mRNA Expression of Ovine Angiopoietin-like Protein 4 Gene in Adipose Tissues

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Jing Zhang

2016-05-01

Full Text Available Angiopoietin-like protein 4 (ANGPTL4 is involved in a variety of functions, including lipoprotein metabolism and angiogenesis. To reveal the role of ANGPTL4 in fat metabolism of sheep, ovine ANGPTL4 mRNA expression was analyzed in seven adipose tissues from two breeds with distinct tail types. Forty-eight animals with the gender ratio of 1:1 for both Guangling Large Tailed (GLT and Small Tailed Han (STH sheep were slaughtered at 2, 4, 6, 8, 10, and 12 months of age, respectively. Adipose tissues were collected from greater and lesser omental, subcutaneous, retroperitoneal, perirenal, mesenteric, and tail fats. Ontogenetic mRNA expression of ANGPTL4 in these adipose tissues from GTL and STH was studied by quantitative real time polymerase chain reaction. The results showed that ANGPTL4 mRNA expressed in all adipose tissues studied with the highest in subcutaneous and the lowest in mesenteric fat depots. Months of age, tissue and breed are the main factors that significantly influence the mRNA expression. These results provide new insights into ovine ANGPTL4 gene expression and clues for its function mechanism.

A novel TBP-TAF complex on RNA polymerase II-transcribed snRNA genes.

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Zaborowska, Justyna; Taylor, Alice; Roeder, Robert G; Murphy, Shona

2012-01-01

Initiation of transcription of most human genes transcribed by RNA polymerase II (RNAP II) requires the formation of a preinitiation complex comprising TFIIA, B, D, E, F, H and RNAP II. The general transcription factor TFIID is composed of the TATA-binding protein and up to 13 TBP-associated factors. During transcription of snRNA genes, RNAP II does not appear to make the transition to long-range productive elongation, as happens during transcription of protein-coding genes. In addition, recognition of the snRNA gene-type specific 3' box RNA processing element requires initiation from an snRNA gene promoter. These characteristics may, at least in part, be driven by factors recruited to the promoter. For example, differences in the complement of TAFs might result in differential recruitment of elongation and RNA processing factors. As precedent, it already has been shown that the promoters of some protein-coding genes do not recruit all the TAFs found in TFIID. Although TAF5 has been shown to be associated with RNAP II-transcribed snRNA genes, the full complement of TAFs associated with these genes has remained unclear. Here we show, using a ChIP and siRNA-mediated approach, that the TBP/TAF complex on snRNA genes differs from that found on protein-coding genes. Interestingly, the largest TAF, TAF1, and the core TAFs, TAF10 and TAF4, are not detected on snRNA genes. We propose that this snRNA gene-specific TAF subset plays a key role in gene type-specific control of expression.

Heterologous and endogenous U6 snRNA promoters enable CRISPR/Cas9 mediated genome editing in Aspergillus niger.

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Zheng, Xiaomei; Zheng, Ping; Sun, Jibin; Kun, Zhang; Ma, Yanhe

2018-01-01

U6 promoters have been used for single guide RNA (sgRNA) transcription in the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas9) genome editing system. However, no available U6 promoters have been identified in Aspergillus niger, which is an important industrial platform for organic acid and protein production. Two CRISPR/Cas9 systems established in A. niger have recourse to the RNA polymerase II promoter or in vitro transcription for sgRNA synthesis, but these approaches generally increase cloning efforts and genetic manipulation. The validation of functional RNA polymerase II promoters is therefore an urgent need for A. niger . Here, we developed a novel CRISPR/Cas9 system in A. niger for sgRNA expression, based on one endogenous U6 promoter and two heterologous U6 promoters. The three tested U6 promoters enabled sgRNA transcription and the disruption of the polyketide synthase albA gene in A. niger . Furthermore, this system enabled highly efficient gene insertion at the targeted genome loci in A. niger using donor DNAs with homologous arms as short as 40-bp. This study demonstrated that both heterologous and endogenous U6 promoters were functional for sgRNA expression in A. niger . Based on this result, a novel and simple CRISPR/Cas9 toolbox was established in A. niger, that will benefit future gene functional analysis and genome editing.

Assembly and dynamics of the U4/U6 di-snRNP by single-molecule FRET

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Hardin, John W.; Warnasooriya, Chandani; Kondo, Yasushi; Nagai, Kiyoshi; Rueda, David

2015-01-01

In large ribonucleoprotein machines, such as ribosomes and spliceosomes, RNA functions as an assembly scaffold as well as a critical catalytic component. Protein binding to the RNA scaffold can induce structural changes, which in turn modulate subsequent binding of other components. The spliceosomal U4/U6 di-snRNP contains extensively base paired U4 and U6 snRNAs, Snu13, Prp31, Prp3 and Prp4, seven Sm and seven LSm proteins. We have studied successive binding of all protein components to the snRNA duplex during di-snRNP assembly by electrophoretic mobility shift assay and accompanying conformational changes in the U4/U6 RNA 3-way junction by single-molecule FRET. Stems I and II of the duplex were found to co-axially stack in free RNA and function as a rigid scaffold during the entire assembly, but the U4 snRNA 5′ stem-loop adopts alternative orientations each stabilized by Prp31 and Prp3/4 binding accounting for altered Prp3/4 binding affinities in presence of Prp31. PMID:26503251

Phage T4 SegB protein is a homing endonuclease required for the preferred inheritance of T4 tRNA gene region occurring in co-infection with a related phage.

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Brok-Volchanskaya, Vera S; Kadyrov, Farid A; Sivogrivov, Dmitry E; Kolosov, Peter M; Sokolov, Andrey S; Shlyapnikov, Michael G; Kryukov, Valentine M; Granovsky, Igor E

2008-04-01

Homing endonucleases initiate nonreciprocal transfer of DNA segments containing their own genes and the flanking sequences by cleaving the recipient DNA. Bacteriophage T4 segB gene, which is located in a cluster of tRNA genes, encodes a protein of unknown function, homologous to homing endonucleases of the GIY-YIG family. We demonstrate that SegB protein is a site-specific endonuclease, which produces mostly 3' 2-nt protruding ends at its DNA cleavage site. Analysis of SegB cleavage sites suggests that SegB recognizes a 27-bp sequence. It contains 11-bp conserved sequence, which corresponds to a conserved motif of tRNA TpsiC stem-loop, whereas the remainder of the recognition site is rather degenerate. T4-related phages T2L, RB1 and RB3 contain tRNA gene regions that are homologous to that of phage T4 but lack segB gene and several tRNA genes. In co-infections of phages T4 and T2L, segB gene is inherited with nearly 100% of efficiency. The preferred inheritance depends absolutely on the segB gene integrity and is accompanied by the loss of the T2L tRNA gene region markers. We suggest that SegB is a homing endonuclease that functions to ensure spreading of its own gene and the surrounding tRNA genes among T4-related phages.

Mutations in the gene for EF-G reduce the requirement for 4.5S RNA in the growth of E. coli

DEFF Research Database (Denmark)

Brown, S

1987-01-01

A general strategy is described for the isolation of suppressors of essential genes whose functions are unknown. This strategy was used to analyze the role of 4.5S RNA, an essential RNA of E. coli. In this strategy, the structural gene for 4.5S RNA is fused to the Ptac promoter in such a way...... that the strain becomes dependent upon inducers of lac for growth. Mutants mapping to fus, the structural gene for protein synthesis elongation factor G, appear as spontaneous, inducer-independent revertants. These mutants alter the intracellular distribution of 4.5S RNA such that it sediments at 70S or greater....... Furthermore, the increased sedimentation velocity is sensitive to the antibiotic puromycin. These results show that 4.5S RNA physically associates with the ribosome in performing its essential function, and that this association is mediated by elongation factor G....

Developmental regulation of Xenopus 5S RNA genes

International Nuclear Information System (INIS)

Wormington, W.M.; Schlissel, M.; Brown, D.D.

1983-01-01

In this paper it is demonstrated that the actively transcribed fraction of somatic 5S RNA genes in somatic-cell chromatin is complexed stably with all required factors, so that the addition of only purified RNA polymerase III is needed to support somatic 5S RNA synthesis in vitro. Oocyte 5S RNA genes in somatic-cell chromatin appear to lack these factors, since their activation in salt-washed somatic-cell chromatin depends on exogeneous transciption factors in addition to RNA polymerase III. The developmental control of 5S RNA genes is established over a period beginning with the onset of 5S RNA synthesis in late blastula embryos, and this control is reproduced in vitro using chromatin templates isolated from appropriate stages. We propose that a decreasing concentration of the 5S-specific transcription factor during embryogenesis contributes to the inactivation of oocyte 5S RNA genes. 12 references, 4 figures, 1 table

Species-independent MicroRNA Gene Discovery

KAUST Repository

Kamanu, Timothy K.

2012-12-01

MicroRNA (miRNA) are a class of small endogenous non-coding RNA that are mainly negative transcriptional and post-transcriptional regulators in both plants and animals. Recent studies have shown that miRNA are involved in different types of cancer and other incurable diseases such as autism and Alzheimer’s. Functional miRNAs are excised from hairpin-like sequences that are known as miRNA genes. There are about 21,000 known miRNA genes, most of which have been determined using experimental methods. miRNA genes are classified into different groups (miRNA families). This study reports about 19,000 unknown miRNA genes in nine species whereby approximately 15,300 predictions were computationally validated to contain at least one experimentally verified functional miRNA product. The predictions are based on a novel computational strategy which relies on miRNA family groupings and exploits the physics and geometry of miRNA genes to unveil the hidden palindromic signals and symmetries in miRNA gene sequences. Unlike conventional computational miRNA gene discovery methods, the algorithm developed here is species-independent: it allows prediction at higher accuracy and resolution from arbitrary RNA/DNA sequences in any species and thus enables examination of repeat-prone genomic regions which are thought to be non-informative or ’junk’ sequences. The information non-redundancy of uni-directional RNA sequences compared to information redundancy of bi-directional DNA is demonstrated, a fact that is overlooked by most pattern discovery algorithms. A novel method for computing upstream and downstream miRNA gene boundaries based on mathematical/statistical functions is suggested, as well as cutoffs for annotation of miRNA genes in different miRNA families. Another tool is proposed to allow hypotheses generation and visualization of data matrices, intra- and inter-species chromosomal distribution of miRNA genes or miRNA families. Our results indicate that: miRNA and miRNA

Expressions of interferon-inducible genes IFIT1 and IFIT4 mRNA in PBMCs of patients with systemic lupus erythematosus

International Nuclear Information System (INIS)

Liu Chunyan; Chen Xingguo; Wang Zizheng

2009-01-01

To investigate the expression levels of interferon-inducible genes (IFIT1, IFIT4) in the peripheral blood mononuclear cells (PBMCs) of patients with systemic lupus erythematosus (SLE), and the relations between these genes expression levels and disease activity, the expression levels of IFIT1 and IFIT4 mRNA in the 95 patients with SLE and 48 normal controls were detected by Sybr green dye based real-time quantitative PCR method, and these genes expression levels were compared with anti-double strand DNA antibody. The associations between the expression levels of IFIT1, IFIT4 mRNA, anti-double strand DNA antibody and SLEDAI scores in patients with SLE were analyzed. The results showed that the expression levels of IFIT1, IFIT4 mRNA in the SLE patients were significantly higher than those of the normal controls (P<0.01). The expression levels of IFIT1, IFIT4 mRNA in the active SLE patients were higher than those of the inactive SLE patients (P<0.05). The real time expression levels of IFIT1 and IFIT4 mRNA showed positive correlations with each other (P<0.05) in patients with SLE. There was positively correlation between the expression levels of IFIT1, IFIT4 mRNA and the anti-double strand DNA antibody (P<0.05). The expression levels of IFIT1, IFIT4 mRNA in patients with SLE were significantly higher than those of the normal controls, and positively associated with SLEDAI scores, so they were helpful in evaluating SLE disease activity and severity. To inhibit the expressions of IFIT1, IFIT4 mRNA may provide a novel target for SLE treatment. (authors)

Diversity of 23S rRNA genes within individual prokaryotic genomes.

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Anna Pei

Full Text Available BACKGROUND: The concept of ribosomal constraints on rRNA genes is deduced primarily based on the comparison of consensus rRNA sequences between closely related species, but recent advances in whole-genome sequencing allow evaluation of this concept within organisms with multiple rRNA operons. METHODOLOGY/PRINCIPAL FINDINGS: Using the 23S rRNA gene as an example, we analyzed the diversity among individual rRNA genes within a genome. Of 184 prokaryotic species containing multiple 23S rRNA genes, diversity was observed in 113 (61.4% genomes (mean 0.40%, range 0.01%-4.04%. Significant (1.17%-4.04% intragenomic variation was found in 8 species. In 5 of the 8 species, the diversity in the primary structure had only minimal effect on the secondary structure (stem versus loop transition. In the remaining 3 species, the diversity significantly altered local secondary structure, but the alteration appears minimized through complex rearrangement. Intervening sequences (IVS, ranging between 9 and 1471 nt in size, were found in 7 species. IVS in Deinococcus radiodurans and Nostoc sp. encode transposases. T. tengcongensis was the only species in which intragenomic diversity >3% was observed among 4 paralogous 23S rRNA genes. CONCLUSIONS/SIGNIFICANCE: These findings indicate tight ribosomal constraints on individual 23S rRNA genes within a genome. Although classification using primary 23S rRNA sequences could be erroneous, significant diversity among paralogous 23S rRNA genes was observed only once in the 184 species analyzed, indicating little overall impact on the mainstream of 23S rRNA gene-based prokaryotic taxonomy.

Functional characterization of endogenous siRNA target genes in Caenorhabditis elegans

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Heikkinen Liisa

2008-06-01

Full Text Available Abstract Background Small interfering RNA (siRNA molecules mediate sequence specific silencing in RNA interference (RNAi, a gene regulatory phenomenon observed in almost all organisms. Large scale sequencing of small RNA libraries obtained from C. elegans has revealed that a broad spectrum of siRNAs is endogenously transcribed from genomic sequences. The biological role and molecular diversity of C. elegans endogenous siRNA (endo-siRNA molecules, nonetheless, remain poorly understood. In order to gain insight into their biological function, we annotated two large libraries of endo-siRNA sequences, identified their cognate targets, and performed gene ontology analysis to identify enriched functional categories. Results Systematic trends in categorization of target genes according to the specific length of siRNA sequences were observed: 18- to 22-mer siRNAs were associated with genes required for embryonic development; 23-mers were associated uniquely with post-embryonic development; 24–26-mers were associated with phosphorus metabolism or protein modification. Moreover, we observe that some argonaute related genes associate with siRNAs with multiple reads. Sequence frequency graphs suggest that different lengths of siRNAs share similarities in overall sequence structure: the 5' end begins with G, while the body predominates with U and C. Conclusion These results suggest that the lengths of endogenous siRNA molecules are consequential to their biological functions since the gene ontology categories for their cognate mRNA targets vary depending upon their lengths.

Detecting microRNA activity from gene expression data

LENUS (Irish Health Repository)

Madden, Stephen F

2010-05-18

Abstract Background MicroRNAs (miRNAs) are non-coding RNAs that regulate gene expression by binding to the messenger RNA (mRNA) of protein coding genes. They control gene expression by either inhibiting translation or inducing mRNA degradation. A number of computational techniques have been developed to identify the targets of miRNAs. In this study we used predicted miRNA-gene interactions to analyse mRNA gene expression microarray data to predict miRNAs associated with particular diseases or conditions. Results Here we combine correspondence analysis, between group analysis and co-inertia analysis (CIA) to determine which miRNAs are associated with differences in gene expression levels in microarray data sets. Using a database of miRNA target predictions from TargetScan, TargetScanS, PicTar4way PicTar5way, and miRanda and combining these data with gene expression levels from sets of microarrays, this method produces a ranked list of miRNAs associated with a specified split in samples. We applied this to three different microarray datasets, a papillary thyroid carcinoma dataset, an in-house dataset of lipopolysaccharide treated mouse macrophages, and a multi-tissue dataset. In each case we were able to identified miRNAs of biological importance. Conclusions We describe a technique to integrate gene expression data and miRNA target predictions from multiple sources.

Detecting microRNA activity from gene expression data.

LENUS (Irish Health Repository)

Madden, Stephen F

2010-01-01

BACKGROUND: MicroRNAs (miRNAs) are non-coding RNAs that regulate gene expression by binding to the messenger RNA (mRNA) of protein coding genes. They control gene expression by either inhibiting translation or inducing mRNA degradation. A number of computational techniques have been developed to identify the targets of miRNAs. In this study we used predicted miRNA-gene interactions to analyse mRNA gene expression microarray data to predict miRNAs associated with particular diseases or conditions. RESULTS: Here we combine correspondence analysis, between group analysis and co-inertia analysis (CIA) to determine which miRNAs are associated with differences in gene expression levels in microarray data sets. Using a database of miRNA target predictions from TargetScan, TargetScanS, PicTar4way PicTar5way, and miRanda and combining these data with gene expression levels from sets of microarrays, this method produces a ranked list of miRNAs associated with a specified split in samples. We applied this to three different microarray datasets, a papillary thyroid carcinoma dataset, an in-house dataset of lipopolysaccharide treated mouse macrophages, and a multi-tissue dataset. In each case we were able to identified miRNAs of biological importance. CONCLUSIONS: We describe a technique to integrate gene expression data and miRNA target predictions from multiple sources.

Functional characterization of Pol III U6 promoters for gene knockdown and knockout in Plutella xylostella.

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Huang, Yuping; Wang, Yajun; Zeng, Baosheng; Liu, Zhaoxia; Xu, Xuejiao; Meng, Qian; Huang, Yongping; Yang, Guang; Vasseur, Liette; Gurr, Geoff M; You, Minsheng

2017-10-01

RNA polymerase type III (Pol-III) promoters such as U6 are commonly used to express small RNAs, including short hairpin RNAs (shRNAs) and single guide RNAs (sgRNAs). Functional U6 promoters are widely used in CRISPR systems, and their characterization can facilitate genome editing of non-model organisms. In the present study, six U6 small nuclear RNA (snRNA) promoters containing two conserved elements of a proximal sequence element (PSEA) and a TATA box, were identified and characterized in the diamondback moth (Plutella xylostella) genome. Relative efficiency of the U6 promoters to express shRNA induced EGFP knockdown was tested in a P. xylostella cell line, revealing that the PxU6:3 promoter had the strongest expression effect. Further work with the PxU6:3 promoter showed its efficacy in EGFP knockout using CRISPR/Cas9 system in the cells. The expression plasmids with versatile Pxabd-A gene specific sgRNA driven by the PxU6:3 promoter, combined with Cas9 mRNA, could induce mutagenesis at specific genomic loci in vivo. The phenotypes induced by sgRNA expression plasmids were similar to those done in vitro transcription sgRNAs. A plasmid with two tandem arranged PxU6:3:sgRNA expression cassettes targeting Pxabd-A loci was generated, which caused a 28,856 bp fragment deletion, suggesting that the multi-sgRNA expression plasmid can be used for multi-targeting. Our work indicates that U6 snRNA promoters can be used for functional studies of genes with the approach of reverse genetics in P. xylostella. These essential promoters also provide valuable potential for CRISPR-derived gene drive as a tactic for population control in this globally significant pest. Copyright © 2017 Elsevier Ltd. All rights reserved.

Nucleotide sequence of a human tRNA gene heterocluster

International Nuclear Information System (INIS)

Chang, Y.N.; Pirtle, I.L.; Pirtle, R.M.

1986-01-01

Leucine tRNA from bovine liver was used as a hybridization probe to screen a human gene library harbored in Charon-4A of bacteriophage lambda. The human DNA inserts from plaque-pure clones were characterized by restriction endonuclease mapping and Southern hybridization techniques, using both [3'- 32 P]-labeled bovine liver leucine tRNA and total tRNA as hybridization probes. An 8-kb Hind III fragment of one of these γ-clones was subcloned into the Hind III site of pBR322. Subsequent fine restriction mapping and DNA sequence analysis of this plasmid DNA indicated the presence of four tRNA genes within the 8-kb DNA fragment. A leucine tRNA gene with an anticodon of AAG and a proline tRNA gene with an anticodon of AGG are in a 1.6-kb subfragment. A threonine tRNA gene with an anticodon of UGU and an as yet unidentified tRNA gene are located in a 1.1-kb subfragment. These two different subfragments are separated by 2.8 kb. The coding regions of the three sequenced genes contain characteristic internal split promoter sequences and do not have intervening sequences. The 3'-flanking region of these three genes have typical RNA polymerase III termination sites of at least four consecutive T residues

Methylation of miRNA genes and oncogenesis.

