available athttp://microrna.gr/microT-ANN.

  7. Splicing Machinery Facilitates Post-Transcriptional Regulation by FBFs and Other RNA-Binding Proteins in Caenorhabditis elegans Germline.

    Science.gov (United States)

    Novak, Preston; Wang, Xiaobo; Ellenbecker, Mary; Feilzer, Sara; Voronina, Ekaterina

    2015-08-11

    Genetic interaction screens are an important approach for understanding complex regulatory networks governing development. We used a genetic interaction screen to identify cofactors of FBF-1 and FBF-2, RNA-binding proteins that regulate germline stem cell proliferation in Caenorhabditis elegans. We found that components of splicing machinery contribute to FBF activity as splicing factor knockdowns enhance sterility of fbf-1 and fbf-2 single mutants. This sterility phenocopied multiple aspects of loss of fbf function, suggesting that splicing factors contribute to stem cell maintenance. However, previous reports indicate that splicing factors instead promote the opposite cell fate, namely, differentiation. We explain this discrepancy by proposing that splicing factors facilitate overall RNA regulation in the germline. Indeed, we find that loss of splicing factors produces synthetic phenotypes with a mutation in another RNA regulator, FOG-1, but not with a mutation in a gene unrelated to posttranscriptional regulation (dhc-1). We conclude that inefficient pre-mRNA splicing may interfere with multiple posttranscriptional regulatory events, which has to be considered when interpreting results of genetic interaction screens.

  8. MicroRNA genes preferentially expressed in dendritic cells contain sites for conserved transcription factor binding motifs in their promoters

    Directory of Open Access Journals (Sweden)

    Huynen Martijn A

    2011-06-01

    Full Text Available Abstract Background MicroRNAs (miRNAs play a fundamental role in the regulation of gene expression by translational repression or target mRNA degradation. Regulatory elements in miRNA promoters are less well studied, but may reveal a link between their expression and a specific cell type. Results To explore this link in myeloid cells, miRNA expression profiles were generated from monocytes and dendritic cells (DCs. Differences in miRNA expression among monocytes, DCs and their stimulated progeny were observed. Furthermore, putative promoter regions of miRNAs that are significantly up-regulated in DCs were screened for Transcription Factor Binding Sites (TFBSs based on TFBS motif matching score, the degree to which those TFBSs are over-represented in the promoters of the up-regulated miRNAs, and the extent of conservation of the TFBSs in mammals. Conclusions Analysis of evolutionarily conserved TFBSs in DC promoters revealed preferential clustering of sites within 500 bp upstream of the precursor miRNAs and that many mRNAs of cognate TFs of the conserved TFBSs were indeed expressed in the DCs. Taken together, our data provide evidence that selected miRNAs expressed in DCs have evolutionarily conserved TFBSs relevant to DC biology in their promoters.

  9. POF regulates the expression of genes on the fourth chromosome in Drosophila melanogaster by binding to nascent RNA.

    Science.gov (United States)

    Johansson, Anna-Mia; Stenberg, Per; Allgardsson, Anders; Larsson, Jan

    2012-06-01

    In Drosophila, two chromosome-wide compensatory systems have been characterized: the dosage compensation system that acts on the male X chromosome and the chromosome-specific regulation of genes located on the heterochromatic fourth chromosome. Dosage compensation in Drosophila is accomplished by hypertranscription of the single male X chromosome mediated by the male-specific lethal (MSL) complex. The mechanism of this compensation is suggested to involve enhanced transcriptional elongation mediated by the MSL complex, while the mechanism of compensation mediated by the painting of fourth (POF) protein on the fourth chromosome has remained elusive. Here, we show that POF binds to nascent RNA, and this binding is associated with increased transcription output from chromosome 4. We also show that genes located in heterochromatic regions spend less time in transition from the site of transcription to the nuclear envelope. These results provide useful insights into the means by which genes in heterochromatic regions can overcome the repressive influence of their hostile environment.

  10. La-related protein 4 binds poly(A), interacts with the poly(A)-binding protein MLLE domain via a variant PAM2w motif, and can promote mRNA stability.