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Loginov, V I; Rykov, S V; Fridman, M V; Braga, E A

2015-02-01

Interaction between microRNA (miRNA) and messenger RNA of target genes at the posttranscriptional level provides fine-tuned dynamic regulation of cell signaling pathways. Each miRNA can be involved in regulating hundreds of protein-coding genes, and, conversely, a number of different miRNAs usually target a structural gene. Epigenetic gene inactivation associated with methylation of promoter CpG-islands is common to both protein-coding genes and miRNA genes. Here, data on functions of miRNAs in development of tumor-cell phenotype are reviewed. Genomic organization of promoter CpG-islands of the miRNA genes located in inter- and intragenic areas is discussed. The literature and our own results on frequency of CpG-island methylation in miRNA genes from tumors are summarized, and data regarding a link between such modification and changed activity of miRNA genes and, consequently, protein-coding target genes are presented. Moreover, the impact of miRNA gene methylation on key oncogenetic processes as well as affected signaling pathways is discussed.

Down-Regulation of Gene Expression by RNA-Induced Gene Silencing

Science.gov (United States)

Travella, Silvia; Keller, Beat

Down-regulation of endogenous genes via post-transcriptional gene silencing (PTGS) is a key to the characterization of gene function in plants. Many RNA-based silencing mechanisms such as post-transcriptional gene silencing, co-suppression, quelling, and RNA interference (RNAi) have been discovered among species of different kingdoms (plants, fungi, and animals). One of the most interesting discoveries was RNAi, a sequence-specific gene-silencing mechanism initiated by the introduction of double-stranded RNA (dsRNA), homologous in sequence to the silenced gene, which triggers degradation of mRNA. Infection of plants with modified viruses can also induce RNA silencing and is referred to as virus-induced gene silencing (VIGS). In contrast to insertional mutagenesis, these emerging new reverse genetic approaches represent a powerful tool for exploring gene function and for manipulating gene expression experimentally in cereal species such as barley and wheat. We examined how RNAi and VIGS have been used to assess gene function in barley and wheat, including molecular mechanisms involved in the process and available methodological elements, such as vectors, inoculation procedures, and analysis of silenced phenotypes.

Gene expression profiles in BCL11B-siRNA treated malignant T cells

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Grabarczyk Piotr

2011-05-01

Full Text Available Abstract Background Downregulation of the B-cell chronic lymphocytic leukemia (CLL/lymphoma11B (BCL11B gene by small interfering RNA (siRNA leads to growth inhibition and apoptosis of the human T-cell acute lymphoblastic leukemia (T-ALL cell line Molt-4. To further characterize the molecular mechanism, a global gene expression profile of BCL11B-siRNA -treated Molt-4 cells was established. The expression profiles of several genes were further validated in the BCL11B-siRNA -treated Molt-4 cells and primary T-ALL cells. Results 142 genes were found to be upregulated and 109 genes downregulated in the BCL11B-siRNA -treated Molt-4 cells by microarray analysis. Among apoptosis-related genes, three pro-apoptotic genes, TNFSF10, BIK, BNIP3, were upregulated and one anti-apoptotic gene, BCL2L1 was downregulated. Moreover, the expression of SPP1 and CREBBP genes involved in the transforming growth factor (TGF-β pathway was down 16-fold. Expression levels of TNFSF10, BCL2L1, SPP1, and CREBBP were also examined by real-time PCR. A similar expression pattern of TNFSF10, BCL2L1, and SPP1 was identified. However, CREBBP was not downregulated in the BLC11B-siRNA -treated Molt-4 cells. Conclusion BCL11B-siRNA treatment altered expression profiles of TNFSF10, BCL2L1, and SPP1 in both Molt-4 T cell line and primary T-ALL cells.

dPORE-miRNA: Polymorphic regulation of microRNA genes

KAUST Repository

Schmeier, Sebastian; Schaefer, Ulf; MacPherson, Cameron R.; Bajic, Vladimir B.

2011-01-01

Background: MicroRNAs (miRNAs) are short non-coding RNA molecules that act as post-transcriptional regulators and affect the regulation of protein-coding genes. Mostly transcribed by PolII, miRNA genes are regulated at the transcriptional level similarly to protein-coding genes. In this study we focus on human miRNAs. These miRNAs are involved in a variety of pathways and can affect many diseases. Our interest is on possible deregulation of the transcription initiation of the miRNA encoding genes, which is facilitated by variations in the genomic sequence of transcriptional control regions (promoters). Methodology: Our aim is to provide an online resource to facilitate the investigation of the potential effects of single nucleotide polymorphisms (SNPs) on miRNA gene regulation. We analyzed SNPs overlapped with predicted transcription factor binding sites (TFBSs) in promoters of miRNA genes. We also accounted for the creation of novel TFBSs due to polymorphisms not present in the reference genome. The resulting changes in the original TFBSs and potential creation of new TFBSs were incorporated into the Dragon Database of Polymorphic Regulation of miRNA genes (dPORE-miRNA). Conclusions: The dPORE-miRNA database enables researchers to explore potential effects of SNPs on the regulation of miRNAs. dPORE-miRNA can be interrogated with regards to: a/miRNAs (their targets, or involvement in diseases, or biological pathways), b/SNPs, or c/transcription factors. dPORE-miRNA can be accessed at http://cbrc.kaust.edu.sa/dpore and http://apps.sanbi.ac.za/dpore/. Its use is free for academic and non-profit users. © 2011 Schmeier et al.

dPORE-miRNA: Polymorphic regulation of microRNA genes

KAUST Repository

Schmeier, Sebastian

2011-02-04

Background: MicroRNAs (miRNAs) are short non-coding RNA molecules that act as post-transcriptional regulators and affect the regulation of protein-coding genes. Mostly transcribed by PolII, miRNA genes are regulated at the transcriptional level similarly to protein-coding genes. In this study we focus on human miRNAs. These miRNAs are involved in a variety of pathways and can affect many diseases. Our interest is on possible deregulation of the transcription initiation of the miRNA encoding genes, which is facilitated by variations in the genomic sequence of transcriptional control regions (promoters). Methodology: Our aim is to provide an online resource to facilitate the investigation of the potential effects of single nucleotide polymorphisms (SNPs) on miRNA gene regulation. We analyzed SNPs overlapped with predicted transcription factor binding sites (TFBSs) in promoters of miRNA genes. We also accounted for the creation of novel TFBSs due to polymorphisms not present in the reference genome. The resulting changes in the original TFBSs and potential creation of new TFBSs were incorporated into the Dragon Database of Polymorphic Regulation of miRNA genes (dPORE-miRNA). Conclusions: The dPORE-miRNA database enables researchers to explore potential effects of SNPs on the regulation of miRNAs. dPORE-miRNA can be interrogated with regards to: a/miRNAs (their targets, or involvement in diseases, or biological pathways), b/SNPs, or c/transcription factors. dPORE-miRNA can be accessed at http://cbrc.kaust.edu.sa/dpore and http://apps.sanbi.ac.za/dpore/. Its use is free for academic and non-profit users. © 2011 Schmeier et al.

Multifunctional triblock co-polymer mP3/4HB-b-PEG-b-lPEI for efficient intracellular siRNA delivery and gene silencing.

Science.gov (United States)

Zhou, Li; Chen, Zhifei; Wang, Feifei; Yang, Xiuqun; Zhang, Biliang

2013-04-01

A non-viral siRNA carrier composed of mono-methoxy-poly (3-hydroxybutyrate-co-4-hydroxybutyrate)-block-polyethylene glycol-block-linear polyethyleneimine (mP3/4HB-b-PEG-b-lPEI) was synthesized using 1800 Da linear polyethyleneimine and evaluated for siRNA delivery. Our study demonstrated that siRNA could be efficiently combined with mP3/4HB-b-PEG-b-lPEI (mAG) co-polymer and was protected from nuclease degradation. The combined siRNA were released from the complexes easily under heparin competition. The particle size of the mAG/siRNA complexes was 158 nm, with a ζ-potential of around 28 mV. Atomic force microscopy images displayed spherical and homogeneously distributed complexes. The mAG block co-polymer displayed low cytotoxicity and efficient cellular uptake of Cy3-siRNA in A549 cells by flow cytometry and confocal microscopy. In vitro transfection efficiency of the block co-polymer was assessed using siRNA against luciferase in cultured A549-Luc, HeLa-Luc, HLF-Luc, A375-Luc and MCF-7-Luc cells. A higher transfection efficiency and lower cytotoxicity was obtained by mAG block co-polymer in five cell lines. Furthermore, a remarkable improvement in luciferase gene silencing efficiency of the mAG complex (up to 90-95%) over that of Lipofectamine™ 2000 (70-82%) was observed in HLF-Luc and A375-Luc cells. Additionally, a mAG/p65-siRNA complex also showed a better capability than Lipofectamine™ 2000/p65-siRNA complex to drastically reduce the p65 mRNA level down to 10-16% in HeLa, U251 and HUVEC cells at an N/P ratio of 70. Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.

Investigation of histone H4 hyperacetylation dynamics in the 5S rRNA genes family by chromatin immunoprecipitation assay.

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Burlibașa, Liliana; Suciu, Ilinca

2015-12-01

Oogenesis is a critical event in the formation of female gamete, whose role in development is to transfer genomic information to the next generation. During this process, the gene expression pattern changes dramatically concomitant with genome remodelling, while genomic information is stably maintained. The aim of the present study was to investigate the presence of H4 acetylation of the oocyte and somatic 5S rRNA genes in Triturus cristatus, using chromatin immunoprecipitation assay (ChIP). Our findings suggest that some epigenetic mechanisms such as histone acetylation could be involved in the transcriptional regulation of 5S rRNA gene families.

Genetic selection and DNA sequences of 4.5S RNA homologs

DEFF Research Database (Denmark)

Brown, S; Thon, G; Tolentino, E

1989-01-01

A general strategy for cloning the functional homologs of an Escherichia coli gene was used to clone homologs of 4.5S RNA from other bacteria. The genes encoding these homologs were selected by their ability to complement a deletion of the gene for 4.5S RNA. DNA sequences of the regions encoding...

Stable RNA interference of ErbB-2 gene synergistic with epirubicin suppresses breast cancer growth in vitro and in vivo

International Nuclear Information System (INIS)

Hu Xiaoqu; Su Fengxi; Qin Li; Jia Weijuan; Gong Chang; Yu Fengyan; Guo Jujiang; Song Erwei

2006-01-01

Overexpression of human epidermal growth factor receptor-2 (Her2, ErbB-2) contributes to the progression and metastasis of breast cancer, implying that Her2 gene is a suitable target of RNA interference (RNAi) for breast cancer therapy. Here, we employed plasmid-mediated expression of 2 different Her2-shRNAs (pU6-Her2shRNAs) efficiently silenced the target gene expression on Her2 expressing SKBR-3 breast cancer cells in both mRNA and protein levels. Consequently, pU6-Her2shRNA increased apoptosis and reduced proliferation of SKBR-3 cells assayed by TUNEL and MTT, respectively. In vivo, intra-tumor injection of pU6-Her2shRNA inhibited the growth of SKBR-3 tumors inoculated subcutaneously in nude mice. Furthermore, pU6-Her2shRNA synergized the tumor suppression effect of epirubicin to SKBR-3 cells in vitro and implanted subcutaneously in nude mice. Therefore, we concluded that stable silencing of Her2 gene expression with plasmid expressing shRNA may hold great promise as a novel therapy for Her2 expressing breast cancers alone or in combination with anthracycline chemotherapy

In vitro base modification of Escherichia coli glutamate 2 transfer-RNA and phenylalanine transfer-RNA gene transcripts

International Nuclear Information System (INIS)

Shahan, M.N.

1989-01-01

Plasmids were constructed that contain an E. Coli tRNA 2 Glu or tRNA Phe gene in a system transcribable by T7 or SP6 RNA polymerase. Selectively 32 P-labeled transcripts of these plasmids were used to study tRNA base modification in vitro in crude extracts by nearest neighbor analysis. The synthesis of 5-methyl-aminomethyl-2-thiouridine (mnm 5 s 2 U) was studied. Complete synthesis of mnm 5 s 2 2U is not observed. Instead, 2-thiouridine (s 2 U) is synthesized. Synthesis requires ATP, cysteine, Mg + , and monovalent cation concentrations below 50 mM. The reaction has a pH optimum above 7.0. Sulfide ion will substitute for cysteine in the reaction but sulfate, sulfite, methionine, homocysteine, and β-mercaptopyruvate will not. Extracts from E. coli strains carrying either the asuE or asuF mutations have reduced s 2 U synthetic activity which supports in vivo evidence that the wild type genes are involved in 2-thiolation of uridine. The enzyme is shown to be unstable both upon storage at -80 degree C and during the modification reaction. A method was developed to study the synthesis of any one of four pseudouridines ψ found at different positions of the tRNA cloverleaf. Synthesis of ψ is observed at three of the four positions-positions 32, 39, and 55. The asuC mutation is shown to affect ψ synthesis only at position 39 confirming that it is an allele of hisT and that the hisT mutations do not affect ψ synthesis at position 32 in E. coli. Synthesis of ψ32, ψ39, and ψ55 does not require any prior modification. Synthesis of dihydrouridine, 7-methylguanosine, and 3(3-amino-3-carboxypropyl)uridine is also observed. Synthesis of 2-methyladenosine and ψ 13 is not seen. Removal of part of the aminoacyl stem reduces synthesis of all modifications examined by 3' fold or more

RNA Chimeras as a Gene Signature of Breast Cancer

Science.gov (United States)

2013-05-01

www.plosone.org 11 August 2012 | Volume 7 | Issue 8 | e41659 Human genes Human ACTB mRNA: >gi|168480144|ref|NM_001101.3| Homo sapiens actin, beta...TCCCCCTTTTTTGTCCCCCAACTTGAGATGTATGAAGGCTTTTGGTCTCCCTGGGAGTGGGTGGAGGCAGCCAGGGCTTACCTGTACACTGACTTGAGACCAGTTGAATAAA AGTGCACACCTTAAAAATGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA Human GAPDH mRNA: >gi|83641890|ref|NM_002046.4| Homo sapiens ...Homo sapiens hypoxanthine phosphoribosyltransferase 1 (HPRT1), mRNA

Oligonucleotides targeting TCF4 triplet repeat expansion inhibit RNA foci and mis-splicing in Fuchs' dystrophy.

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Hu, Jiaxin; Rong, Ziye; Gong, Xin; Zhou, Zhengyang; Sharma, Vivek K; Xing, Chao; Watts, Jonathan K; Corey, David R; Mootha, V Vinod

2018-03-15

Fuchs' endothelial corneal dystrophy (FECD) is the most common repeat expansion disorder. FECD impacts 4% of U.S. population and is the leading indication for corneal transplantation. Most cases are caused by an expanded intronic CUG tract in the TCF4 gene that forms nuclear foci, sequesters splicing factors and impairs splicing. We investigated the sense and antisense RNA landscape at the FECD gene and find that the sense-expanded repeat transcript is the predominant species in patient corneas. In patient tissue, sense foci number were negatively correlated with age and showed no correlation with sex. Each endothelial cell has ∼2 sense foci and each foci is single RNA molecule. We designed antisense oligonucleotides (ASOs) to target the mutant-repetitive RNA and demonstrated potent inhibition of foci in patient-derived cells. Ex vivo treatment of FECD human corneas effectively inhibits foci and reverses pathological changes in splicing. FECD has the potential to be a model for treating many trinucleotide repeat diseases and targeting the TCF4 expansion with ASOs represents a promising therapeutic strategy to prevent and treat FECD.

Molecular phylogenetic studies on an unnamed bovine Babesia sp. based on small subunit ribosomal RNA gene sequences.

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Luo, Jianxun; Yin, Hong; Liu, Zhijie; Yang, Dongying; Guan, Guiquan; Liu, Aihong; Ma, Miling; Dang, Shengzhi; Lu, Bingyi; Sun, Caiqin; Bai, Qi; Lu, Wenshun; Chen, Puyan

2005-10-10

The 18S small subunit ribosomal RNA (18S rRNA) gene of an unnamed Babesia species (designated B. U sp.) was sequenced and analyzed in an attempt to distinguish it from other Babesia species in China. The target DNA segment was amplified by polymerase chain reaction (PCR). The PCR product was ligated to the pGEM-T Easy vector for sequencing. It was found that the length of the 18S rRNA gene of all B. U sp. Kashi 1 and B. U sp. Kashi 2 was 1699 bp and 1689 bp. Two phylogenetic trees were, respectively, inferred based on 18S rRNA sequence of the Chinese bovine Babesia isolates and all of Babesia species available in GenBank. The first tree showed that B. U sp. was situated in the branch between B. major Yili and B. bovis Shannxian, and the second tree revealed that B. U sp. was confined to the same group as B. caballi. The percent identity of B. U sp. with other Chinese Babesia species was between 74.2 and 91.8, while the percent identity between two B. U sp. isolates was 99.7. These results demonstrated that this B. U sp. is different from other Babesia species, but that two B. U sp. isolates obtained with nymphal and adultal Hyalomma anatolicum anatolicum tick belong to the same species.

Ribonucleoprotein organization of eukaryotic RNA. XXXII. U2 small nuclear RNA precursors and their accurate 3' processing in vitro as ribonucleoprotein particles.

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Wieben, E D; Nenninger, J M; Pederson, T

1985-05-05

Biosynthetic precursors of U2 small nuclear RNA have been identified in cultured human cells by hybrid-selection of pulse-labeled RNA with cloned U2 DNA. These precursor molecules are one to approximately 16 nucleotides longer than mature U2 RNA and contain 2,2,7-trimethylguanosine "caps". The U2 RNA precursors are associated with proteins that react with a monoclonal antibody for antigens characteristic of small nuclear ribonucleoprotein particles. Like previously described precursors of U1 and U4 small nuclear RNAs, the pre-U2 RNAs are recovered in cytoplasmic fractions, although it is not known if this is their location in vivo. The precursors are processed to mature-size U2 RNA when cytoplasmic extracts are incubated in vitro at 37 degrees C. Mg2+ is required but ATP is not. The ribonucleoprotein structure of the pre-U2 RNA is maintained during the processing reaction in vitro, as are the 2,2,7-trimethylguanosine caps. The ribonucleoprotein organization is of major importance, as exogenous, protein-free U2 RNA precursors are degraded rapidly in the in vitro system. Two lines of evidence indicate that the conversion of U2 precursors to mature-size U2 RNA involves a 3' processing reaction. First, the reaction is unaffected by a large excess of mature U2 small nuclear RNP, whose 5' trimethylguanosine caps would be expected to compete for a 5' processing activity. Second, when pre-U2 RNA precursors are first stoichiometrically decorated with an antibody specific for 2,2,7-trimethylguanosine, the extent of subsequent processing in vitro is unaffected. These results provide the first demonstration of a eukaryotic RNA processing reaction in vitro occurring within a ribonucleoprotein particle.

U6 snRNA expression prevents toxicity in TDP-43-knockdown cells.

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Masao Yahara

Full Text Available Depletion of amyotrophic lateral sclerosis (ALS-associated transactivation response (TAR RNA/DNA-binding protein 43 kDa (TDP-43 alters splicing efficiency of multiple transcripts and results in neuronal cell death. TDP-43 depletion can also disturb expression levels of small nuclear RNAs (snRNAs as spliceosomal components. Despite this knowledge, the relationship between cell death and alteration of snRNA expression during TDP-43 depletion remains unclear. Here, we knocked down TDP-43 in murine neuroblastoma Neuro2A cells and found a time lag between efficient TDP-43 depletion and appearance of cell death, suggesting that several mechanisms mediate between these two events. The amount of U6 snRNA was significantly decreased during TDP-43 depletion prior to increase of cell death, whereas that of U1, U2, and U4 snRNAs was not. Downregulation of U6 snRNA led to cell death, whereas transient exogenous expression of U6 snRNA counteracted the effect of TDP-43 knockdown on cell death, and slightly decreased the mis-splicing rate of Dnajc5 and Sortilin 1 transcripts, which are assisted by TDP-43. These results suggest that regulation of the U6 snRNA expression level by TDP-43 is a key factor in the increase in cell death upon TDP-43 loss-of-function.

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

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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.

Antisense targeting of 3' end elements involved in DUX4 mRNA processing is an efficient therapeutic strategy for facioscapulohumeral dystrophy: a new gene-silencing approach.

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Marsollier, Anne-Charlotte; Ciszewski, Lukasz; Mariot, Virginie; Popplewell, Linda; Voit, Thomas; Dickson, George; Dumonceaux, Julie

2016-04-15

Defects in mRNA 3'end formation have been described to alter transcription termination, transport of the mRNA from the nucleus to the cytoplasm, stability of the mRNA and translation efficiency. Therefore, inhibition of polyadenylation may lead to gene silencing. Here, we choose facioscapulohumeral dystrophy (FSHD) as a model to determine whether or not targeting key 3' end elements involved in mRNA processing using antisense oligonucleotide drugs can be used as a strategy for gene silencing within a potentially therapeutic context. FSHD is a gain-of-function disease characterized by the aberrant expression of the Double homeobox 4 (DUX4) transcription factor leading to altered pathogenic deregulation of multiple genes in muscles. Here, we demonstrate that targeting either the mRNA polyadenylation signal and/or cleavage site is an efficient strategy to down-regulate DUX4 expression and to decrease the abnormally high-pathological expression of genes downstream of DUX4. We conclude that targeting key functional 3' end elements involved in pre-mRNA to mRNA maturation with antisense drugs can lead to efficient gene silencing and is thus a potentially effective therapeutic strategy for at least FSHD. Moreover, polyadenylation is a crucial step in the maturation of almost all eukaryotic mRNAs, and thus all mRNAs are virtually eligible for this antisense-mediated knockdown strategy. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Thiol-linked alkylation of RNA to assess expression dynamics.

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Herzog, Veronika A; Reichholf, Brian; Neumann, Tobias; Rescheneder, Philipp; Bhat, Pooja; Burkard, Thomas R; Wlotzka, Wiebke; von Haeseler, Arndt; Zuber, Johannes; Ameres, Stefan L

2017-12-01

Gene expression profiling by high-throughput sequencing reveals qualitative and quantitative changes in RNA species at steady state but obscures the intracellular dynamics of RNA transcription, processing and decay. We developed thiol(SH)-linked alkylation for the metabolic sequencing of RNA (SLAM seq), an orthogonal-chemistry-based RNA sequencing technology that detects 4-thiouridine (s 4 U) incorporation in RNA species at single-nucleotide resolution. In combination with well-established metabolic RNA labeling protocols and coupled to standard, low-input, high-throughput RNA sequencing methods, SLAM seq enabled rapid access to RNA-polymerase-II-dependent gene expression dynamics in the context of total RNA. We validated the method in mouse embryonic stem cells by showing that the RNA-polymerase-II-dependent transcriptional output scaled with Oct4/Sox2/Nanog-defined enhancer activity, and we provide quantitative and mechanistic evidence for transcript-specific RNA turnover mediated by post-transcriptional gene regulatory pathways initiated by microRNAs and N 6 -methyladenosine. SLAM seq facilitates the dissection of fundamental mechanisms that control gene expression in an accessible, cost-effective and scalable manner.

Intrinsic noise of microRNA-regulated genes and the ceRNA hypothesis.

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Javad Noorbakhsh

Full Text Available MicroRNAs are small noncoding RNAs that regulate genes post-transciptionally by binding and degrading target eukaryotic mRNAs. We use a quantitative model to study gene regulation by inhibitory microRNAs and compare it to gene regulation by prokaryotic small non-coding RNAs (sRNAs. Our model uses a combination of analytic techniques as well as computational simulations to calculate the mean-expression and noise profiles of genes regulated by both microRNAs and sRNAs. We find that despite very different molecular machinery and modes of action (catalytic vs stoichiometric, the mean expression levels and noise profiles of microRNA-regulated genes are almost identical to genes regulated by prokaryotic sRNAs. This behavior is extremely robust and persists across a wide range of biologically relevant parameters. We extend our model to study crosstalk between multiple mRNAs that are regulated by a single microRNA and show that noise is a sensitive measure of microRNA-mediated interaction between mRNAs. We conclude by discussing possible experimental strategies for uncovering the microRNA-mRNA interactions and testing the competing endogenous RNA (ceRNA hypothesis.

Effect of Genetic Variability in the CYP4F2, CYP4F11, and CYP4F12 Genes on Liver mRNA Levels and Warfarin Response

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J. E. Zhang

2017-05-01

Full Text Available Genetic polymorphisms in the gene encoding cytochrome P450 (CYP 4F2, a vitamin K oxidase, affect stable warfarin dose requirements and time to therapeutic INR. CYP4F2 is part of the CYP4F gene cluster, which is highly polymorphic and exhibits a high degree of linkage disequilibrium, making it difficult to define causal variants. Our objective was to examine the effect of genetic variability in the CYP4F gene cluster on expression of the individual CYP4F genes and warfarin response. mRNA levels of the CYP4F gene cluster were quantified in human liver samples (n = 149 obtained from a well-characterized liver bank and fine mapping of the CYP4F gene cluster encompassing CYP4F2, CYP4F11, and CYP4F12 was performed. Genome-wide association study (GWAS data from a prospective cohort of warfarin-treated patients (n = 711 was also analyzed for genetic variations across the CYP4F gene cluster. In addition, SNP-gene expression in human liver tissues and interactions between CYP4F genes were explored in silico using publicly available data repositories. We found that SNPs in CYP4F2, CYP4F11, and CYP4F12 were associated with mRNA expression in the CYP4F gene cluster. In particular, CYP4F2 rs2108622 was associated with increased CYP4F2 expression while CYP4F11 rs1060467 was associated with decreased CYP4F2 expression. Interestingly, these CYP4F2 and CYP4F11 SNPs showed similar effects with warfarin stable dose where CYP4F11 rs1060467 was associated with a reduction in daily warfarin dose requirement (∼1 mg/day, Pc = 0.017, an effect opposite to that previously reported with CYP4F2 (rs2108622. However, inclusion of either or both of these SNPs in a pharmacogenetic algorithm consisting of age, body mass index (BMI, gender, baseline clotting factor II level, CYP2C9∗2 rs1799853, CYP2C9∗3 rs1057910, and VKORC1 rs9923231 improved warfarin dose variability only by 0.5–0.7% with an improvement in dose prediction accuracy of ∼1–2%. Although there is complex

Deletion analysis of the expression of rRNA genes and associated tRNA genes carried by a lambda transducing bacteriophage

International Nuclear Information System (INIS)

Morgan, E.A.; Nomura, M.

1979-01-01

Transducing phage lambda ilv5 carries genes for rRNA's, spacer tRNA's (tRNA 1 /sup Ile/ and tRNA/sub 1B//sup Ala/), and two other tRNA's (tRNA 1 /sup Asp/ and tRNA/sup Trp/). We have isolated a mutant of lambda ilv5, lambda ilv5su7, which carries an amber suppressor mutation in the tRNA/sup Trp/ gene. A series of deletion mutants were isolated from the lambda ilv5su7 phage. Genetic and biochemical analyses of these deletion mutants have confirmed our previous conclusion that the genes for tRNA 1 /sup Asp/ and tRNA/sup Trp/ located at the distal end of the rRNA operon (rrnC) are cotranscribed with other rRNA genes in that operon. In addition, these deletions were used to define roughly the physical location of the promoter(s) of the rRNA operon carried by the lambda ilv5su7 transducing phage

Trans-suppression of host CDH3 and LOXL4 genes during Cryptosporidium parvum infection involves nuclear delivery of parasite Cdg7_FLc_1000 RNA.

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Ming, Zhenping; Gong, Ai-Yu; Wang, Yang; Zhang, Xin-Tian; Li, Min; Li, Yao; Pang, Jing; Dong, Stephanie; Strauss-Soukup, Juliane K; Chen, Xian-Ming

2018-05-01

Intestinal infection by Cryptosporidium parvum causes significant alterations in the gene expression profile in host epithelial cells. Previous studies demonstrate that a panel of parasite RNA transcripts of low protein-coding potential are delivered into infected host cells and may modulate host gene transcription. Using in vitro models of human intestinal cryptosporidiosis, we report here that trans-suppression of the cadherin 3 (CDH3) and lysyl oxidase like 4 (LOXL4) genes in human intestinal epithelial cells following C. parvum infection involves host delivery of the Cdg7_FLc_1000 RNA, a C. parvum RNA that has been previously demonstrated to be delivered into the nuclei of infected host cells. Downregulation of CDH3 and LOXL4 genes was detected in host epithelial cells following C. parvum infection or in cells expressing the parasite Cdg7_FLc_1000 RNA. Knockdown of Cdg7_FLc_1000 attenuated the trans-suppression of CDH3 and LOXL4 genes in host cells induced by infection. Interestingly, Cdg7_FLc_1000 was detected to be recruited to the promoter regions of both CDH3 and LOXL4 gene loci in host cells following C. parvum infection. Host delivery of Cdg7_FLc_1000 promoted the PH domain zinc finger protein 1 (PRDM1)-mediated H3K9 methylation associated with trans-suppression in the CDH3 gene locus, but not the LOXL4 gene. Therefore, our data suggest that host delivery of Cdg7_FLc_1000 causes CDH3 trans-suppression in human intestinal epithelial cells following C. parvum infection through PRDM1-mediated H3K9 methylation in the CDH3 gene locus, whereas Cdg7_FLc_1000 induces trans-suppression of the host LOXL4 gene through H3K9/H3K27 methylation-independent mechanisms. Copyright © 2018 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.

Strategies for Improving siRNA-Induced Gene Silencing Efficiency.

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Safari, Fatemeh; Rahmani Barouji, Solmaz; Tamaddon, Ali Mohammad

2017-12-01

Purpose: Human telomerase reverse transcriptase (hTERT) plays a crucial role in tumorigenesis and progression of cancers. Gene silencing of hTERT by short interfering RNA (siRNA) is considered as a promising strategy for cancer gene therapy. Various algorithms have been devised for designing a high efficient siRNA which is a significant issue in the clinical usage. Thereby, in the present study, the relation of siRNA designing criteria and the gene silencing efficiency was evaluated. Methods: The siRNA sequences were designed and characterized by using on line soft wares. Cationic co-polymer (polyethylene glycol-g-polyethylene imine (PEG-g-PEI)) was used for the construction of polyelectrolyte complexes (PECs) containing siRNAs. The cellular uptake of the PECs was evaluated. The gene silencing efficiency of different siRNA sequences was investigated and the effect of observing the rational designing on the functionality of siRNAs was assessed. Results: The size of PEG-g-PEI siRNA with N/P (Nitrogen/Phosphate) ratio of 2.5 was 114 ± 0.645 nm. The transfection efficiency of PECs was desirable (95.5% ± 2.4%.). The results of Real-Time PCR showed that main sequence (MS) reduced the hTERT expression up to 90% and control positive sequence (CPS) up to 63%. These findings demonstrated that the accessibility to the target site has priority than the other criteria such as sequence preferences and thermodynamic features. Conclusion: siRNA opens a hopeful window in cancer therapy which provides a convenient and tolerable therapeutic approach. Thereby, using the set of criteria and rational algorithms in the designing of siRNA remarkably affect the gene silencing efficiency.

Novel RNA hybridization method for the in situ detection of ETV1, ETV4, and ETV5 gene fusions in prostate cancer.

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Kunju, Lakshmi P; Carskadon, Shannon; Siddiqui, Javed; Tomlins, Scott A; Chinnaiyan, Arul M; Palanisamy, Nallasivam

2014-09-01

The genetic basis of 50% to 60% of prostate cancer (PCa) is attributable to rearrangements in E26 transformation-specific (ETS) (ERG, ETV1, ETV4, and ETV5), BRAF, and RAF1 genes and overexpression of SPINK1. The development and validation of reliable detection methods are warranted to classify various molecular subtypes of PCa for diagnostic and prognostic purposes. ETS gene rearrangements are typically detected by fluorescence in situ hybridization and reverse-transcription polymerase chain reaction methods. Recently, monoclonal antibodies against ERG have been developed that detect the truncated ERG protein in immunohistochemical assays where staining levels are strongly correlated with ERG rearrangement status by fluorescence in situ hybridization. However, specific antibodies for ETV1, ETV4, and ETV5 are unavailable, challenging their clinical use. We developed a novel RNA in situ hybridization-based assay for the in situ detection of ETV1, ETV4, and ETV5 in formalin-fixed paraffin-embedded tissues from prostate needle biopsies, prostatectomy, and metastatic PCa specimens using RNA probes. Further, with combined RNA in situ hybridization and immunohistochemistry we identified a rare subset of PCa with dual ETS gene rearrangements in collisions of independent tumor foci. The high specificity and sensitivity of RNA in situ hybridization provides an alternate method enabling bright-field in situ detection of ETS gene aberrations in routine clinically available PCa specimens.

Small Interference RNA Targeting TLR4 Gene Effectively Attenuates Pulmonary Inflammation in a Rat Model

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Feixiang Wu

2012-01-01

Full Text Available Objective. The present study was to investigate the feasibility of adenovirus-mediated small interference RNA (siRNA targeting Toll-like receptor 4 (TLR4 gene in ameliorating lipopolysaccharide- (LPS- induced acute lung injury (ALI. Methods. In vitro, alveolar macrophages (AMs were treated with Ad-siTLR4 and Ad-EFGP, respectively, for 12 h, 24 h, and 48 h, and then with LPS (100 ng/mL for 2 h, and the function and expression of TLR4 were evaluated. In vivo, rats received intratracheal injection of 300 μL of normal saline (control group, 300 μL of Ad-EGFP (Ad-EGFP group, or 300 μL of Ad-siTLR4 (Ad-siTLR4 group and then were intravenously treated with LPS (50 mg/kg to induce ALI. Results. Ad-siTLR4 treatment significantly reduced TLR4 expression and production of proinflammatory cytokines following LPS treatment both in vitro and in vivo. Significant alleviation of tissue edema, microvascular protein leakage, and neutrophil infiltration was observed in the AdsiTLR4-treated animals. Conclusion. TLR4 plays a critical role in LPS-induced ALI, and transfection of Ad-siTLR4 can effectively downregulate TLR4 expression in vitro and in vivo, accompanied by alleviation of LPS-induced lung injury. These findings suggest that TLR4 may serve as a potential target in the treatment of ALI and RNA interfering targeting TLR4 expression represents a therapeutic strategy.

The hypoxic proteome is influenced by gene-specific changes in mRNA translation

International Nuclear Information System (INIS)

Koritzinsky, Marianne; Seigneuric, Renaud; Magagnin, Michael G.; Beucken, Twan van den; Lambin, Philippe; Wouters, Bradly G.

2005-01-01

Background and purpose: Hypoxia causes a rapid reduction in mRNA translation efficiency. This inhibition does not affect all mRNA species to the same extent and can therefore contribute significantly to hypoxia-induced differential protein expression. Our aim in this study was to characterize changes in gene expression during acute hypoxia and evaluate the contribution of regulation via mRNA translation on these changes. For each gene, the contribution of changes in mRNA abundance versus mRNA translation was determined. Materials and methods: DU145 prostate carcinoma cells were exposed to 4 h of hypoxia ( 2 ). Efficiently translated mRNAs were isolated by sedimentation through a sucrose gradient. Affymetrix microarray technology was used to evaluate both the transcriptional and translational contribution to gene expression. Results were validated by quantitative PCR. Results: One hundred and twenty genes were more than 4-fold upregulated by hypoxia in the efficiently translated fraction of mRNA, in comparison to only 76 genes at the level of transcription. Of the 50 genes demonstrating the largest changes in translation, 11 were found to be more than 2-fold over represented in the translated fraction in comparison to their overall transcriptional level. The gene with the highest translational contribution to its induction was CITED-2, which is a negative regulator of HIF-1 transcriptional activity. Conclusions: Gene-specific regulation of mRNA translation contributes significantly to differential gene expression during hypoxia

Using RNA-Seq Data to Evaluate Reference Genes Suitable for Gene Expression Studies in Soybean.

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Aldrin Kay-Yuen Yim

Full Text Available Differential gene expression profiles often provide important clues for gene functions. While reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR is an important tool, the validity of the results depends heavily on the choice of proper reference genes. In this study, we employed new and published RNA-sequencing (RNA-Seq datasets (26 sequencing libraries in total to evaluate reference genes reported in previous soybean studies. In silico PCR showed that 13 out of 37 previously reported primer sets have multiple targets, and 4 of them have amplicons with different sizes. Using a probabilistic approach, we identified new and improved candidate reference genes. We further performed 2 validation tests (with 26 RNA samples on 8 commonly used reference genes and 7 newly identified candidates, using RT-qPCR. In general, the new candidate reference genes exhibited more stable expression levels under the tested experimental conditions. The three newly identified candidate reference genes Bic-C2, F-box protein2, and VPS-like gave the best overall performance, together with the commonly used ELF1b. It is expected that the proposed probabilistic model could serve as an important tool to identify stable reference genes when more soybean RNA-Seq data from different growth stages and treatments are used.

Global Analysis of miRNA Gene Clusters and Gene Families Reveals Dynamic and Coordinated Expression

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Li Guo

2014-01-01

Full Text Available To further understand the potential expression relationships of miRNAs in miRNA gene clusters and gene families, a global analysis was performed in 4 paired tumor (breast cancer and adjacent normal tissue samples using deep sequencing datasets. The compositions of miRNA gene clusters and families are not random, and clustered and homologous miRNAs may have close relationships with overlapped miRNA species. Members in the miRNA group always had various expression levels, and even some showed larger expression divergence. Despite the dynamic expression as well as individual difference, these miRNAs always indicated consistent or similar deregulation patterns. The consistent deregulation expression may contribute to dynamic and coordinated interaction between different miRNAs in regulatory network. Further, we found that those clustered or homologous miRNAs that were also identified as sense and antisense miRNAs showed larger expression divergence. miRNA gene clusters and families indicated important biological roles, and the specific distribution and expression further enrich and ensure the flexible and robust regulatory network.

A fast, simple method for screening radiation susceptibility genes by RNA interference

International Nuclear Information System (INIS)

Tsuji, Atsushi B.; Sudo, Hitomi; Sugyo, Aya; Otsuki, Marika; Miyagishi, Makoto; Taira, Kazunari; Imai, Takashi; Harada, Yoshi-nobu

2005-01-01

Radiotherapy can cause unacceptable levels of damage to normal tissues in some cancer patients. To understand the molecular mechanisms underlying radiation-induced physiological responses, and to be able to predict the radiation susceptibility of normal tissues in individual patients, it is important to identify a comprehensive set of genes responsible for radiation susceptibility. We have developed a simple and rapid 96-well screening protocol using cell proliferation assays and RNA interference to identify genes associated with radiation susceptibility. We evaluated the performance of alamarBlue-, BrdU-, and sulforhodamine B-based cell proliferation assays using the 96-well format. Each proliferation assay detected the known radiation susceptibility gene, PRKDC. In a trial screen using 28 shRNA vectors, another known gene, CDKN1A, and one new radiation susceptibility gene, ATP5G3, were identified. Our results indicate that this method may be useful for large-scale screens designed to identify novel radiation susceptibility genes

Selection and Verification of Candidate Reference Genes for Mature MicroRNA Expression by Quantitative RT-PCR in the Tea Plant (Camellia sinensis

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Hui Song

2016-05-01

Full Text Available Quantitative reverse transcription-polymerase chain reaction (qRT-PCR is a rapid and sensitive method for analyzing microRNA (miRNA expression. However, accurate qRT-PCR results depend on the selection of reliable reference genes as internal positive controls. To date, few studies have identified reliable reference genes for differential expression analysis of miRNAs among tissues, and among experimental conditions in plants. In this study, three miRNAs and four non-coding small RNAs (ncRNA were selected as reference candidates, and the stability of their expression was evaluated among different tissues and under different experimental conditions in the tea plant (Camellia sinensis using the geNorm and NormFinder programs. It was shown that miR159a was the best single reference gene in the bud to the fifth leaf, 5S rRNA was the most suitable gene in different organs, miR6149 was the most stable gene when the leaves were attacked by Ectropis oblique and U4, miR5368n and miR159a were the best genes when the leaves were treated by methyl jasmonate (MeJA, salicylic acid (SA and abscisic acid (ABA, respectively. Our results provide suitable reference genes for future investigations on miRNA functions in tea plants.