    Science.gov (United States)

    Yang, Ruiqing; Gaidamakov, Sergei A; Xie, Jingwei; Lee, Joowon; Martino, Luigi; Kozlov, Guennadi; Crawford, Amanda K; Russo, Amy N; Conte, Maria R; Gehring, Kalle; Maraia, Richard J

    2011-02-01

    The conserved RNA binding protein La recognizes UUU-3'OH on its small nuclear RNA ligands and stabilizes them against 3'-end-mediated decay. We report that newly described La-related protein 4 (LARP4) is a factor that can bind poly(A) RNA and interact with poly(A) binding protein (PABP). Yeast two-hybrid analysis and reciprocal immunoprecipitations (IPs) from HeLa cells revealed that LARP4 interacts with RACK1, a 40S ribosome- and mRNA-associated protein. LARP4 cosediments with 40S ribosome subunits and polyribosomes, and its knockdown decreases translation. Mutagenesis of the RNA binding or PABP interaction motifs decrease LARP4 association with polysomes. Several translation and mRNA metabolism-related proteins use a PAM2 sequence containing a critical invariant phenylalanine to make direct contact with the MLLE domain of PABP, and their competition for the MLLE is thought to regulate mRNA homeostasis. Unlike all ∼150 previously analyzed PAM2 sequences, LARP4 contains a variant PAM2 (PAM2w) with tryptophan in place of the phenylalanine. Binding and nuclear magnetic resonance (NMR) studies have shown that a peptide representing LARP4 PAM2w interacts with the MLLE of PABP within the affinity range measured for other PAM2 motif peptides. A cocrystal of PABC bound to LARP4 PAM2w shows tryptophan in the pocket in PABC-MLLE otherwise occupied by phenylalanine. We present evidence that LARP4 expression stimulates luciferase reporter activity by promoting mRNA stability, as shown by mRNA decay analysis of luciferase and cellular mRNAs. We propose that LARP4 activity is integrated with other PAM2 protein activities by PABP as part of mRNA homeostasis.

  11. Site-directed mutagenesis of Arg58 and Asp86 of elongation factor Tu from Escherichia coli: effects on the GTPase reaction and aminoacyl-tRNA binding

    DEFF Research Database (Denmark)

    Knudsen, Charlotte Rohde; Clark, Brian F. C.

    1996-01-01

    Elongation factor Tu from Escherichia coli was mutated separately at positions Asp86 and Arg58, in order to shed light both on the GTPase mechanism of elongation factor Tu and on the binding of aminoacyl-tRNA. In addition, the binding of guanine nucleotides was investigated by determination...

  12. Site-directed mutagenesis of Arg58 and Asp86 of elongation factor Tu from Escherichia coli: effects on the GTPase reaction and aminoacyl-tRNA binding

    DEFF Research Database (Denmark)

    Knudsen, Charlotte Rohde; Clark, Brian F. C.

    1996-01-01

    Elongation factor Tu from Escherichia coli was mutated separately at positions Asp86 and Arg58, in order to shed light both on the GTPase mechanism of elongation factor Tu and on the binding of aminoacyl-tRNA. In addition, the binding of guanine nucleotides was investigated by determination of th...

  13. Functional characterization of two paralogs that are novel RNA binding proteins influencing mitochondrial transcripts of Trypanosoma brucei.

    Science.gov (United States)

    Kafková, Lucie; Ammerman, Michelle L; Faktorová, Drahomíra; Fisk, John C; Zimmer, Sara L; Sobotka, Roman; Read, Laurie K; Lukes, Julius; Hashimi, Hassan