Engineering and Validation of a Vector for Concomitant Expression of Rare Transfer RNA (tRNA and HIV-1 nef Genes in Escherichia coli.

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Siti Aisyah Mualif

Full Text Available Relative ease in handling and manipulation of Escherichia coli strains make them primary candidate to express proteins heterologously. Overexpression of heterologous genes that contain codons infrequently used by E. coli is related with difficulties such as mRNA instability, early termination of transcription and/or translation, deletions and/or misincorporation, and cell growth inhibition. These codon bias -associated problems are addressed by co-expressing ColE1-compatible, rare tRNA expressing helper plasmids. However, this approach has inadequacies, which we have addressed by engineering an expression vector that concomitantly expresses the heterologous protein of interest, and rare tRNA genes in E. coli. The expression vector contains three (argU, ileY, leuW rare tRNA genes and a useful multiple cloning site for easy in-frame cloning. To maintain the overall size of the parental plasmid vector, the rare tRNA genes replaced the non-essential DNA segments in the vector. The cloned gene is expressed under the control of T7 promoter and resulting recombinant protein has a C-terminal 6His tag for IMAC-mediated purification. We have evaluated the usefulness of this expression vector by expressing three HIV-1 genes namely HIV-1 p27 (nef, HIV-1 p24 (ca, and HIV-1 vif in NiCo21(DE3 E.coli and demonstrated the advantages of using expression vector that concomitantly expresses rare tRNA and heterologous genes.

Locus-specific ribosomal RNA gene silencing in nucleolar dominance.

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Michelle S Lewis

2007-08-01

Full Text Available The silencing of one parental set of rRNA genes in a genetic hybrid is an epigenetic phenomenon known as nucleolar dominance. We showed previously that silencing is restricted to the nucleolus organizer regions (NORs, the loci where rRNA genes are tandemly arrayed, and does not spread to or from neighboring protein-coding genes. One hypothesis is that nucleolar dominance is the net result of hundreds of silencing events acting one rRNA gene at a time. A prediction of this hypothesis is that rRNA gene silencing should occur independent of chromosomal location. An alternative hypothesis is that the regulatory unit in nucleolar dominance is the NOR, rather than each individual rRNA gene, in which case NOR localization may be essential for rRNA gene silencing. To test these alternative hypotheses, we examined the fates of rRNA transgenes integrated at ectopic locations. The transgenes were accurately transcribed in all independent transgenic Arabidopsis thaliana lines tested, indicating that NOR localization is not required for rRNA gene expression. Upon crossing the transgenic A. thaliana lines as ovule parents with A. lyrata to form F1 hybrids, a new system for the study of nucleolar dominance, the endogenous rRNA genes located within the A. thaliana NORs are silenced. However, rRNA transgenes escaped silencing in multiple independent hybrids. Collectively, our data suggest that rRNA gene activation can occur in a gene-autonomous fashion, independent of chromosomal location, whereas rRNA gene silencing in nucleolar dominance is locus-dependent.

Complex organisation and structure of the ghrelin antisense strand gene GHRLOS, a candidate non-coding RNA gene

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Herington Adrian C

2008-10-01

Full Text Available Abstract Background The peptide hormone ghrelin has many important physiological and pathophysiological roles, including the stimulation of growth hormone (GH release, appetite regulation, gut motility and proliferation of cancer cells. We previously identified a gene on the opposite strand of the ghrelin gene, ghrelinOS (GHRLOS, which spans the promoter and untranslated regions of the ghrelin gene (GHRL. Here we further characterise GHRLOS. Results We have described GHRLOS mRNA isoforms that extend over 1.4 kb of the promoter region and 106 nucleotides of exon 4 of the ghrelin gene, GHRL. These GHRLOS transcripts initiate 4.8 kb downstream of the terminal exon 4 of GHRL and are present in the 3' untranslated exon of the adjacent gene TATDN2 (TatD DNase domain containing 2. Interestingly, we have also identified a putative non-coding TATDN2-GHRLOS chimaeric transcript, indicating that GHRLOS RNA biogenesis is extremely complex. Moreover, we have discovered that the 3' region of GHRLOS is also antisense, in a tail-to-tail fashion to a novel terminal exon of the neighbouring SEC13 gene, which is important in protein transport. Sequence analyses revealed that GHRLOS is riddled with stop codons, and that there is little nucleotide and amino-acid sequence conservation of the GHRLOS gene between vertebrates. The gene spans 44 kb on 3p25.3, is extensively spliced and harbours multiple variable exons. We have also investigated the expression of GHRLOS and found evidence of differential tissue expression. It is highly expressed in tissues which are emerging as major sites of non-coding RNA expression (the thymus, brain, and testis, as well as in the ovary and uterus. In contrast, very low levels were found in the stomach where sense, GHRL derived RNAs are highly expressed. Conclusion GHRLOS RNA transcripts display several distinctive features of non-coding (ncRNA genes, including 5' capping, polyadenylation, extensive splicing and short open reading

Evaluation of gene expression data generated from expired Affymetrix GeneChip® microarrays using MAQC reference RNA samples

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Tong Weida

2010-10-01

Full Text Available Abstract Background The Affymetrix GeneChip® system is a commonly used platform for microarray analysis but the technology is inherently expensive. Unfortunately, changes in experimental planning and execution, such as the unavailability of previously anticipated samples or a shift in research focus, may render significant numbers of pre-purchased GeneChip® microarrays unprocessed before their manufacturer’s expiration dates. Researchers and microarray core facilities wonder whether expired microarrays are still useful for gene expression analysis. In addition, it was not clear whether the two human reference RNA samples established by the MAQC project in 2005 still maintained their transcriptome integrity over a period of four years. Experiments were conducted to answer these questions. Results Microarray data were generated in 2009 in three replicates for each of the two MAQC samples with either expired Affymetrix U133A or unexpired U133Plus2 microarrays. These results were compared with data obtained in 2005 on the U133Plus2 microarray. The percentage of overlap between the lists of differentially expressed genes (DEGs from U133Plus2 microarray data generated in 2009 and in 2005 was 97.44%. While there was some degree of fold change compression in the expired U133A microarrays, the percentage of overlap between the lists of DEGs from the expired and unexpired microarrays was as high as 96.99%. Moreover, the microarray data generated using the expired U133A microarrays in 2009 were highly concordant with microarray and TaqMan® data generated by the MAQC project in 2005. Conclusions Our results demonstrated that microarray data generated using U133A microarrays, which were more than four years past the manufacturer’s expiration date, were highly specific and consistent with those from unexpired microarrays in identifying DEGs despite some appreciable fold change compression and decrease in sensitivity. Our data also suggested that the

Species-independent MicroRNA Gene Discovery

KAUST Repository

Kamanu, Timothy K.

2012-01-01

and other incurable diseases such as autism and Alzheimer’s. Functional miRNAs are excised from hairpin-like sequences that are known as miRNA genes. There are about 21,000 known miRNA genes, most of which have been determined using experimental methods. mi

C to U RNA editing mediated by APOBEC1 requires RNA-binding protein RBM47.

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Fossat, Nicolas; Tourle, Karin; Radziewic, Tania; Barratt, Kristen; Liebhold, Doreen; Studdert, Joshua B; Power, Melinda; Jones, Vanessa; Loebel, David A F; Tam, Patrick P L

2014-08-01

Cytidine (C) to Uridine (U) RNA editing is a post-transcriptional modification that is accomplished by the deaminase APOBEC1 and its partnership with the RNA-binding protein A1CF. We identify and characterise here a novel RNA-binding protein, RBM47, that interacts with APOBEC1 and A1CF and is expressed in tissues where C to U RNA editing occurs. RBM47 can substitute for A1CF and is necessary and sufficient for APOBEC1-mediated editing in vitro. Editing is further impaired in Rbm47-deficient mutant mice. These findings suggest that RBM47 and APOBEC1 constitute the basic machinery for C to U RNA editing. © 2014 The Authors.

Impact of pEGFP mediated ING4 gene on growth of glioma U251 ...

African Journals Online (AJOL)

Jane

2011-06-22

Jun 22, 2011 ... Full Length Research Paper. Impact of pEGFP mediated ING4 gene on growth of glioma U251 cells and its potential molecular mechanism. Yuefei Deng*, Bingxi Lei and Yiying Zhao. Department of Neurosurgery, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou 510120, China.

Gene expression analysis of FABP4 in gastric cancer

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Abdulkarim Yasin Karim

2016-06-01

Full Text Available Purpose: Gastric cancer has high incidence and mortality rate in several countries and is still one of the most frequent and lethal disease. In this study, we aimed to determine diagnostic markers in gastric cancer by molecular techniques; include mRNA expression analysis of FABP4 gene. Fatty acid binding protein 4 (FABP4 gene encodes the fatty acid binding protein found in adipocytes. The protein encoded by FABP4 are a family of small, highly conserved, cytoplasmic proteins that bind long-chain fatty acids and other hydrophobic ligands. It is thought that FABPs roles include fatty acid uptake, transport, and metabolism. Material and Methods: Total RNA were extracted from paired tumor and normal tissues of 47 gastric cancer. The mRNA expression level of FABP4 was measured employing semi- quantitative reverse transcription- polymerase chain reaction (RT- PCR. Results: The mRNA expression level of FABP4 was significantly decreased (down- regulated. Conclusion: Down-regulation of FABP4 gene seems to occur at the initial steps of gastric cancer development. In order to confirm the relationship between the gastric tumor and FABP4 gene, further analysis like immunohistochemistry and epigenetc techniques are necessary. [Cukurova Med J 2016; 41(2.000: 248-252

miRNA-mediated 'tug-of-war' model reveals ceRNA propensity of genes in cancers.

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Swain, Arpit Chandan; Mallick, Bibekanand

2018-06-01

Competing endogenous RNA (ceRNA) are transcripts that cross-regulate each other at the post-transcriptional level by competing for shared microRNA response elements (MREs). These have been implicated in various biological processes impacting cell-fate decisions and diseases including cancer. There are several studies that predict possible ceRNA pairs by adopting various machine-learning and mathematical approaches; however, there is no method that enables us to gauge as well as compare the propensity of the ceRNA of a gene and precisely envisages which among a pair exerts a stronger pull on the shared miRNA pool. In this study, we developed a method that uses the 'tug of war of genes' concept to predict and quantify ceRNA potential of a gene for the shared miRNA pool in cancers based on a score represented by SoCeR (score of competing endogenous RNA). The method was executed on the RNA-Seq transcriptional profiles of genes and miRNA available at TCGA along with CLIP-supported miRNA-target sites to predict ceRNA in 32 cancer types which were validated with already reported cases. The proposed method can be used to determine the sequestering capability of the gene of interest as well as in ranking the probable ceRNA candidates of a gene. Finally, we developed standalone applications (SoCeR tool) to aid researchers in easier implementation of the method in analysing different data sets or diseases. © 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

mRNA/microRNA gene expression profile in microsatellite unstable colorectal cancer

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Calin George A

2007-08-01

Full Text Available Abstract Background Colorectal cancer develops through two main genetic instability pathways characterized by distinct pathologic features and clinical outcome. Results We investigated colon cancer samples (23 characterized by microsatellite stability, MSS, and 16 by high microsatellite instability, MSI-H for genome-wide expression of microRNA (miRNA and mRNA. Based on combined miRNA and mRNA gene expression, a molecular signature consisting of twenty seven differentially expressed genes, inclusive of 8 miRNAs, could correctly distinguish MSI-H versus MSS colon cancer samples. Among the differentially expressed miRNAs, various members of the oncogenic miR-17-92 family were significantly up-regulated in MSS cancers. The majority of protein coding genes were also up-regulated in MSS cancers. Their functional classification revealed that they were most frequently associated with cell cycle, DNA replication, recombination, repair, gastrointestinal disease and immune response. Conclusion This is the first report that indicates the existence of differences in miRNA expression between MSS versus MSI-H colorectal cancers. In addition, the work suggests that the combination of mRNA/miRNA expression signatures may represent a general approach for improving bio-molecular classification of human cancer.

Integration analysis of microRNA and mRNA paired expression profiling identifies deregulated microRNA-transcription factor-gene regulatory networks in ovarian endometriosis.

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Zhao, Luyang; Gu, Chenglei; Ye, Mingxia; Zhang, Zhe; Li, Li'an; Fan, Wensheng; Meng, Yuanguang

2018-01-22

The etiology and pathophysiology of endometriosis remain unclear. Accumulating evidence suggests that aberrant microRNA (miRNA) and transcription factor (TF) expression may be involved in the pathogenesis and development of endometriosis. This study therefore aims to survey the key miRNAs, TFs and genes and further understand the mechanism of endometriosis. Paired expression profiling of miRNA and mRNA in ectopic endometria compared with eutopic endometria were determined by high-throughput sequencing techniques in eight patients with ovarian endometriosis. Binary interactions and circuits among the miRNAs, TFs, and corresponding genes were identified by the Pearson correlation coefficients. miRNA-TF-gene regulatory networks were constructed using bioinformatic methods. Eleven selected miRNAs and TFs were validated by quantitative reverse transcription-polymerase chain reaction in 22 patients. Overall, 107 differentially expressed miRNAs and 6112 differentially expressed mRNAs were identified by comparing the sequencing of the ectopic endometrium group and the eutopic endometrium group. The miRNA-TF-gene regulatory network consists of 22 miRNAs, 12 TFs and 430 corresponding genes. Specifically, some key regulators from the miR-449 and miR-34b/c cluster, miR-200 family, miR-106a-363 cluster, miR-182/183, FOX family, GATA family, and E2F family as well as CEBPA, SOX9 and HNF4A were suggested to play vital regulatory roles in the pathogenesis of endometriosis. Integration analysis of the miRNA and mRNA expression profiles presents a unique insight into the regulatory network of this enigmatic disorder and possibly provides clues regarding replacement therapy for endometriosis.

Drosha regulates gene expression independently of RNA cleavage function

DEFF Research Database (Denmark)

Gromak, Natalia; Dienstbier, Martin; Macias, Sara

2013-01-01

Drosha is the main RNase III-like enzyme involved in the process of microRNA (miRNA) biogenesis in the nucleus. Using whole-genome ChIP-on-chip analysis, we demonstrate that, in addition to miRNA sequences, Drosha specifically binds promoter-proximal regions of many human genes in a transcription......-dependent manner. This binding is not associated with miRNA production or RNA cleavage. Drosha knockdown in HeLa cells downregulated nascent gene transcription, resulting in a reduction of polyadenylated mRNA produced from these gene regions. Furthermore, we show that this function of Drosha is dependent on its N......-terminal protein-interaction domain, which associates with the RNA-binding protein CBP80 and RNA Polymerase II. Consequently, we uncover a previously unsuspected RNA cleavage-independent function of Drosha in the regulation of human gene expression....

Short Hairpin RNA (shRNA): Design, Delivery, and Assessment of Gene Knockdown

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Moore, Chris B.; Guthrie, Elizabeth H.; Huang, Max Tze-Han; Taxman, Debra J.

2013-01-01

Shortly after the cellular mechanism of RNA interference (RNAi) was first described, scientists began using this powerful technique to study gene function. This included designing better methods for the successful delivery of small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs) into mammalian cells. While the simplest method for RNAi is the cytosolic delivery of siRNA oligonucleotides, this technique is limited to cells capable of transfection and is primarily utilized during transient in vitro studies. The introduction of shRNA into mammalian cells through infection with viral vectors allows for stable integration of shRNA and long-term knockdown of the targeted gene; however, several challenges exist with the implementation of this technology. Here we describe some well-tested protocols which should increase the chances of successful design, delivery, and assessment of gene knockdown by shRNA. We provide suggestions for designing shRNA targets and controls, a protocol for sequencing through the secondary structure of the shRNA hairpin structure, and protocols for packaging and delivery of shRNA lentiviral particles. Using real-time PCR and functional assays we demonstrate the successful knockdown of ASC, an inflammatory adaptor molecule. These studies demonstrate the practicality of including two shRNAs with different efficacies of knockdown to provide an additional level of control and to verify dose dependency of functional effects. Along with the methods described here, as new techniques and algorithms are designed in the future, shRNA is likely to include further promising application and continue to be a critical component of gene discovery. PMID:20387148

Changes in expression of the long noncoding RNA FMR4 associate with altered gene expression during differentiation of human neural precursor cells

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Veronica Julia Peschansky

2015-08-01

Full Text Available CGG repeat expansions in the Fragile X mental retardation 1 (FMR1 gene are responsible for a family of associated disorders characterized by either intellectual disability and autism (Fragile X Syndrome, FXS, or adult-onset neurodegeneration (Fragile X-associated Tremor/Ataxia Syndrome, FXTAS. However, the FMR1 locus is complex and encodes several long noncoding RNAs (lncRNAs, whose expression is altered by repeat expansion mutations.The role of these lncRNAs is thus far unknown; therefore we investigated the functionality of FMR4, which we previously identified. Full-length expansions of the FMR1 triplet repeat cause silencing of both FMR1 and FMR4, thus we are interested in potential loss-of-function that may add to phenotypic manifestation of FXS. Since the two transcripts do not exhibit cis-regulation of one another, we examined the potential for FMR4 to regulate target genes at distal genomic loci using gene expression microarrays. We identified FMR4-responsive genes, including the methyl-CpG-binding domain protein 4 (MBD4. Furthermore, we found that in differentiating human neural precursor cells (hNPCs, FMR4 expression is developmentally regulated in opposition to expression of both FMR1 (which is expected to share a bidirectional promoter with FMR4 and MBD4.We therefore propose that FMR4’s function is as a gene-regulatory lncRNA and that this transcript may function in normal development. Closer examination of FMR4 increases our understanding of the role of regulatory lncRNA and the consequences of FMR1 repeat expansions.

lncRNA-Induced Nucleosome Repositioning Reinforces Transcriptional Repression of rRNA Genes upon Hypotonic Stress

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Zhongliang Zhao

2016-03-01

Full Text Available The activity of rRNA genes (rDNA is regulated by pathways that target the transcription machinery or alter the epigenetic state of rDNA. Previous work has established that downregulation of rRNA synthesis in quiescent cells is accompanied by upregulation of PAPAS, a long noncoding RNA (lncRNA that recruits the histone methyltransferase Suv4-20h2 to rDNA, thus triggering trimethylation of H4K20 (H4K20me3 and chromatin compaction. Here, we show that upregulation of PAPAS in response to hypoosmotic stress does not increase H4K20me3 because of Nedd4-dependent ubiquitinylation and proteasomal degradation of Suv4-20h2. Loss of Suv4-20h2 enables PAPAS to interact with CHD4, a subunit of the chromatin remodeling complex NuRD, which shifts the promoter-bound nucleosome into the transcriptional “off” position. Thus, PAPAS exerts a “stress-tailored” dual function in rDNA silencing, facilitating either Suv4-20h2-dependent chromatin compaction or NuRD-dependent changes in nucleosome positioning.

mRNA expression of genes regulating lipid metabolism in ringed seals (Pusa hispida) from differently polluted areas

International Nuclear Information System (INIS)

Castelli, Martina Galatea; Rusten, Marte; Goksøyr, Anders; Routti, Heli

2014-01-01

Highlights: •Genes regulating lipid metabolism were studied in ringed seals. •We compared highly contaminated Baltic seals and less contaminated Svalbard seals. •mRNA expression of hepatic PPARγ was higher in the Baltic seals. •mRNA expression of adipose PPARγ target genes was higher in the Baltic seals. •Contaminant exposure may affect lipid metabolism in the Baltic ringed seals. -- Abstract: There is a growing concern about the ability of persistent organic pollutants (POPs) to influence lipid metabolism. Although POPs are found at high concentrations in some populations of marine mammals, for example in the ringed seal (Pusa hispida) from the Baltic Sea, little is known about the effects of POPs on their lipid metabolism. An optimal regulation of lipid metabolism is crucial for ringed seals during the fasting/molting season. This is a physiologically stressful period, during which they rely on the energy stored in their fat reserves. The mRNA expression levels for seven genes involved in lipid metabolism were analyzed in liver and/or blubber tissue from molting ringed seals from the polluted Baltic Sea and a less polluted reference location, Svalbard (Norway). mRNA expression of genes encoding peroxisome proliferator-activated receptors (PPAR) α and γ and their target genes acyl-coenzyme A oxidase 1 (ACOX1) and cluster of differentiation 36 (CD36) were analyzed in liver. mRNA expression level of genes encoding PPARβ, PPARγ and their target genes encoding fatty acid binding protein 4 (FABP4) and adiponectin (ADIPOQ) were measured in inner and middle blubber layers. In addition, we evaluated the influence of molting status on hepatic mRNA expression of genes encoding PPARs and their target genes in ringed seals from Svalbard. Our results show higher mRNA expression of genes encoding hepatic PPARγ and adipose PPARβ, FABP4, and ADIPOQ in the Baltic seals compared to the Svalbard seals. A positive relationship between mRNA expressions of genes

mRNA expression of genes regulating lipid metabolism in ringed seals (Pusa hispida) from differently polluted areas

Energy Technology Data Exchange (ETDEWEB)

Castelli, Martina Galatea [Norwegian Polar Institute, Fram Centre, 9296 Tromsø (Norway); University of Bergen, Department of Biology, 5020 Bergen (Norway); Rusten, Marte; Goksøyr, Anders [University of Bergen, Department of Biology, 5020 Bergen (Norway); Routti, Heli, E-mail: heli.routti@npolar.no [Norwegian Polar Institute, Fram Centre, 9296 Tromsø (Norway)

2014-01-15

Highlights: •Genes regulating lipid metabolism were studied in ringed seals. •We compared highly contaminated Baltic seals and less contaminated Svalbard seals. •mRNA expression of hepatic PPARγ was higher in the Baltic seals. •mRNA expression of adipose PPARγ target genes was higher in the Baltic seals. •Contaminant exposure may affect lipid metabolism in the Baltic ringed seals. -- Abstract: There is a growing concern about the ability of persistent organic pollutants (POPs) to influence lipid metabolism. Although POPs are found at high concentrations in some populations of marine mammals, for example in the ringed seal (Pusa hispida) from the Baltic Sea, little is known about the effects of POPs on their lipid metabolism. An optimal regulation of lipid metabolism is crucial for ringed seals during the fasting/molting season. This is a physiologically stressful period, during which they rely on the energy stored in their fat reserves. The mRNA expression levels for seven genes involved in lipid metabolism were analyzed in liver and/or blubber tissue from molting ringed seals from the polluted Baltic Sea and a less polluted reference location, Svalbard (Norway). mRNA expression of genes encoding peroxisome proliferator-activated receptors (PPAR) α and γ and their target genes acyl-coenzyme A oxidase 1 (ACOX1) and cluster of differentiation 36 (CD36) were analyzed in liver. mRNA expression level of genes encoding PPARβ, PPARγ and their target genes encoding fatty acid binding protein 4 (FABP4) and adiponectin (ADIPOQ) were measured in inner and middle blubber layers. In addition, we evaluated the influence of molting status on hepatic mRNA expression of genes encoding PPARs and their target genes in ringed seals from Svalbard. Our results show higher mRNA expression of genes encoding hepatic PPARγ and adipose PPARβ, FABP4, and ADIPOQ in the Baltic seals compared to the Svalbard seals. A positive relationship between mRNA expressions of genes

RNA polymerase II mediated transcription from the polymerase III promoters in short hairpin RNA expression vector

International Nuclear Information System (INIS)

Rumi, Mohammad; Ishihara, Shunji; Aziz, Monowar; Kazumori, Hideaki; Ishimura, Norihisa; Yuki, Takafumi; Kadota, Chikara; Kadowaki, Yasunori; Kinoshita, Yoshikazu

2006-01-01

RNA polymerase III promoters of human ribonuclease P RNA component H1, human U6, and mouse U6 small nuclear RNA genes are commonly used in short hairpin RNA (shRNA) expression vectors due their precise initiation and termination sites. During transient transfection of shRNA vectors, we observed that H1 or U6 promoters also express longer transcripts enough to express several reporter genes including firefly luciferase, green fluorescent protein EGFP, and red fluorescent protein JRed. Expression of such longer transcripts was augmented by upstream RNA polymerase II enhancers and completely inhibited by downstream polyA signal sequences. Moreover, the transcription of firefly luciferase from human H1 promoter was sensitive to RNA polymerase II inhibitor α-amanitin. Our findings suggest that commonly used polymerase III promoters in shRNA vectors are also prone to RNA polymerase II mediated transcription, which may have negative impacts on their targeted use

HIV-1 resistance conferred by siRNA cosuppression of CXCR4 and CCR5 coreceptors by a bispecific lentiviral vector

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Akkina Ramesh

2005-01-01

Full Text Available Abstract Background RNA interference (RNAi mediated by small interfering RNAs (siRNAs has proved to be a highly effective gene silencing mechanism with great potential for HIV/AIDS gene therapy. Previous work with siRNAs against cellular coreceptors CXCR4 and CCR5 had shown that down regulation of these surface molecules could prevent HIV-1 entry and confer viral resistance. Since monospecific siRNAs targeting individual coreceptors are inadequate in protecting against both T cell tropic (X4 and monocyte tropic (R5 viral strains simultaneously, bispecific constructs with dual specificity are required. For effective long range therapy, the bispecific constructs need to be stably transduced into HIV-1 target cells via integrating viral vectors. Results To achieve this goal, lentiviral vectors incorporating both CXCR4 and CCR5 siRNAs of short hairpin design were constructed. The CXCR4 siRNA was driven by a U6 promoter whereas the CCR5 siRNA was driven by an H1 promoter. A CMV promoter driven EGFP reporter gene is also incorporated in the bispecific construct. High efficiency transduction into coreceptor expressing Magi and Ghost cell lines with a concomitant down regulation of respective coreceptors was achieved with lentiviral vectors. When the siRNA expressing transduced cells were challenged with X4 and R5 tropic HIV-1, they demonstrated marked viral resistance. HIV-1 resistance was also observed in bispecific lentiviral vector transduced primary PBMCs. Conclusions Both CXCR4 and CCR5 coreceptors could be simultaneously targeted for down regulation by a single combinatorial lentiviral vector incorporating respective anti-coreceptor siRNAs. Stable down regulation of both the coreceptors protects cells against infection by both X4 and R5 tropic HIV-1. Stable down regulation of cellular molecules that aid in HIV-1 infection will be an effective strategy for long range HIV gene therapy.

Re-editing the paradigm of Cytidine (C) to Uridine (U) RNA editing.

Science.gov (United States)

Fossat, Nicolas; Tam, Patrick P L

2014-01-01

Cytidine (C) to Uridine (U) RNA editing is a post-trancriptional modification that until recently was known to only affect Apolipoprotein b (Apob) RNA and minimally require 2 components of the C to U editosome, the deaminase APOBEC1 and the RNA-binding protein A1CF. Our latest work has identified a novel RNA-binding protein, RBM47, as a core component of the editosome, which can substitute A1CF for the editing of ApoB mRNA. In addition, new RNA species that are subjected to C to U editing have been identified. Here, we highlight these recent discoveries and discuss how they change our view of the composition of the C to U editing machinery and expand our knowledge of the functional attributes of C to U RNA editing.

An RNA polymerase II-and AGO4-associated protein acts in RNA-directed DNA methylation

KAUST Repository

Gao, Zhihuan

2010-04-21

DNA methylation is an important epigenetic mark in many eukaryotes. In plants, 24-nucleotide small interfering RNAs (siRNAs) bound to the effector protein, Argonaute 4 (AGO4), can direct de novo DNA methylation by the methyltransferase DRM2 (refs 2, 4-6). Here we report a new regulator of RNA-directed DNA methylation (RdDM) in Arabidopsis: RDM1. Loss-of-function mutations in the RDM1 gene impair the accumulation of 24-nucleotide siRNAs, reduce DNA methylation, and release transcriptional gene silencing at RdDM target loci. RDM1 encodes a small protein that seems to bind single-stranded methyl DNA, and associates and co-localizes with RNA polymerase II (Pol II, also known as NRPB), AGO4 and DRM2 in the nucleus. Our results indicate that RDM1 is a component of the RdDM effector complex and may have a role in linking siRNA production with pre-existing or de novo cytosine methylation. Our results also indicate that, although RDM1 and Pol V (also known as NRPE) may function together at some RdDM target sites in the peri-nucleolar siRNA processing centre, Pol II rather than Pol V is associated with the RdDM effector complex at target sites in the nucleoplasm. © 2010 Macmillan Publishers Limited. All rights reserved.

DNA sequence analysis of the mutational specificity of u.v. light in the SUP4-o gene of yeast

International Nuclear Information System (INIS)

Kunz, B.A.; Mis, J.R.A.; Pierce, M.K.; Giroux, C.N.

1987-01-01

Mutations induced in the SUP4-o gene of Saccharomyces cerevisiae by u.v. irradiation have been characterized. DNA sequence analysis of 120 mutants revealed that u.v. induced all types of base substitutions, although transitions, in particular G:C → A:T events predominated. In addition, a small number of single base pair deletions and double mutations, occurring in tandem or separated by a few base pairs, were recovered. The base pair substitutions were not distributed randomly in the SUP4-o gene and, with one exception, were all located at sites of adjacent pyrimidines, suggesting they were targeted by u.v. photolesions. A substantial fraction of the mutations were detected at hotspots for u.v. mutagenesis. The majority of changes occurred at the 3' base of dipyrimidine sequences where both cyclobutane dimers and [6-4]-photoproducts could form. Approximately one-third of the induced base substitutions were found at potential pyrimidine dimer sites where [6-4]-photoproducts would be expected to occur rarely. Possible origins of the induced mutations and the role of cyclobutane dimers as premutational u.v. lesions in yeast are considered. (author)

Novel sequence variations in LAMA2 and SGCG genes modulating cis-acting regulatory elements and RNA secondary structure

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Olfa Siala

2010-01-01

Full Text Available In this study, we detected new sequence variations in LAMA2 and SGCG genes in 5 ethnic populations, and analysed their effect on enhancer composition and mRNA structure. PCR amplification and DNA sequencing were performed and followed by bioinformatics analyses using ESEfinder as well as MFOLD software. We found 3 novel sequence variations in the LAMA2 (c.3174+22_23insAT and c.6085 +12delA and SGCG (c.*102A/C genes. These variations were present in 210 tested healthy controls from Tunisian, Moroccan, Algerian, Lebanese and French populations suggesting that they represent novel polymorphisms within LAMA2 and SGCG genes sequences. ESEfinder showed that the c.*102A/C substitution created a new exon splicing enhancer in the 3'UTR of SGCG genes, whereas the c.6085 +12delA deletion was situated in the base pairing region between LAMA2 mRNA and the U1snRNA spliceosomal components. The RNA structure analyses showed that both variations modulated RNA secondary structure. Our results are suggestive of correlations between mRNA folding and the recruitment of spliceosomal components mediating splicing, including SR proteins. The contribution of common sequence variations to mRNA structural and functional diversity will contribute to a better study of gene expression.

A viral microRNA down-regulates multiple cell cycle genes through mRNA 5'UTRs.

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Finn Grey

2010-06-01

Full Text Available Global gene expression data combined with bioinformatic analysis provides strong evidence that mammalian miRNAs mediate repression of gene expression primarily through binding sites within the 3' untranslated region (UTR. Using RNA induced silencing complex immunoprecipitation (RISC-IP techniques we have identified multiple cellular targets for a human cytomegalovirus (HCMV miRNA, miR-US25-1. Strikingly, this miRNA binds target sites primarily within 5'UTRs, mediating significant reduction in gene expression. Intriguingly, many of the genes targeted by miR-US25-1 are associated with cell cycle control, including cyclin E2, BRCC3, EID1, MAPRE2, and CD147, suggesting that miR-US25-1 is targeting genes within a related pathway. Deletion of miR-US25-1 from HCMV results in over expression of cyclin E2 in the context of viral infection. Our studies demonstrate that a viral miRNA mediates translational repression of multiple cellular genes by targeting mRNA 5'UTRs.

Antiviral RNA silencing initiated in the absence of RDE-4, a double-stranded RNA binding protein, in Caenorhabditis elegans.

Science.gov (United States)

Guo, Xunyang; Zhang, Rui; Wang, Jeffrey; Lu, Rui

2013-10-01

Small interfering RNAs (siRNAs) processed from double-stranded RNA (dsRNA) of virus origins mediate potent antiviral defense through a process referred to as RNA interference (RNAi) or RNA silencing in diverse organisms. In the simple invertebrate Caenorhabditis elegans, the RNAi process is initiated by a single Dicer, which partners with the dsRNA binding protein RDE-4 to process dsRNA into viral siRNAs (viRNAs). Notably, in C. elegans this RNA-directed viral immunity (RDVI) also requires a number of worm-specific genes for its full antiviral potential. One such gene is rsd-2 (RNAi spreading defective 2), which was implicated in RDVI in our previous studies. In the current study, we first established an antiviral role by showing that rsd-2 null mutants permitted higher levels of viral RNA accumulation, and that this enhanced viral susceptibility was reversed by ectopic expression of RSD-2. We then examined the relationship of rsd-2 with other known components of RNAi pathways and established that rsd-2 functions in a novel pathway that is independent of rde-4 but likely requires the RNA-dependent RNA polymerase RRF-1, suggesting a critical role for RSD-2 in secondary viRNA biogenesis, likely through coordinated action with RRF-1. Together, these results suggest that RDVI in the single-Dicer organism C. elegans depends on the collective actions of both RDE-4-dependent and RDE-4-independent mechanisms to produce RNAi-inducing viRNAs. Our study reveals, for the first time, a novel siRNA-producing mechanism in C. elegans that bypasses the need for a dsRNA-binding protein.

In silico analysis of miRNA-mediated gene regulation in OCA and OA genes.

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Kamaraj, Balu; Gopalakrishnan, Chandrasekhar; Purohit, Rituraj

2014-12-01

Albinism is an autosomal recessive genetic disorder due to low secretion of melanin. The oculocutaneous albinism (OCA) and ocular albinism (OA) genes are responsible for melanin production and also act as a potential targets for miRNAs. The role of miRNA is to inhibit the protein synthesis partially or completely by binding with the 3'UTR of the mRNA thus regulating gene expression. In this analysis, we predicted the genetic variation that occurred in 3'UTR of the transcript which can be a reason for low melanin production thus causing albinism. The single nucleotide polymorphisms (SNPs) in 3'UTR cause more new binding sites for miRNA which binds with mRNA which leads to inhibit the translation process either partially or completely. The SNPs in the mRNA of OCA and OA genes can create new binding sites for miRNA which may control the gene expression and lead to hypopigmentation. We have developed a computational procedure to determine the SNPs in the 3'UTR region of mRNA of OCA (TYR, OCA2, TYRP1 and SLC45A2) and OA (GPR143) genes which will be a potential cause for albinism. We identified 37 SNPs in five genes that are predicted to create 87 new binding sites on mRNA, which may lead to abrogation of the translation process. Expression analysis confirms that these genes are highly expressed in skin and eye regions. It is well supported by enrichment analysis that these genes are mainly involved in eye pigmentation and melanin biosynthesis process. The network analysis also shows how the genes are interacting and expressing in a complex network. This insight provides clue to wet-lab researches to understand the expression pattern of OCA and OA genes and binding phenomenon of mRNA and miRNA upon mutation, which is responsible for inhibition of translation process at genomic levels.

Concerted evolution of the tandemly repeated genes encoding primate U2 small nuclear RNA (the RNU2 locus) does not prevent rapid diversification of the (CT){sub n} {center_dot} (GA){sub n} microsatellite embedded within the U2 repeat unit

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Liao, D.; Weiner, A.M. [Yale Univ., New Haven, CT (United States)

1995-12-10

The RNU2 locus encoding human U2 small nuclear RNA (snRNA) is organized as a nearly perfect tandem array containing 5 to 22 copies of a 5.8-kb repeat unit. Just downstream of the U2 snRNA gene in each 5.8-kb repeat unit lies a large (CT){sub n}{center_dot}(GA){sub n} dinucleotide repeat (n {approx} 70). This form of genomic organization, in which one repeat is embedded within another, provides an unusual opportunity to study the balance of forces maintaining the homogeneity of both kinds of repeats. Using a combination of field inversion gel electrophoresis and polymerase chain reaction, we have been able to study the CT microsatellites within individual U2 tandem arrays. We find that the CT microsatellites within an RNU2 allele exhibit significant length polymorphism, despite the remarkable homogeneity of the surrounding U2 repeat units. Length polymorphism is due primarily to loss or gain of CT dinucleotide repeats, but other types of deletions, insertions, and substitutions are also frequent. Polymorphism is greatly reduced in regions where pure (CT){sub n} tracts are interrupted by occasional G residues, suggesting that irregularities stabilize both the length and the sequence of the dinucleotide repeat. We further show that the RNU2 loci of other catarrhine primates (gorilla, chimpanzee, ogangutan, and baboon) contain orthologous CT microsatellites; these also exhibit length polymorphism, but are highly divergent from each other. Thus, although the CT microsatellite is evolving far more rapidly than the rest of the U2 repeat unit, it has persisted through multiple speciation events spanning >35 Myr. The persistence of the CT microsatellite, despite polymorphism and rapid evolution, suggests that it might play a functional role in concerted evolution of the RNU2 loci, perhaps as an initiation site for recombination and/or gene conversion. 70 refs., 5 figs.

A genome-wide characterization of microRNA genes in maize.

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Lifang Zhang

2009-11-01

Full Text Available MicroRNAs (miRNAs are small, non-coding RNAs that play essential roles in plant growth, development, and stress response. We conducted a genome-wide survey of maize miRNA genes, characterizing their structure, expression, and evolution. Computational approaches based on homology and secondary structure modeling identified 150 high-confidence genes within 26 miRNA families. For 25 families, expression was verified by deep-sequencing of small RNA libraries that were prepared from an assortment of maize tissues. PCR-RACE amplification of 68 miRNA transcript precursors, representing 18 families conserved across several plant species, showed that splice variation and the use of alternative transcriptional start and stop sites is common within this class of genes. Comparison of sequence variation data from diverse maize inbred lines versus teosinte accessions suggest that the mature miRNAs are under strong purifying selection while the flanking sequences evolve equivalently to other genes. Since maize is derived from an ancient tetraploid, the effect of whole-genome duplication on miRNA evolution was examined. We found that, like protein-coding genes, duplicated miRNA genes underwent extensive gene-loss, with approximately 35% of ancestral sites retained as duplicate homoeologous miRNA genes. This number is higher than that observed with protein-coding genes. A search for putative miRNA targets indicated bias towards genes in regulatory and metabolic pathways. As maize is one of the principal models for plant growth and development, this study will serve as a foundation for future research into the functional roles of miRNA genes.

Evolutionary relationships between miRNA genes and their activity.

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Zhu, Yan; Skogerbø, Geir; Ning, Qianqian; Wang, Zhen; Li, Biqing; Yang, Shuang; Sun, Hong; Li, Yixue

2012-12-22

The emergence of vertebrates is characterized by a strong increase in miRNA families. MicroRNAs interact broadly with many transcripts, and the evolution of such a system is intriguing. However, evolutionary questions concerning the origin of miRNA genes and their subsequent evolution remain unexplained. In order to systematically understand the evolutionary relationship between miRNAs gene and their function, we classified human known miRNAs into eight groups based on their evolutionary ages estimated by maximum parsimony method. New miRNA genes with new functional sequences accumulated more dynamically in vertebrates than that observed in Drosophila. Different levels of evolutionary selection were observed over miRNA gene sequences with different time of origin. Most genic miRNAs differ from their host genes in time of origin, there is no particular relationship between the age of a miRNA and the age of its host genes, genic miRNAs are mostly younger than the corresponding host genes. MicroRNAs originated over different time-scales are often predicted/verified to target the same or overlapping sets of genes, opening the possibility of substantial functional redundancy among miRNAs of different ages. Higher degree of tissue specificity and lower expression level was found in young miRNAs. Our data showed that compared with protein coding genes, miRNA genes are more dynamic in terms of emergence and decay. Evolution patterns are quite different between miRNAs of different ages. MicroRNAs activity is under tight control with well-regulated expression increased and targeting decreased over time. Our work calls attention to the study of miRNA activity with a consideration of their origin time.