    2012-10-01

    A majority of Trypanosoma brucei proteins have unknown functions, a consequence of its independent evolutionary history within the order Kinetoplastida that allowed for the emergence of several unique biological properties. Among these is RNA editing, needed for expression of mitochondrial-encoded genes. The recently discovered mitochondrial RNA binding complex 1 (MRB1) is composed of proteins with several functions in processing organellar RNA. We characterize two MRB1 subunits, referred to herein as MRB8170 and MRB4160, which are paralogs arisen from a large chromosome duplication occurring only in T. brucei. As with many other MRB1 proteins, both have no recognizable domains, motifs, or orthologs outside the order. We show that they are both novel RNA binding proteins, possibly representing a new class of these proteins. They associate with a similar subset of MRB1 subunits but not directly with each other. We generated cell lines that either individually or simultaneously target the mRNAs encoding both proteins using RNAi. Their dual silencing results in a differential effect on moderately and pan-edited RNAs, suggesting a possible functional separation of the two proteins. Cell growth persists upon RNAi silencing of each protein individually in contrast to the dual knockdown. Yet, their apparent redundancy in terms of cell viability is at odds with the finding that only one of these knockdowns results in the general degradation of pan-edited RNAs. While MRB8170 and MRB4160 share a considerable degree of conservation, our results suggest that their recent sequence divergence has led to them influencing mitochondrial mRNAs to differing degrees.

  14. Expression and RNA-binding of human zinc-finger antiviral protein

    Energy Technology Data Exchange (ETDEWEB)

    Jeong, Mi Suk; Kim, Eun Jung [Department of Molecular Biology, College of Natural Sciences, Pusan National University, Jangjeon-dong, Geumjeong-gu, Busan 609-735 (Korea, Republic of); Jang, Se Bok, E-mail: sbjang@pusan.ac.kr [Department of Molecular Biology, College of Natural Sciences, Pusan National University, Jangjeon-dong, Geumjeong-gu, Busan 609-735 (Korea, Republic of)

    2010-06-04

    Zinc-finger antiviral protein (ZAP) is a recently isolated host antiviral factor that inhibits the replication of many viruses such as Moloney murine leukemia virus (MLV) and Sindbis virus (SIN) by preventing the accumulation of viral mRNA in the cytoplasm. ZAP comprises four CCCH zinc-finger motifs, the second and fourth of which are responsible for protein activity based on their integrity. Thus far, there have been no reports on whether or not ZAP expressed in Escherichia coli is soluble. Therefore, we expressed N-terminal ZAP (NZAP, 254 amino acids) in E. coli as a fusion protein with several different cleavage sites and protein tags. Cleaved ZAP in soluble form strongly bound to RNA through its four CCCH zinc-finger motifs. Here, we provide evidence indicating that ZAP directly interacted with viral RNA. Each conserved zinc-finger motif of ZAP coordinates a zinc ion using three cysteines and one histidine. These findings suggest that ZAP recruits the cellular RNA degradation machinery for the degradation of viral RNA.

  15. Structure-activity studies on the fluorescent indicator in a displacement assay for the screening of small molecules binding to RNA.

    Science.gov (United States)

    Umemoto, Shiori; Im, Seongwang; Zhang, Jinhua; Hagihara, Masaki; Murata, Asako; Harada, Yasue; Fukuzumi, Takeo; Wazaki, Takahiro; Sasaoka, Shin-ichi; Nakatani, Kazuhiko

    2012-08-06

    A series of xanthone and thioxanthone derivatives with aminoalkoxy substituents were synthesized as fluorescent indicators for a displacement assay in the study of small-molecule-RNA interactions. The RNA-binding properties of these molecules were investigated in terms of the improved binding selectivity to the loop region in the RNA secondary structure relative to 2,7-bis(2-aminoethoxy)xanthone (X2S) by fluorimetric titration and displacement assay. An 11-mer double-stranded RNA and a hairpin RNA mimicking the stem loop IIB of Rev response element (RRE) RNA of HIV-1 mRNA were used. The X2S derivatives with longer aminoalkyl substituents showed a higher affinity to the double-stranded RNA than the parent molecule. Introduction of a methyl group on the aminoethoxy moiety of X2S effectively modulated the selectivity to the RNA secondary structure. Methyl group substitution at the C1' position suppressed the binding to the loop regions. Substitution with two methyl groups on the amino nitrogen atom resulted in reducing the affinity to the double-stranded region by a factor of 40%. The effect of methyl substitution on the amino nitrogen atom was also observed for a thioxanthone derivative. Titration experiments, however, suggested that thioxanthone derivatives showed a more prominent tendency of multiple binding to RNA than xanthone derivatives. The selectivity index calculated from the affinity to the double-stranded and loop regions suggested that the N,N-dimethyl derivative of X2S would be suitable for the screening of small molecules binding to RRE. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Telomerase RNA stem terminus element affects template boundary element function, telomere sequence, and shelterin binding.