MiR-30e suppresses proliferation of hepatoma cells via targeting prolyl 4-hydroxylase subunit alpha-1 (P4HA1) mRNA

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Feng, Guoxing [State Key Laboratory of Medicinal Chemical Biology, Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin (China); Shi, Hui [State Key Laboratory of Medicinal Chemical Biology, Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin (China); Li, Jiong; Yang, Zhe [State Key Laboratory of Medicinal Chemical Biology, Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin (China); Fang, Runping; Ye, Lihong [State Key Laboratory of Medicinal Chemical Biology, Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin (China); Zhang, Weiying, E-mail: zhwybao@nankai.edu.cn [State Key Laboratory of Medicinal Chemical Biology, Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin (China); Zhang, Xiaodong, E-mail: zhangxd@nankai.edu.cn [State Key Laboratory of Medicinal Chemical Biology, Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin (China)

2016-04-08

Aberrant microRNA expression has been shown to be characteristic of many cancers. It has been reported that the expression levels of miR-30e are decreased in liver cancer tissues. However, the role of miR-30e in hepatocellular carcinoma remains poorly understood. In the present study, we investigated the significance of miR-30e in hepatocarcinogenesis. Bioinformatics analysis reveals a putative target site of miR-30e in the 3′-untranslated region (3′UTR) of prolyl 4-hydroxylase subunit alpha-1 (P4HA1) mRNA. Moreover, luciferase reporter gene assays verified that miR-30e directly targeted 3′UTR of P4HA1 mRNA. Then, we demonstrated that miR-30e was able to reduce the expression of P4HA1 at the levels of mRNA and protein using reverse transcription-polymerase chain reaction and Western blot analysis. Enforced expression of miR-30e suppressed proliferation of HepG2 cells by 5-ethynyl-2-deoxyuridine (EdU) assay and reduced colony formation of these cells by colony formation analysis. Conversely, anti-miR-30e enhanced the proliferation of hepatoma cells in vitro. Interestingly, the ectopic expression of P4HA1 could efficiently rescue the inhibition of cell proliferation mediated by miR-30e in HepG2 cells. Meanwhile, silencing of P4HA1 abolished the anti-miR-30e-induced proliferation of cells. Clinically, quantitative real-time PCR showed that miR-30e was down-regulated in liver tumor tissues relative to their peritumor tissues. The expression levels of miR-30e were negatively correlated to those of P4HA1 mRNA in clinical liver tumor tissues. Thus, we conclude that miR-30e suppresses proliferation of hepatoma cells through targeting P4HA1 mRNA. Our finding provides new insights into the mechanism of hepatocarcinogenesis. - Highlights: • P4HA1 is a novel target gene of miR-30e. • P4HA1 is increased in clinical HCC tissues. • MiR-30e is negatively correlated with P4HA1 in clinical HCC tissues. • MiR-30e suppresses the proliferation of HCC cells through

Transcription factor trapping by RNA in gene regulatory elements.

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Sigova, Alla A; Abraham, Brian J; Ji, Xiong; Molinie, Benoit; Hannett, Nancy M; Guo, Yang Eric; Jangi, Mohini; Giallourakis, Cosmas C; Sharp, Phillip A; Young, Richard A

2015-11-20

Transcription factors (TFs) bind specific sequences in promoter-proximal and -distal DNA elements to regulate gene transcription. RNA is transcribed from both of these DNA elements, and some DNA binding TFs bind RNA. Hence, RNA transcribed from regulatory elements may contribute to stable TF occupancy at these sites. We show that the ubiquitously expressed TF Yin-Yang 1 (YY1) binds to both gene regulatory elements and their associated RNA species across the entire genome. Reduced transcription of regulatory elements diminishes YY1 occupancy, whereas artificial tethering of RNA enhances YY1 occupancy at these elements. We propose that RNA makes a modest but important contribution to the maintenance of certain TFs at gene regulatory elements and suggest that transcription of regulatory elements produces a positive-feedback loop that contributes to the stability of gene expression programs. Copyright © 2015, American Association for the Advancement of Science.

Functional gene silencing mediated by chitosan/siRNA nanocomplexes

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Ji, A M; Su, D; Che, O; Li, W S; Sun, L; Zhang, Z Y; Xu, F [Department of Pharmaceutical Science, Zhujiang Hospital, Southern Medical University, Guangzhou 510282 (China); Yang, B, E-mail: andrewfxu1998@gmail.co [Department of Chemistry, Indiana University-Bloomington, Bloomington, IN 47405 (United States)

2009-10-07

Chitosan/siRNA nanoparticles to knock down FHL2 gene expression were reported in this work. The physicochemical properties such as particle size, surface charge, morphology and complex stability of chitosan nanoparticle-incorporated siRNA were evaluated. Nanoparticles which were formulated with chitosan/siRNA exhibited irregular, lamellar and dendritic structures with a hydrodynamic radius size of about 148 nm and net positive charges with zeta-potential value of 58.5 mV. The knockdown effect of the chitosan/siRNA nanoparticles on gene expression in FHL2 over-expressed human colorectal cancer Lovo cells was investigated. The result showed that FHL2 siRNA formulated within chitosan nanoparticles could knock down about 69.6% FHL2 gene expression, which is very similar to the 68.8% reduced gene expression when siRNA was transfected with liposome Lipofectamine. Western analysis further showed significant FHL-2 protein expression reduced by the chitosan/siRNA nanoparticles. The results also showed that blocking FHL2 expression by siRNA could also inhibit the growth and proliferation of human colorectal cancer Lovo cells. The current results demonstrated that chitosan-based siRNA nanoparticles were a very efficient delivery system for siRNA in vivo as previously reported.

Functional gene silencing mediated by chitosan/siRNA nanocomplexes

International Nuclear Information System (INIS)

Ji, A M; Su, D; Che, O; Li, W S; Sun, L; Zhang, Z Y; Xu, F; Yang, B

2009-01-01

Chitosan/siRNA nanoparticles to knock down FHL2 gene expression were reported in this work. The physicochemical properties such as particle size, surface charge, morphology and complex stability of chitosan nanoparticle-incorporated siRNA were evaluated. Nanoparticles which were formulated with chitosan/siRNA exhibited irregular, lamellar and dendritic structures with a hydrodynamic radius size of about 148 nm and net positive charges with zeta-potential value of 58.5 mV. The knockdown effect of the chitosan/siRNA nanoparticles on gene expression in FHL2 over-expressed human colorectal cancer Lovo cells was investigated. The result showed that FHL2 siRNA formulated within chitosan nanoparticles could knock down about 69.6% FHL2 gene expression, which is very similar to the 68.8% reduced gene expression when siRNA was transfected with liposome Lipofectamine. Western analysis further showed significant FHL-2 protein expression reduced by the chitosan/siRNA nanoparticles. The results also showed that blocking FHL2 expression by siRNA could also inhibit the growth and proliferation of human colorectal cancer Lovo cells. The current results demonstrated that chitosan-based siRNA nanoparticles were a very efficient delivery system for siRNA in vivo as previously reported.

The effects of quercetin on microRNA and inflammatory gene expression in lipopolysaccharide-stimulated bovine neutrophils

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Phongsakorn Chuammitri

2017-04-01

Full Text Available Aim: To investigate gene expression of microRNA (miRNA milieus (MIRLET7E, MIR17, MIR24-2, MIR146A, and MIR181C, inflammatory cytokine genes (interleukin 1β [IL1B], IL6, CXCL8, and tumor necrosis factor [TNF], and the pathogen receptor toll-like receptor (TLR4 in bovine neutrophils under quercetin supplementation. Materials and Methods: Isolated bovine neutrophils were incubated with bacterial lipopolysaccharide under quercetin treatment or left untreated. Real-time polymerase chain reaction was performed to determine the expression of the miRNAs and messenger RNA (mRNA transcripts in neutrophils. Results: Quercetin-treated neutrophils exhibited a remarkable suppression in MIR24-2, MIR146A, and MIR181C expression. Similarly, mRNA expression of IL1B, IL6, CXCL8, TLR4, and TNF genes noticeably declined in the quercetin group. Many proinflammatory genes (IL1B, IL6, and CXCL8 and the pathogen receptor TLR4 had a negative correlation with MIR146A and MIR181C as revealed by Pearson correlation. Conclusion: Interaction between cognate mRNAs and miRNAs under quercetin supplementation can be summarized as a positive or negative correlation. This finding may help understand the effects of quercetin either on miRNA or gene expression during inflammation, especially as a potentially applicable indicator in bovine mastitis.

tRNA gene diversity in the three domains of life

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Kosuke eFujishima

2014-05-01

Full Text Available Transfer RNA (tRNA is widely known for its key role in decoding mRNA into protein. Despite their necessity and relatively short nucleotide sequences, a large diversity of gene structures and RNA secondary structures of pre-tRNAs and mature tRNAs have recently been discovered in the three domains of life. Growing evidences of disrupted tRNA genes in the genomes of Archaea reveals unique gene structures such as, intron-containing tRNA, split tRNA, and permuted tRNA. Coding sequence for these tRNAs are either separated with introns, fragmented, or permuted at the genome level. Although evolutionary scenario behind the tRNA gene disruption is still unclear, diversity of tRNA structure seems to be co-evolved with their processing enzyme, so-called RNA splicing endonuclease. Metazoan mitochondrial tRNAs (mtRNAs are known for their unique lack of either one or two arms from the typical tRNA cloverleaf structure, while still maintaining functionality. Recently identified nematode-specific V-arm containing tRNAs (nev-tRNAs possess long variable arms that are specific to eukaryotic class II tRNASer and tRNALeu but also decode class I tRNA codons. Moreover, many tRNA-like sequences have been found in the genomes of different organisms and viruses. Thus this review is aimed to cover the latest knowledge on tRNA gene diversity and further recapitulate the evolutionary and biological aspects that caused such uniqueness.

Pseudogenes regulate parental gene expression via ceRNA network.

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An, Yang; Furber, Kendra L; Ji, Shaoping

2017-01-01

The concept of competitive endogenous RNA (ceRNA) was first proposed by Salmena and colleagues. Evidence suggests that pseudogene RNAs can act as a 'sponge' through competitive binding of common miRNA, releasing or attenuating repression through sequestering miRNAs away from parental mRNA. In theory, ceRNAs refer to all transcripts such as mRNA, tRNA, rRNA, long non-coding RNA, pseudogene RNA and circular RNA, because all of them may become the targets of miRNA depending on spatiotemporal situation. As binding of miRNA to the target RNA is not 100% complementary, it is possible that one miRNA can bind to multiple target RNAs and vice versa. All RNAs crosstalk through competitively binding to miRNAvia miRNA response elements (MREs) contained within the RNA sequences, thus forming a complex regulatory network. The ratio of a subset of miRNAs to the corresponding number of MREs determines repression strength on a given mRNA translation or stability. An increase in pseudogene RNA level can sequester miRNA and release repression on the parental gene, leading to an increase in parental gene expression. A massive number of transcripts constitute a complicated network that regulates each other through this proposed mechanism, though some regulatory significance may be mild or even undetectable. It is possible that the regulation of gene and pseudogene expression occurring in this manor involves all RNAs bearing common MREs. In this review, we will primarily discuss how pseudogene transcripts regulate expression of parental genes via ceRNA network and biological significance of regulation. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Applying the breaks on gene expression - mRNA deadenylation by Pop2p

DEFF Research Database (Denmark)

Andersen, Kasper Røjkjær; Jonstrup, Anette Thyssen; Van, Lan Bich

When driving a car, control of the brakes is just as important as control of the accelerator pedal. Likewise, in gene expression, regulation of mRNA degradation is as important as regulation of its synthesis (Mühlemann, 2005). The rate-determining step of mRNA decay in eukaryotes seems to be the ......When driving a car, control of the brakes is just as important as control of the accelerator pedal. Likewise, in gene expression, regulation of mRNA degradation is as important as regulation of its synthesis (Mühlemann, 2005). The rate-determining step of mRNA decay in eukaryotes seems...... to be the shortening of the poly(A) tail (deadenylation), as this step is slower than the subsequent decapping and degradation of the mRNA body. The Mega-Dalton Ccr4-Not complex contains two exonucleases, Ccr4p and Pop2p, responsible for this process. It is not known at present why two conserved nucleases are needed...

Predicted RNA Binding Proteins Pes4 and Mip6 Regulate mRNA Levels, Translation, and Localization during Sporulation in Budding Yeast.

Science.gov (United States)

Jin, Liang; Zhang, Kai; Sternglanz, Rolf; Neiman, Aaron M

2017-05-01

In response to starvation, diploid cells of Saccharomyces cerevisiae undergo meiosis and form haploid spores, a process collectively referred to as sporulation. The differentiation into spores requires extensive changes in gene expression. The transcriptional activator Ndt80 is a central regulator of this process, which controls many genes essential for sporulation. Ndt80 induces ∼300 genes coordinately during meiotic prophase, but different mRNAs within the NDT80 regulon are translated at different times during sporulation. The protein kinase Ime2 and RNA binding protein Rim4 are general regulators of meiotic translational delay, but how differential timing of individual transcripts is achieved was not known. This report describes the characterization of two related NDT80 -induced genes, PES4 and MIP6 , encoding predicted RNA binding proteins. These genes are necessary to regulate the steady-state expression, translational timing, and localization of a set of mRNAs that are transcribed by NDT80 but not translated until the end of meiosis II. Mutations in the predicted RNA binding domains within PES4 alter the stability of target mRNAs. PES4 and MIP6 affect only a small portion of the NDT80 regulon, indicating that they act as modulators of the general Ime2/Rim4 pathway for specific transcripts. Copyright © 2017 American Society for Microbiology.

Restoration of u.v.-induced excision repair in Xeroderma D cells transfected with the denV gene of bacteriophage T4

International Nuclear Information System (INIS)

Arrand, J.E.; Squires, S.; Bone, N.M.; Johnson, R.T.

1987-01-01

The heritable DNA repair defect in human Xeroderma D cells, resulting in failure to incise at u.v. light-induced pyrimidine dimers, has been partially but stably corrected by transfection of immortalised cells with the denV pyrimidine dimer glycosylase gene of bacteriophage T4. Transfectants selected either for a dominant marker on the mammalian vector carrying the prokaryotic gene or for dominant marker plus resistance to killing by u.v. light, were shown to express the denV gene to varying degrees. denV expression results in significant phenotypic change in the initially repair-deficient, u.v.-hypersensitive cells. Increased resistance to u.v. light and more rapid recovery of replicative DNA synthesis following u.v. irradiation were correlated with improved repair DNA synthesis and with a novel dimer incision capability present in denV transfected Xeroderma cells but not as evident in transfected normal cells. Most transfectants contain a single integrated copy of the denV gene; increase in denV copy number does not result in either increased gene expression or enhanced survival to u.v. light. Results show that expression of a heterologous prokaryotic repair gene can partially compensate for the genetic defect in a human Xeroderma D cell. (author)

Multiple independent insertions of 5S rRNA genes in the spliced-leader gene family of trypanosome species.

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Beauparlant, Marc A; Drouin, Guy

2014-02-01

Analyses of the 5S rRNA genes found in the spliced-leader (SL) gene repeat units of numerous trypanosome species suggest that such linkages were not inherited from a common ancestor, but were the result of independent 5S rRNA gene insertions. In trypanosomes, 5S rRNA genes are found either in the tandemly repeated units coding for SL genes or in independent tandemly repeated units. Given that trypanosome species where 5S rRNA genes are within the tandemly repeated units coding for SL genes are phylogenetically related, one might hypothesize that this arrangement is the result of an ancestral insertion of 5S rRNA genes into the tandemly repeated SL gene family of trypanosomes. Here, we use the types of 5S rRNA genes found associated with SL genes, the flanking regions of the inserted 5S rRNA genes and the position of these insertions to show that most of the 5S rRNA genes found within SL gene repeat units of trypanosome species were not acquired from a common ancestor but are the results of independent insertions. These multiple 5S rRNA genes insertion events in trypanosomes are likely the result of frequent founder events in different hosts and/or geographical locations in species having short generation times.

Photochemistry of U(BH4)4 and U(BD4)4

International Nuclear Information System (INIS)

Paine, R.T.; Schonberg, P.R.; Light, R.W.; Danen, W.C.; Freund, S.M.

1979-01-01

U(BH 4 ) 4 and U(BD 4 ) 4 are observed to undergo complex degradation reactions promoted by broadband UV radiation. The primary products of these reactions appear to be U(BH 4 ) 3 , B 2 H 6 , H 2 , U(BD 4 ) 3 , B 2 D 6 and D 2 . Further, U(BD 4 ) 4 undergoes a related decomposition reaction under the influence of CO 2 laser irradiation at 924.97 cm -1 . (author)

Reconstruction of ribosomal RNA genes from metagenomic data.

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Lu Fan

Full Text Available Direct sequencing of environmental DNA (metagenomics has a great potential for describing the 16S rRNA gene diversity of microbial communities. However current approaches using this 16S rRNA gene information to describe community diversity suffer from low taxonomic resolution or chimera problems. Here we describe a new strategy that involves stringent assembly and data filtering to reconstruct full-length 16S rRNA genes from metagenomicpyrosequencing data. Simulations showed that reconstructed 16S rRNA genes provided a true picture of the community diversity, had minimal rates of chimera formation and gave taxonomic resolution down to genus level. The strategy was furthermore compared to PCR-based methods to determine the microbial diversity in two marine sponges. This showed that about 30% of the abundant phylotypes reconstructed from metagenomic data failed to be amplified by PCR. Our approach is readily applicable to existing metagenomic datasets and is expected to lead to the discovery of new microbial phylotypes.

Characterization of hydrocortisone bioconversion and 16S RNA gene in Synechococcus nidulans cultures.

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Rasoul-Amini, S; Ghasemi, Y; Morowvat, M H; Ghoshoon, M B; Raee, M J; Mosavi-Azam, S B; Montazeri-Najafabady, N; Nouri, F; Parvizi, R; Negintaji, N; Khoubani, S

2010-01-01

A unicellular cyanobacterium, Synechococcus nidulans (Pringsheim) Komárek, was isolated from paddy-fields and applied in the biotransformation experiment of hydrocortisone (1). This strain has not been previously tested for steroid bioconversion. Fermentation was carried out in BG-11 medium supplemented with 0.05% substrate at 25 degrees C for 14 days of incubation. The obtained products were chromatographically purified followed by their characterization using spectroscopic methods. 11beta,17beta-dihydroxyandrost-4-en-3-one (2), 11beta-hydroxyandrost-4-en-3,17-dione (3), and androst-4-ene-3,17-dione (4) were the main bioproducts in the hydrocortisone bioconversion. The observed bioreaction characteristics were the side chain degradation of the substrate to prepare compounds (2) and (3) following the 11beta-dehydroxylation for accumulation of the compound (4). Time course study showed the accumulation of the product (2) from the second day of the fermentation and compounds (3) and (4) from the third day. All the metabolites reached their maximum concentration in seven days. Cyanobacterial 16S rRNA gene was also amplified by PCR. Sequences were amplified using the universal prokaryotic primers which amplify a approximately 400-bp region of the 16S rRNA gene. PCR products were sequenced to confirm their authenticity as 16S rRNA gene of cyanobacteria. The result of PCR blasted with other sequenced cyanobacteria in NCBI showed 99% identity to the 16S small subunit rRNA of seven Synechococcus species.

Activation of the ribosomal RNA genes late in the third cell cycle of porcine embryos

DEFF Research Database (Denmark)

Viuff, Dorthe; Greve, Torben; Holm, Peter

2002-01-01

; there was no silver staining at the sites of the rRNA genes and nucleolus precursor bodies. From 30 hpc onwards, most 4-cell embryos had medium size to large clusters of FITC-labeled areas colocalized with silver staining of rRNA gene clusters and fibrillogranular nucleoli. These observations indicate that r...

Microcephalic osteodysplastic primordial dwarfism type I with biallelic mutations in the RNU4ATAC gene.

Science.gov (United States)

Nagy, R; Wang, H; Albrecht, B; Wieczorek, D; Gillessen-Kaesbach, G; Haan, E; Meinecke, P; de la Chapelle, A; Westman, J A

2012-08-01

Microcephalic osteodysplastic primordial dwarfism type I (MOPD I) is a rare autosomal recessive developmental disorder characterized by extreme intrauterine growth retardation, severe microcephaly, central nervous system abnormalities, dysmorphic facial features, skin abnormalities, skeletal changes, limb deformations, and early death. Recently, mutations in the RNU4ATAC gene, which encodes U4atac, a small nuclear RNA that is a crucial component of the minor spliceosome, were found to cause MOPD I. MOPD I is the first disease known to be associated with a defect in small nuclear RNAs. We describe here the clinical and molecular data for 17 cases of MOPD I, including 15 previously unreported cases, all carrying biallelic mutations in the RNU4ATAC gene. © 2011 John Wiley & Sons A/S.

Mitochondrial tRNA gene translocations in highly eusocial bees

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Daniela Silvestre

2006-01-01

Full Text Available Mitochondrial gene rearrangement events, especially involving tRNA genes, have been described more frequently as more complete mitochondrial genome sequences are becoming available. In the present work, we analyzed mitochondrial tRNA gene rearrangements between two bee species belonging to the tribes Apini and Meliponini within the "corbiculate Apidae". Eleven tRNA genes are in different genome positions or strands. The molecular events responsible for each translocation are explained. Considering the high number of rearrangements observed, the data presented here contradict the general rule of high gene order conservation among closely related organisms, and also represent a powerful molecular tool to help solve questions about phylogeny and evolution in bees.

Analysis of the complement and molecular evolution of tRNA genes in cow

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Barris Wesley C

2009-04-01

Full Text Available Abstract Background Detailed information regarding the number and organization of transfer RNA (tRNA genes at the genome level is becoming readily available with the increase of DNA sequencing of whole genomes. However the identification of functional tRNA genes is challenging for species that have large numbers of repetitive elements containing tRNA derived sequences, such as Bos taurus. Reliable identification and annotation of entire sets of tRNA genes allows the evolution of tRNA genes to be understood on a genomic scale. Results In this study, we explored the B. taurus genome using bioinformatics and comparative genomics approaches to catalogue and analyze cow tRNA genes. The initial analysis of the cow genome using tRNAscan-SE identified 31,868 putative tRNA genes and 189,183 pseudogenes, where 28,830 of the 31,868 predicted tRNA genes were classified as repetitive elements by the RepeatMasker program. We then used comparative genomics to further discriminate between functional tRNA genes and tRNA-derived sequences for the remaining set of 3,038 putative tRNA genes. For our analysis, we used the human, chimpanzee, mouse, rat, horse, dog, chicken and fugu genomes to predict that the number of active tRNA genes in cow lies in the vicinity of 439. Of this set, 150 tRNA genes were 100% identical in their sequences across all nine vertebrate genomes studied. Using clustering analyses, we identified a new tRNA-GlyCCC subfamily present in all analyzed mammalian genomes. We suggest that this subfamily originated from an ancestral tRNA-GlyGCC gene via a point mutation prior to the radiation of the mammalian lineages. Lastly, in a separate analysis we created phylogenetic profiles for each putative cow tRNA gene using a representative set of genomes to gain an overview of common evolutionary histories of tRNA genes. Conclusion The use of a combination of bioinformatics and comparative genomics approaches has allowed the confident identification of a

Taxonomic resolutions based on 18S rRNA genes: a case study of subclass copepoda.

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Shu Wu

Full Text Available Biodiversity studies are commonly conducted using 18S rRNA genes. In this study, we compared the inter-species divergence of variable regions (V1-9 within the copepod 18S rRNA gene, and tested their taxonomic resolutions at different taxonomic levels. Our results indicate that the 18S rRNA gene is a good molecular marker for the study of copepod biodiversity, and our conclusions are as follows: 1 18S rRNA genes are highly conserved intra-species (intra-species similarities are close to 100%; and could aid in species-level analyses, but with some limitations; 2 nearly-whole-length sequences and some partial regions (around V2, V4, and V9 of the 18S rRNA gene can be used to discriminate between samples at both the family and order levels (with a success rate of about 80%; 3 compared with other regions, V9 has a higher resolution at the genus level (with an identification success rate of about 80%; and 4 V7 is most divergent in length, and would be a good candidate marker for the phylogenetic study of Acartia species. This study also evaluated the correlation between similarity thresholds and the accuracy of using nuclear 18S rRNA genes for the classification of organisms in the subclass Copepoda. We suggest that sample identification accuracy should be considered when a molecular sequence divergence threshold is used for taxonomic identification, and that the lowest similarity threshold should be determined based on a pre-designated level of acceptable accuracy.

Down-regulation of HSP40 gene family following OCT4B1 suppression in human tumor cell lines

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Mohammad Reza Mirzaei

2016-02-01

Full Text Available Objective(s: The OCT4B1, as one of OCT4 variants, is expressed in cancer cell lines and tissues more than other variants and plays an important role in apoptosis and stress (heat shock protein pathways. The present study was designed to determine the effects of OCT4B1 silencing on expressional profile of HSP40 gene family expression in three different human tumor cell lines. Materials and Methods: The OCT4B1 expression was suppressed by specific siRNA transfection in AGS (gastric adenocarcinoma, 5637 (bladder tumor and U-87MG (brain tumor cell lines employing Lipofectamine reagent. Real-time PCR array technique was employed for RNA qualification. The fold changes were calculated using RT2 Profiler PCR array data analysis software version 3.5. Results: Our results indicated that fifteen genes (from 36 studied genes were down-regulated and two genes (DNAJC11 and DNAJC5B were up-regulated in all three studied tumor cell lines by approximately more than two folds. The result of other studied genes (19 genes showed different expressional pattern (up or down-expression based on tumor cell lines. Conclusion: According to the findings of the present study, we may suggest that there is a direct correlation between OCT4B1 expression in tumor cell lines (and tissues and HSP40 family gene expressions to escape from apoptosis and cancer expansion.

Domestication of transposable elements into MicroRNA genes in plants.

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Yang Li

Full Text Available Transposable elements (TE usually take up a substantial portion of eukaryotic genome. Activities of TEs can cause genome instability or gene mutations that are harmful or even disastrous to the host. TEs also contribute to gene and genome evolution at many aspects. Part of miRNA genes in mammals have been found to derive from transposons while convincing evidences are absent for plants. We found that a considerable number of previously annotated plant miRNAs are identical or homologous to transposons (TE-MIR, which include a small number of bona fide miRNA genes that conform to generally accepted plant miRNA annotation rules, and hairpin derived siRNAs likely to be pre-evolved miRNAs. Analysis of these TE-MIRs indicate that transitions from the medium to high copy TEs into miRNA genes may undergo steps such as inverted repeat formation, sequence speciation and adaptation to miRNA biogenesis. We also identified initial target genes of the TE-MIRs, which contain homologous sequences in their CDS as consequence of cognate TE insertions. About one-third of the initial target mRNAs are supported by publicly available degradome sequencing data for TE-MIR sRNA induced cleavages. Targets of the TE-MIRs are biased to non-TE related genes indicating their penchant to acquire cellular functions during evolution. Interestingly, most of these TE insertions span boundaries between coding and non-coding sequences indicating their incorporation into CDS through alteration of splicing or translation start or stop signals. Taken together, our findings suggest that TEs in gene rich regions can form foldbacks in non-coding part of transcripts that may eventually evolve into miRNA genes or be integrated into protein coding sequences to form potential targets in a "temperate" manner. Thus, transposons may supply as resources for the evolution of miRNA-target interactions in plants.

Use of bacterially expressed dsRNA to downregulate Entamoeba histolytica gene expression.

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Carlos F Solis

Full Text Available BACKGROUND: Modern RNA interference (RNAi methodologies using small interfering RNA (siRNA oligonucleotide duplexes or episomally synthesized hairpin RNA are valuable tools for the analysis of gene function in the protozoan parasite Entamoeba histolytica. However, these approaches still require time-consuming procedures including transfection and drug selection, or costly synthetic molecules. PRINCIPAL FINDINGS: Here we report an efficient and handy alternative for E. histolytica gene down-regulation mediated by bacterial double-stranded RNA (dsRNA targeting parasite genes. The Escherichia coli strain HT115 which is unable to degrade dsRNA, was genetically engineered to produce high quantities of long dsRNA segments targeting the genes that encode E. histolytica beta-tubulin and virulence factor KERP1. Trophozoites cultured in vitro were directly fed with dsRNA-expressing bacteria or soaked with purified dsRNA. Both dsRNA delivery methods resulted in significant reduction of protein expression. In vitro host cell-parasite assays showed that efficient downregulation of kerp1 gene expression mediated by bacterial dsRNA resulted in significant reduction of parasite adhesion and lytic capabilities, thus supporting a major role for KERP1 in the pathogenic process. Furthermore, treatment of trophozoites cultured in microtiter plates, with a repertoire of eighty-five distinct bacterial dsRNA segments targeting E. histolytica genes with unknown function, led to the identification of three genes potentially involved in the growth of the parasite. CONCLUSIONS: Our results showed that the use of bacterial dsRNA is a powerful method for the study of gene function in E. histolytica. This dsRNA delivery method is also technically suitable for the study of a large number of genes, thus opening interesting perspectives for the identification of novel drug and vaccine targets.

U1 small nuclear RNA variants differentially form ribonucleoprotein particles in vitro.

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Somarelli, Jason A; Mesa, Annia; Rodriguez, Carol E; Sharma, Shalini; Herrera, Rene J

2014-04-25

The U1 small nuclear (sn)RNA participates in splicing of pre-mRNAs by recognizing and binding to 5' splice sites at exon/intron boundaries. U1 snRNAs associate with 5' splice sites in the form of ribonucleoprotein particles (snRNPs) that are comprised of the U1 snRNA and 10 core components, including U1A, U1-70K, U1C and the 'Smith antigen', or Sm, heptamer. The U1 snRNA is highly conserved across a wide range of taxa; however, a number of reports have identified the presence of expressed U1-like snRNAs in multiple species, including humans. While numerous U1-like molecules have been shown to be expressed, it is unclear whether these variant snRNAs have the capacity to form snRNPs and participate in splicing. The purpose of the present study was to further characterize biochemically the ability of previously identified human U1-like variants to form snRNPs and bind to U1 snRNP proteins. A bioinformatics analysis provided support for the existence of multiple expressed variants. In vitro gel shift assays, competition assays, and immunoprecipitations (IPs) revealed that the variants formed high molecular weight assemblies to varying degrees and associated with core U1 snRNP proteins to a lesser extent than the canonical U1 snRNA. Together, these data suggest that the human U1 snRNA variants analyzed here are unable to efficiently bind U1 snRNP proteins. The current work provides additional biochemical insights into the ability of the variants to assemble into snRNPs. Copyright © 2014 Elsevier B.V. All rights reserved.

Functional characterization of the Drosophila MRP (mitochondrial RNA processing) RNA gene.

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Schneider, Mary D; Bains, Anupinder K; Rajendra, T K; Dominski, Zbigniew; Matera, A Gregory; Simmonds, Andrew J

2010-11-01

MRP RNA is a noncoding RNA component of RNase mitochondrial RNA processing (MRP), a multi-protein eukaryotic endoribonuclease reported to function in multiple cellular processes, including ribosomal RNA processing, mitochondrial DNA replication, and cell cycle regulation. A recent study predicted a potential Drosophila ortholog of MRP RNA (CR33682) by computer-based genome analysis. We have confirmed the expression of this gene and characterized the phenotype associated with this locus. Flies with mutations that specifically affect MRP RNA show defects in growth and development that begin in the early larval period and end in larval death during the second instar stage. We present several lines of evidence demonstrating a role for Drosophila MRP RNA in rRNA processing. The nuclear fraction of Drosophila MRP RNA localizes to the nucleolus. Further, a mutant strain shows defects in rRNA processing that include a defect in 5.8S rRNA processing, typical of MRP RNA mutants in other species, as well as defects in early stages of rRNA processing.

The phosphotransferase VanU represses expression of four qrr genes antagonizing VanO-mediated quorum-sensing regulation in Vibrio anguillarum.

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Weber, Barbara; Lindell, Kristoffer; El Qaidi, Samir; Hjerde, Erik; Willassen, Nils-Peder; Milton, Debra L

2011-12-01

Vibrio anguillarum utilizes quorum sensing to regulate stress responses required for survival in the aquatic environment. Like other Vibrio species, V. anguillarum contains the gene qrr1, which encodes the ancestral quorum regulatory RNA Qrr1, and phosphorelay quorum-sensing systems that modulate the expression of small regulatory RNAs (sRNAs) that destabilize mRNA encoding the transcriptional regulator VanT. In this study, three additional Qrr sRNAs were identified. All four sRNAs were positively regulated by σ(54) and the σ(54)-dependent response regulator VanO, and showed a redundant activity. The Qrr sRNAs, together with the RNA chaperone Hfq, destabilized vanT mRNA and modulated expression of VanT-regulated genes. Unexpectedly, expression of all four qrr genes peaked at high cell density, and exogenously added N-acylhomoserine lactone molecules induced expression of the qrr genes at low cell density. The phosphotransferase VanU, which phosphorylates and activates VanO, repressed expression of the Qrr sRNAs and stabilized vanT mRNA. A model is presented proposing that VanU acts as a branch point, aiding cross-regulation between two independent phosphorelay systems that activate or repress expression of the Qrr sRNAs, giving flexibility and precision in modulating VanT expression and inducing a quorum-sensing response to stresses found in a constantly changing aquatic environment.

miRNA-Processing Gene Methylation and Cancer Risk.

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Joyce, Brian T; Zheng, Yinan; Zhang, Zhou; Liu, Lei; Kocherginsky, Masha; Murphy, Robert; Achenbach, Chad J; Musa, Jonah; Wehbe, Firas; Just, Allan; Shen, Jincheng; Vokonas, Pantel; Schwartz, Joel; Baccarelli, Andrea A; Hou, Lifang

2018-05-01

Background: Dysregulation of miRNA and methylation levels are epigenetic hallmarks of cancer, potentially linked via miRNA-processing genes. Studies have found genetic alterations to miRNA-processing genes in cancer cells and human population studies. Our objective was to prospectively examine changes in DNA methylation of miRNA-processing genes and their associations with cancer risk. Methods: We examined cohort data from the Department of Veterans' Affairs Normative Aging Study. Participants were assessed every 3 to 5 years starting in 1999 through 2013 including questionnaires, medical record review, and blood collection. Blood from 686 consenting participants was analyzed using the Illumina 450K BeadChip array to measure methylation at CpG sites throughout the genome. We selected 19 genes based on a literature review, with 519 corresponding CpG sites. We then used Cox proportional hazards models to examine associations with cancer incidence, and generalized estimating equations to examine associations with cancer prevalence. Associations at false discovery rate time to cancer development (positively for cg06751583, inversely for cg23230564 and cg21034183), whereas methylation of one CpG site ( DROSHA : cg16131300) was positively associated with cancer prevalence. Conclusions: DNA methylation of DROSHA , a key miRNA-processing gene, and TNRC6B may play a role in early carcinogenesis. Impact: Changes in miRNA processing may exert multiple effects on cancer development, including protecting against it via altered global miRNAs, and may be a useful early detection biomarker of cancer. Cancer Epidemiol Biomarkers Prev; 27(5); 550-7. ©2018 AACR . ©2018 American Association for Cancer Research.

Evaluation of two main RNA-seq approaches for gene quantification in clinical RNA sequencing: polyA+ selection versus rRNA depletion.

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Zhao, Shanrong; Zhang, Ying; Gamini, Ramya; Zhang, Baohong; von Schack, David

2018-03-19

To allow efficient transcript/gene detection, highly abundant ribosomal RNAs (rRNA) are generally removed from total RNA either by positive polyA+ selection or by rRNA depletion (negative selection) before sequencing. Comparisons between the two methods have been carried out by various groups, but the assessments have relied largely on non-clinical samples. In this study, we evaluated these two RNA sequencing approaches using human blood and colon tissue samples. Our analyses showed that rRNA depletion captured more unique transcriptome features, whereas polyA+ selection outperformed rRNA depletion with higher exonic coverage and better accuracy of gene quantification. For blood- and colon-derived RNAs, we found that 220% and 50% more reads, respectively, would have to be sequenced to achieve the same level of exonic coverage in the rRNA depletion method compared with the polyA+ selection method. Therefore, in most cases we strongly recommend polyA+ selection over rRNA depletion for gene quantification in clinical RNA sequencing. Our evaluation revealed that a small number of lncRNAs and small RNAs made up a large fraction of the reads in the rRNA depletion RNA sequencing data. Thus, we recommend that these RNAs are specifically depleted to improve the sequencing depth of the remaining RNAs.

Developmental and functional expression of miRNA-stability related genes in the nervous system.

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de Sousa, Érica; Walter, Lais Takata; Higa, Guilherme Shigueto Vilar; Casado, Otávio Augusto Nocera; Kihara, Alexandre Hiroaki

2013-01-01

In the nervous system, control of gene expression by microRNAs (miRNAs) has been investigated in fundamental processes, such as development and adaptation to ambient demands. The action of these short nucleotide sequences on specific genes depends on intracellular concentration, which in turn reflects the balance of biosynthesis and degradation. Whereas mechanisms underlying miRNA biogenesis has been investigated in recent studies, little is known about miRNA-stability related proteins. We first detected two genes in the retina that have been associated to miRNA stability, XRN2 and PAPD4. These genes are highly expressed during retinal development, however with distinct subcellular localization. We investigated whether these proteins are regulated during specific phases of the cell cycle. Combined analyses of nuclei position in neuroblastic layer and labeling using anti-cyclin D1 revealed that both proteins do not accumulate in S or M phases of the cell cycle, being poorly expressed in progenitor cells. Indeed, XRN2 and PAPD4 were observed mainly after neuronal differentiation, since low expression was also observed in astrocytes, endothelial and microglial cells. XRN2 and PAPD4 are expressed in a wide variety of neurons, including horizontal, amacrine and ganglion cells. To evaluate the functional role of both genes, we carried out experiments addressed to the retinal adaptation in response to different ambient light conditions. PAPD4 is upregulated after 3 and 24 hours of dark- adaptation, revealing that accumulation of this protein is governed by ambient light levels. Indeed, the fast and functional regulation of PAPD4 was not related to changes in gene expression, disclosing that control of protein levels occurs by post-transcriptional mechanisms. Furthermore, we were able to quantify changes in PAPD4 in specific amacrine cells after dark -adaptation, suggesting for circuitry-related roles in visual perception. In summary, in this study we first described the

Developmental and functional expression of miRNA-stability related genes in the nervous system.

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Érica de Sousa

Full Text Available In the nervous system, control of gene expression by microRNAs (miRNAs has been investigated in fundamental processes, such as development and adaptation to ambient demands. The action of these short nucleotide sequences on specific genes depends on intracellular concentration, which in turn reflects the balance of biosynthesis and degradation. Whereas mechanisms underlying miRNA biogenesis has been investigated in recent studies, little is known about miRNA-stability related proteins. We first detected two genes in the retina that have been associated to miRNA stability, XRN2 and PAPD4. These genes are highly expressed during retinal development, however with distinct subcellular localization. We investigated whether these proteins are regulated during specific phases of the cell cycle. Combined analyses of nuclei position in neuroblastic layer and labeling using anti-cyclin D1 revealed that both proteins do not accumulate in S or M phases of the cell cycle, being poorly expressed in progenitor cells. Indeed, XRN2 and PAPD4 were observed mainly after neuronal differentiation, since low expression was also observed in astrocytes, endothelial and microglial cells. XRN2 and PAPD4 are expressed in a wide variety of neurons, including horizontal, amacrine and ganglion cells. To evaluate the functional role of both genes, we carried out experiments addressed to the retinal adaptation in response to different ambient light conditions. PAPD4 is upregulated after 3 and 24 hours of dark- adaptation, revealing that accumulation of this protein is governed by ambient light levels. Indeed, the fast and functional regulation of PAPD4 was not related to changes in gene expression, disclosing that control of protein levels occurs by post-transcriptional mechanisms. Furthermore, we were able to quantify changes in PAPD4 in specific amacrine cells after dark -adaptation, suggesting for circuitry-related roles in visual perception. In summary, in this study we

The recruitment of the U5 snRNP to nascent transcripts requires internal loop 1 of U5 snRNA.