    Science.gov (United States)

    Webb, Christopher J; Zakian, Virginia A

    2015-09-08

    The stem terminus element (STE), which was discovered 13 y ago in human telomerase RNA, is required for telomerase activity, yet its mode of action is unknown. We report that the Schizosaccharomyces pombe telomerase RNA, TER1 (telomerase RNA 1), also contains a STE, which is essential for telomere maintenance. Cells expressing a partial loss-of-function TER1 STE allele maintained short stable telomeres by a recombination-independent mechanism. Remarkably, the mutant telomere sequence was different from that of wild-type cells. Generation of the altered sequence is explained by reverse transcription into the template boundary element, demonstrating that the STE helps maintain template boundary element function. The altered telomeres bound less Pot1 (protection of telomeres 1) and Taz1 (telomere-associated in Schizosaccharomyces pombe 1) in vivo. Thus, the S. pombe STE, although distant from the template, ensures proper telomere sequence, which in turn promotes proper assembly of the shelterin complex.

  17. The FTD/ALS-associated RNA-binding protein TDP-43 regulates the robustness of neuronal specification through microRNA-9a in Drosophila.

    Science.gov (United States)

    Li, Zhaodong; Lu, Yubing; Xu, Xia-Lian; Gao, Fen-Biao

    2013-01-15

    TDP-43 is an evolutionarily conserved RNA-binding protein currently under intense investigation for its involvement in the molecular pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). TDP-43 is normally localized in the nucleus, but translocated to the cytoplasm in diseased neurons. The endogenous functions of TDP-43 in the nervous system remain poorly understood. Here, we show that the loss of Drosophila TDP-43 (dTDP-43) results in an increased production of sensory bristles and sensory organ precursor (SOP) cells on the notum of some but not all flies. The location of ectopic SOPs varies among mutant flies. The penetrance of this novel phenotype is dependent on the gender and sensitive to environmental influences. A similar SOP phenotype was also observed on the wing and in the embryos. Overexpression of dTDP-43 causes both loss and ectopic production of SOPs. Ectopic expression of ALS-associated mutant human TDP-43 (hTDP-43(M337V) and hTDP-43(Q331K)) produces a less severe SOP phenotype than hTDP-43(WT), indicating a partial loss of function of mutant hTDP-43. In dTDP-43 mutants, miR-9a expression is significantly reduced. Genetic interaction studies further support the notion that dTDP-43 acts through miR-9a to control the precision of SOP specification. These findings reveal a novel role for endogenous TDP-43 in neuronal specification and suggest that the FTD/ALS-associated RNA-binding protein TDP-43 functions to ensure the robustness of genetic control programs.

  18. Ribosomal protein L16 binds to the 3'-end of transfer RNA.

    Science.gov (United States)

    Maimets, T; Remme, J; Villems, R

    1984-01-23

    Escherichia coli 50 S ribosomal subunits were reconstituted with and without protein L16 present. The latter particles, although active in puromycin reaction, were unable to use CACCA-Phe as an acceptor substrate. We also found that L16 interacts directly with this oligonucleotide and, in the complex with tRNA, protects its 3'-end from pancreatic ribonuclease digestion. We suggest that the role of L16 is in the fixation of the aminoacyl stem of tRNA to the ribosome at its A-site.

  19. The sea urchin stem–loop-binding protein: a maternally expressed protein that probably functions in expression of multiple classes of histone mRNA

    Science.gov (United States)

    Robertson, Anthony J.; Howard, Jason T.; Dominski, Zbigniew; Schnackenberg, Bradley J.; Sumerel, Jan L.; McCarthy, John J.; Coffman, James A.; Marzluff, William F.

    2004-01-01

    Following the completion of oogenesis and oocyte maturation, histone mRNAs are synthesized and stored in the sea urchin egg pronucleus. Histone mRNAs are the only mRNAs that are not polyadenylated but instead end in a stem–loop which has been conserved in evolution. The 3′ end binds the stem–loop-binding protein (SLBP), and SLBP is required for histone pre-mRNA processing as well as translation of the histone mRNAs. A cDNA encoding a 59 kDa sea urchin SLBP (suSLBP) has been cloned from an oocyte cDNA library. The suSLBP contains an RNA-binding domain that is similar to the RNA-binding domain found in SLBPs from other species, although there is no similarity between the rest of the suSLBP and other SLBPs. The suSLBP is present at constant levels in eggs and for the first 12 h of development. The levels of suSLBP then decline and remain at a low level for the rest of embryogenesis. The suSLBP is concentrated in the egg pronucleus and is released from the nucleus only when cells enter the first mitosis. SuSLBP expressed by in vitro translation does not bind the stem–loop RNA, suggesting that suSLBP is modified to activate RNA binding in sea urchin embryos. PMID:14762208

  20. Markov State Models Reveal a Two-Step Mechanism of miRNA Loading into the Human Argonaute Protein: Selective Binding followed by Structural Re-arrangement

    KAUST Repository

    Jiang, Hanlun

    2015-07-16

    Argonaute (Ago) proteins and microRNAs (miRNAs) are central components in RNA interference, which is a key cellular mechanism for sequence-specific gene silencing. Despite intensive studies, molecular mechanisms of how Ago recognizes miRNA remain largely elusive. In this study, we propose a two-step mechanism for this molecular recognition: selective binding followed by structural re-arrangement. Our model is based on the results of a combination of Markov State Models (MSMs), large-scale protein-RNA docking, and molecular dynamics (MD) simulations. Using MSMs, we identify an open state of apo human Ago-2 in fast equilibrium with partially open and closed states. Conformations in this open state are distinguished by their largely exposed binding grooves that can geometrically accommodate miRNA as indicated in our protein-RNA docking studies. miRNA may then selectively bind to these open conformations. Upon the initial binding, the complex may perform further structural re-arrangement as shown in our MD simulations and eventually reach the stable binary complex structure. Our results provide novel insights in Ago-miRNA recognition mechanisms and our methodology holds great potential to be widely applied in the studies of other important molecular recognition systems.

  1. Selective translational repression of HIV-1 RNA by Sam68DeltaC occurs by altering PABP1 binding to unspliced viral RNA

    Directory of Open Access Journals (Sweden)

    Soros Vanessa

    2008-10-01

    Full Text Available Abstract HIV-1 structural proteins are translated from incompletely spliced 9 kb and 4 kb mRNAs, which are transported to the cytoplasm by Crm1. It has been assumed that once in the cytoplasm, translation of incompletely spliced HIV-1 mRNAs occurs in the same manner as host mRNAs. Previous analyses have demonstrated that Sam68 and a mutant thereof, Sam68ΔC, have dramatic effects on HIV gene expression, strongly enhancing and inhibiting viral structural protein synthesis, respectively. While investigating the inhibition of incompletely spliced HIV-1 mRNAs by Sam68ΔC, we determined that the effect was independent of the perinuclear bundling of the viral RNA. Inhibition was dependent upon the nuclear export pathway used, as translation of viral RNA exported via the Tap/CTE export pathway was not blocked by Sam68ΔC. We demonstrate that inhibition of HIV expression by Sam68ΔC is correlated with a loss of PABP1 binding with no attendant change in polyadenosine tail length of the affected RNAs. The capacity of Sam68ΔC to selectively inhibit translation of HIV-1 RNAs exported by Crm1 suggests that it is able to recognize unique characteristics of these viral RNPs, a property that could lead to new therapeutic approaches to controlling HIV-1 replication.

  2. Evolutionary Limitation and Opportunities for Developing tRNA Synthetase Inhibitors with 5-Binding-Mode Classification

    Directory of Open Access Journals (Sweden)

    Pengfei Fang

    2015-12-01

    Full Text Available Aminoacyl-tRNA synthetases (aaRSs are enzymes that catalyze the transfer of amino acids to their cognate tRNAs as building blocks for translation. Each of the aaRS families plays a pivotal role in protein biosynthesis and is indispensable for cell growth and survival. In addition, aaRSs in higher species have evolved important non-translational functions. These translational and non-translational functions of aaRS are attractive for developing antibacterial, antifungal, and antiparasitic agents and for treating other human diseases. The interplay between amino acids, tRNA, ATP, EF-Tu and non-canonical binding partners, had shaped each family with distinct pattern of key sites for regulation, with characters varying among species across the path of evolution. These sporadic variations in the aaRSs offer great opportunity to target these essential enzymes for therapy. Up to this day, growing numbers of aaRS inhibitors have been discovered and developed. Here, we summarize the latest developments and structural studies of aaRS inhibitors, and classify them with distinct binding modes into five categories.

  3. Transition of Plasmodium sporozoites into liver stage-like forms is regulated by the RNA binding protein Pumilio

    KAUST Repository

    Gomes-Santos, Carina S. S.

    2011-05-19

    Many eukaryotic developmental and cell fate decisions that are effected post-transcriptionally involve RNA binding proteins as regulators of translation of key mRNAs. In malaria parasites (Plasmodium spp.), the development of round, non-motile and replicating exo-erythrocytic liver stage forms from slender, motile and cell-cycle arrested sporozoites is believed to depend on environmental changes experienced during the transmission of the parasite from the mosquito vector to the vertebrate host. Here we identify a Plasmodium member of the RNA binding protein family PUF as a key regulator of this transformation. In the absence of Pumilio-2 (Puf2) sporozoites initiate EEF development inside mosquito salivary glands independently of the normal transmission-associated environmental cues. Puf2- sporozoites exhibit genome-wide transcriptional changes that result in loss of gliding motility, cell traversal ability and reduction in infectivity, and, moreover, trigger metamorphosis typical of early Plasmodium intra-hepatic development. These data demonstrate that Puf2 is a key player in regulating sporozoite developmental control, and imply that transformation of salivary gland-resident sporozoites into liver stage-like parasites is regulated by a post-transcriptional mechanism. 2011 Gomes-Santos et al.

  4. PiRaNhA: a server for the computational prediction of RNA-binding residues in protein sequences

    Science.gov (United States)

    Murakami, Yoichi; Spriggs, Ruth V.; Nakamura, Haruki; Jones, Susan

    2010-01-01

    The PiRaNhA web server is a publicly available online resource that automatically predicts the location of RNA-binding residues (RBRs) in protein sequences. The goal of functional annotation of sequences in the field of RNA binding is to provide predictions of high accuracy that require only small numbers of targeted mutations for verification. The PiRaNhA server uses a support vector machine (SVM), with position-specific scoring matrices, residue interface propensity, predicted residue accessibility and residue hydrophobicity as features. The server allows the submission of up to 10 protein sequences, and the predictions for each sequence are provided on a web page and via email. The prediction results are provided in sequence format with predicted RBRs highlighted, in text format with the SVM threshold score indicated and as a graph which enables users to quickly identify those residues above any specific SVM threshold. The graph effectively enables the increase or decrease of the false positive rate. When tested on a non-redundant data set of 42 protein sequences not used in training, the PiRaNhA server achieved an accuracy of 85%, specificity of 90% and a Matthews correlation coefficient of 0.41 and outperformed other publicly available servers. The PiRaNhA prediction server is freely available at http://www.bioinformatics.sussex.ac.uk/PIRANHA. PMID:20507911

  5. Transition of Plasmodium sporozoites into liver stage-like forms is regulated by the RNA binding protein Pumilio.

    Directory of Open Access Journals (Sweden)

    Carina S S Gomes-Santos

    2011-05-01

    Full Text Available Many eukaryotic developmental and cell fate decisions that are effected post-transcriptionally involve RNA binding proteins as regulators of translation of key mRNAs. In malaria parasites (Plasmodium spp., the development of round, non-motile and replicating exo-erythrocytic liver stage forms from slender, motile and cell-cycle arrested sporozoites is believed to depend on environmental changes experienced during the transmission of the parasite from the mosquito vector to the vertebrate host. Here we identify a Plasmodium member of the RNA binding protein family PUF as a key regulator of this transformation. In the absence of Pumilio-2 (Puf2 sporozoites initiate EEF development inside