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Kim, Rebecca; Paschedag, Joshua; Novikova, Natalya; Bellini, Michel

2012-12-01

In this study, we take advantage of the high spatial resolution offered by the nucleus and lampbrush chromosomes of the amphibian oocyte to investigate the mechanisms that regulate the intranuclear trafficking of the U5 snRNP and its recruitment to nascent transcripts. We monitor the fate of newly assembled fluorescent U5 snRNP in Xenopus oocytes depleted of U4 and/or U6 snRNAs and demonstrate that the U4/U6.U5 tri-snRNP is not required for the association of U5 snRNP with Cajal bodies, splicing speckles, and nascent transcripts. In addition, using a mutational analysis, we show that a non-functional U5 snRNP can associate with nascent transcripts, and we further characterize internal loop structure 1 of U5 snRNA as a critical element for licensing U5 snRNP to target both nascent transcripts and splicing speckles. Collectively, our data support the model where the recruitment of snRNPs onto pre-mRNAs is independent of spliceosome assembly and suggest that U5 snRNP may promote the association of the U4/U6.U5 tri-snRNP with nascent transcripts.

Identification of a novel Drosophila gene, beltless, using injectable embryonic and adult RNA interference (RNAi

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Manev Hari

2003-08-01

Full Text Available Abstract Background RNA interference (RNAi is a process triggered by a double-stranded RNA that leads to targeted down-regulation/silencing of gene expression and can be used for functional genomics; i.e. loss-of-function studies. Here we report on the use of RNAi in the identification of a developmentally important novel Drosophila (fruit fly gene (corresponding to a putative gene CG5652/GM06434, that we named beltless based on an embryonic loss-of-function phenotype. Results Beltless mRNA is expressed in all developmental stages except in 0–6 h embryos. In situ RT-PCR localized beltless mRNA in the ventral cord and brain of late stage embryos and in the nervous system, ovaries, and the accessory glands of adult flies. RNAi was induced by injection of short (22 bp beltless double-stranded RNAs into embryos or into adult flies. Embryonic RNAi altered cuticular phenotypes ranging from partially-formed to missing denticle belts (thus beltless of the abdominal segments A2–A4. Embryonic beltless RNAi was lethal. Adult RNAi resulted in the shrinkage of the ovaries by half and reduced the number of eggs laid. We also examined Df(1RK4 flies in which deletion removes 16 genes, including beltless. In some embryos, we observed cuticular abnormalities similar to our findings with beltless RNAi. After differentiating Df(1RK4 embryos into those with visible denticle belts and those missing denticle belts, we assayed the presence of beltless mRNA; no beltless mRNA was detectable in embryos with missing denticle belts. Conclusions We have identified a developmentally important novel Drosophila gene, beltless, which has been characterized in loss-of-function studies using RNA interference. The putative beltless protein shares homologies with the C. elegans nose resistant to fluoxetine (NRF NRF-6 gene, as well as with several uncharacterized C. elegans and Drosophila melanogaster genes, some with prominent acyltransferase domains. Future studies should

High prevalence of plasmid-mediated 16S rRNA methylase gene rmtB among Escherichia coli clinical isolates from a Chinese teaching hospital

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Zhang Xue-qing

2010-06-01

Full Text Available Abstract Background Recently, production of 16S rRNA methylases by Gram-negative bacilli has emerged as a novel mechanism for high-level resistance to aminoglycosides by these organisms in a variety of geographic locations. Therefore, the spread of high-level aminoglycoside resistance determinants has become a great concern. Methods Between January 2006 and July 2008, 680 distinct Escherichia coli clinical isolates were collected from a teaching hospital in Wenzhou, China. PCR and DNA sequencing were used to identify 16S rRNA methylase and extended-spectrum β-lactamase (ESBL genes, including armA and rmtB, and in situ hybridization was performed to determine the location of 16S rRNA methylase genes. Conjugation experiments were subsequently performed to determine whether aminoglycoside resistance was transferable from the E. coli isolates via 16S rRNA methylase-bearing plasmids. Homology of the isolates harboring 16S rRNA methylase genes was determined using pulse-field gel electrophoresis (PFGE. Results Among the 680 E. coli isolates, 357 (52.5%, 346 (50.9% and 44 (6.5% isolates were resistant to gentamicin, tobramycin and amikacin, respectively. Thirty-seven of 44 amikacin-resistant isolates harbored 16S rRNA methylase genes, with 36 of 37 harboring the rmtB gene and only one harboring armA. The positive rates of 16S rRNA methylase genes among all isolates and amikacin-resistant isolates were 5.4% (37/680 and 84.1% (37/44, respectively. Thirty-one isolates harboring 16S rRNA methylase genes also produced ESBLs. In addition, high-level aminoglycoside resistance could be transferred by conjugation from four rmtB-positive donors. The plasmids of incompatibility groups IncF, IncK and IncN were detected in 34, 3 and 3 isolates, respectively. Upstream regions of the armA gene contained ISCR1 and tnpU, the latter a putative transposase gene,. Another putative transposase gene, tnpD, was located within a region downstream of armA. Moreover, a

5S rRNA gene arrangements in protists: a case of nonadaptive evolution.

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Drouin, Guy; Tsang, Corey

2012-06-01

Given their high copy number and high level of expression, one might expect that both the sequence and organization of eukaryotic ribosomal RNA genes would be conserved during evolution. Although the organization of 18S, 5.8S and 28S ribosomal RNA genes is indeed relatively well conserved, that of 5S rRNA genes is much more variable. Here, we review the different types of 5S rRNA gene arrangements which have been observed in protists. This includes linkages to the other ribosomal RNA genes as well as linkages to ubiquitin, splice-leader, snRNA and tRNA genes. Mapping these linkages to independently derived phylogenies shows that these diverse linkages have repeatedly been gained and lost during evolution. This argues against such linkages being the primitive condition not only in protists but also in other eukaryote species. Because the only characteristic the diverse genes with which 5S rRNA genes are found linked with is that they are tandemly repeated, these arrangements are unlikely to provide any selective advantage. Rather, the observed high variability in 5S rRNA genes arrangements is likely the result of the fact that 5S rRNA genes contain internal promoters, that these genes are often transposed by diverse recombination mechanisms and that these new gene arrangements are rapidly homogenized by unequal crossingovers and/or by gene conversions events in species with short generation times and frequent founder events.

The mRNA expression of XRCC repair genes in mice after γ-ray radiation

International Nuclear Information System (INIS)

Wang Qin; Yue Jingyin; Li Jin; Mu Chuanjie; Fan Feiyue

2006-01-01

Objective: To investigate the role of XRCC repair genes in radioresistance of IRM-2 inbred mice. Methods: Northern hybridization was used to measure mRNA expression of XRCC1 and XRCC5 genes in IRM-2 inbred mice. ICR/JCL and 615 after exposure to different doses of γ-ray radiation at different postirradiation time. Results: The levels of XRCC1 and XRCC5 mRNA expression in control IRM-2 mice were higher significantly than those in their control parental mice (P<0.01 and P<0.05). The mRNA expression of XRCC genes in ICR/JCL and 615 mice all increased to some extent after exposure 1, 2 and 4 Gy radiation. But the levels were significantly higher at 2h postirradiation (P<0.05) . The levels of XRCC mRNA expression in IRM-2 mice did not increase significnatly compared with the control mice after exposure 1 and 2 Gy radiation. But the levels of XRCC1 and XRCC5 mRNA expression increased markedly at 4Gy 1h postirradiation (P<0.05 and P<0.01). Conclusion: The basal levels of XRCC1 and XRCC5 mRNA expression in IRM-2 mice were high. The high level of XRCC5 mRNA expression was involved in the repair of DNA double strand breaks induced by higher dose radiation, which perhaps was one of radioresistance causes of IRM-2 mice. (authors)

Genome-wide identification and characterization of microRNA genes and their targets in flax (Linum usitatissimum): Characterization of flax miRNA genes.

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Barvkar, Vitthal T; Pardeshi, Varsha C; Kale, Sandip M; Qiu, Shuqing; Rollins, Meaghen; Datla, Raju; Gupta, Vidya S; Kadoo, Narendra Y

2013-04-01

MicroRNAs (miRNAs) are small (20-24 nucleotide long) endogenous regulatory RNAs that play important roles in plant growth and development. They regulate gene expression at the post-transcriptional level by translational repression or target degradation and gene silencing. In this study, we identified 116 conserved miRNAs belonging to 23 families from the flax (Linum usitatissimum L.) genome using a computational approach. The precursor miRNAs varied in length; while most of the mature miRNAs were 21 nucleotide long, intergenic and showed conserved signatures of RNA polymerase II transcripts in their upstream regions. Promoter region analysis of the flax miRNA genes indicated prevalence of MYB transcription factor binding sites. Four miRNA gene clusters containing members of three phylogenetic groups were identified. Further, 142 target genes were predicted for these miRNAs and most of these represent transcriptional regulators. The miRNA encoding genes were expressed in diverse tissues as determined by digital expression analysis as well as real-time PCR. The expression of fourteen miRNAs and nine target genes was independently validated using the quantitative reverse transcription PCR (qRT-PCR). This study suggests that a large number of conserved plant miRNAs are also found in flax and these may play important roles in growth and development of flax.

In vivo targeting of ADAM9 gene expression using lentivirus-delivered shRNA suppresses prostate cancer growth by regulating REG4 dependent cell cycle progression.

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Che-Ming Liu

Full Text Available Cancer cells respond to stress by activating a variety of survival signaling pathways. A disintegrin and metalloproteinase (ADAM 9 is upregulated during cancer progression and hormone therapy, functioning in part through an increase in reactive oxygen species. Here, we present in vitro and in vivo evidence that therapeutic targeting of ADAM9 gene expression by lentivirus-delivered small hairpin RNA (shRNA significantly inhibited proliferation of human prostate cancer cell lines and blocked tumor growth in a murine model of prostate cancer bone metastasis. Cell cycle studies confirmed an increase in the G1-phase and decrease in the S-phase population of cancer cells under starvation stress conditions, which correlated with elevated intracellular superoxide levels. Microarray data showed significantly decreased levels of regenerating islet-derived family member 4 (REG4 expression in prostate cancer cells with knockdown of ADAM9 gene expression. This REG4 downregulation also resulted in induction of expression of p21(Cip1/WAF1, which negatively regulates cyclin D1 and blocks the G1/S transition. Our data reveal a novel molecular mechanism of ADAM9 in the regulation of prostate cancer cell proliferation, and suggests a combined modality of ADAM9 shRNA gene therapy and cytotoxic agents for hormone refractory and bone metastatic prostate cancer.

Non-functional genes repaired at the RNA level.

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Burger, Gertraud

2016-01-01

Genomes and genes continuously evolve. Gene sequences undergo substitutions, deletions or nucleotide insertions; mobile genetic elements invade genomes and interleave in genes; chromosomes break, even within genes, and pieces reseal in reshuffled order. To maintain functional gene products and assure an organism's survival, two principal strategies are used - either repair of the gene itself or of its product. I will introduce common types of gene aberrations and how gene function is restored secondarily, and then focus on systematically fragmented genes found in a poorly studied protist group, the diplonemids. Expression of their broken genes involves restitching of pieces at the RNA-level, and substantial RNA editing, to compensate for point mutations. I will conclude with thoughts on how such a grotesquely unorthodox system may have evolved, and why this group of organisms persists and thrives since tens of millions of years. Copyright © 2016 Académie des sciences. Published by Elsevier SAS. All rights reserved.

The Structure of the RNA m5C Methyltransferase YebU from Escherichia coli Reveals a C-terminal RNA-recruiting PUA Domain

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Hallberg, B. Martin; Ericsson, Ulrika B.; Johnson, Kenneth A

2006-01-01

potential that differ from other RNA-MTase structures, suggesting that YebU interacts with its RNA target in a different manner. Docking of YebU onto the 30 S subunit indicates that the PUA and MTase domains make several contacts with 16 S rRNA as well as with the ribosomal protein S12. The ribosomal...... protein interactions would explain why the assembled 30 S subunit, and not naked 16 S rRNA, is the preferred substrate for YebU....... by X-ray crystallography, and we present a molecular model for how YebU specifically recognizes, binds and methylates its ribosomal substrate. The YebU protein has an N-terminal SAM-binding catalytic domain with structural similarity to the equivalent domains in several other m(5)C RNA MTases including...

Computational prediction of miRNA genes from small RNA sequencing data

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Wenjing eKang

2015-01-01

Full Text Available Next-generation sequencing now for the first time allows researchers to gauge the depth and variation of entire transcriptomes. However, now as rare transcripts can be detected that are present in cells at single copies, more advanced computational tools are needed to accurately annotate and profile them. miRNAs are 22 nucleotide small RNAs (sRNAs that post-transcriptionally reduce the output of protein coding genes. They have established roles in numerous biological processes, including cancers and other diseases. During miRNA biogenesis, the sRNAs are sequentially cleaved from precursor molecules that have a characteristic hairpin RNA structure. The vast majority of new miRNA genes that are discovered are mined from small RNA sequencing (sRNA-seq, which can detect more than a billion RNAs in a single run. However, given that many of the detected RNAs are degradation products from all types of transcripts, the accurate identification of miRNAs remain a non-trivial computational problem. Here we review the tools available to predict animal miRNAs from sRNA sequencing data. We present tools for generalist and specialist use cases, including prediction from massively pooled data or in species without reference genome. We also present wet-lab methods used to validate predicted miRNAs, and approaches to computationally benchmark prediction accuracy. For each tool, we reference validation experiments and benchmarking efforts. Last, we discuss the future of the field.

Design, synthesis and evaluation of VEGF-siRNA/CRS as a novel vector for gene delivery

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Zhao W

2016-11-01

Full Text Available Wen Zhao, Yifan Zhang, Xueyun Jiang, Chunying Cui School of Chemical Biology and Pharmaceutical Sciences, Capital Medical University, Beijing, China Abstract: Small interfering RNA (siRNA delivery is a prospective method in gene therapy, but it has application limitations such as negative charge, water solubility and high molecular weight. In this study, a safe and efficient nano-vector, CRS, was designed and synthesized to facilitate siRNA delivery. Physical and chemical properties of VEGF-siRNA/CRS were characterized by methods including scanning electron microscopy (SEM, transmission electron microscopy, zeta potential (ζ measurement, drug-releasing rate measurement, gel electrophoresis and confocal microscopy. The biological activities were evaluated using cell viability assay, gene-silencing efficacy assay in vitro, real-time polymerase chain reaction, enzyme-linked immunosorbent assay (ELISA and antitumor tests in vivo. The mean nanoparticle size of VEGF-siRNA/CRS was 121.4±0.3 nm with positive ζ potential of 7.69±4.47 mV. The release rate of VEGF-siRNA from VEGF-siRNA/CRS was 82.50% sustained for 48 h in Tris-ethylenediaminetetraacetic acid buffer (pH 8.0. Real-time polymerase chain reaction was used to analyze the efficiency of the transfection, and the result showed that VEGF mRNA expression had been knocked down by 82.36%. The expression of VEGF protein was also recorded to be downregulated to 14.83% using ELISA. The results of cytotoxicity measured by Cell Counting Kit-8 assay showed that VEGF-siRNA/CRS had significant inhibitory effect on HeLa cells. The results of antitumor assays indicated that VEGF-siRNA/CRS exhibited tumor cell growth inhibition in vivo. The results demonstrated that VEGF-siRNA could be delivered and transported by the designed carrier, while siRNA could be released constantly and led to an increasing gene-silencing effect against VEGF gene. In conclusion, VEGF-siRNA/CRS is a promising carrier for siRNA

BAY11 enhances OCT4 synthetic mRNA expression in adult human skin cells.

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Awe, Jason P; Crespo, Agustin Vega; Li, You; Kiledjian, Megerditch; Byrne, James A

2013-02-06

The OCT4 transcription factor is involved in many cellular processes, including development, reprogramming, maintaining pluripotency and differentiation. Synthetic OCT4 mRNA was recently used (in conjunction with other reprogramming factors) to generate human induced pluripotent stem cells. Here, we discovered that BAY 11-7082 (BAY11), at least partially through an NF-κB-inhibition based mechanism, could significantly increase the expression of OCT4 following transfection of synthetic mRNA (synRNA) into adult human skin cells. We tested various chemical and molecular small molecules on their ability to suppress the innate immune response seen upon synthetic mRNA transfection. Three molecules - B18R, BX795, and BAY11 - were used in immunocytochemical and proliferation-based assays. We also utilized global transcriptional meta-analysis coupled with quantitative PCR to identify relative gene expression downstream of OCT4. We found that human skin cells cultured in the presence of BAY11 resulted in reproducible increased expression of OCT4 that did not inhibit normal cell proliferation. The increased levels of OCT4 resulted in significantly increased expression of genes downstream of OCT4, including the previously identified SPP1, DUSP4 and GADD45G, suggesting the expressed OCT4 was functional. We also discovered a novel OCT4 putative downstream target gene SLC16A9 which demonstrated significantly increased expression following elevation of OCT4 levels. For the first time we have shown that small molecule-based stabilization of synthetic mRNA expression can be achieved with use of BAY11. This small molecule-based inhibition of innate immune responses and subsequent robust expression of transfected synthetic mRNAs may have multiple applications for future cell-based research and therapeutics.

TMPyP4 porphyrin distorts RNA G-quadruplex structures of the disease-associated r(GGGGCC)n repeat of the C9orf72 gene and blocks interaction of RNA-binding proteins.

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Zamiri, Bita; Reddy, Kaalak; Macgregor, Robert B; Pearson, Christopher E

2014-02-21

Certain DNA and RNA sequences can form G-quadruplexes, which can affect genetic instability, promoter activity, RNA splicing, RNA stability, and neurite mRNA localization. Amyotrophic lateral sclerosis and frontotemporal dementia can be caused by expansion of a (GGGGCC)n repeat in the C9orf72 gene. Mutant r(GGGGCC)n- and r(GGCCCC)n-containing transcripts aggregate in nuclear foci, possibly sequestering repeat-binding proteins such as ASF/SF2 and hnRNPA1, suggesting a toxic RNA pathogenesis, as occurs in myotonic dystrophy. Furthermore, the C9orf72 repeat RNA was recently demonstrated to undergo the noncanonical repeat-associated non-AUG translation (RAN translation) into pathologic dipeptide repeats in patient brains, a process that is thought to depend upon RNA structure. We previously demonstrated that the r(GGGGCC)n RNA forms repeat tract length-dependent G-quadruplex structures that bind the ASF/SF2 protein. Here we show that the cationic porphyrin (5,10,15,20-tetra(N-methyl-4-pyridyl) porphyrin (TMPyP4)), which can bind some G-quadruplex-forming sequences, can bind and distort the G-quadruplex formed by r(GGGGCC)8, and this ablates the interaction of either hnRNPA1 or ASF/SF2 with the repeat. These findings provide proof of concept that nucleic acid binding small molecules, such as TMPyP4, can distort the secondary structure of the C9orf72 repeat, which may beneficially disrupt protein interactions, which may ablate either protein sequestration and/or RAN translation into potentially toxic dipeptides. Disruption of secondary structure formation of the C9orf72 RNA repeats may be a viable therapeutic avenue, as well as a means to test the role of RNA structure upon RAN translation.

Genetic and epigenetic variation in 5S ribosomal RNA genes reveals genome dynamics in Arabidopsis thaliana.

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Simon, Lauriane; Rabanal, Fernando A; Dubos, Tristan; Oliver, Cecilia; Lauber, Damien; Poulet, Axel; Vogt, Alexander; Mandlbauer, Ariane; Le Goff, Samuel; Sommer, Andreas; Duborjal, Hervé; Tatout, Christophe; Probst, Aline V

2018-04-06

Organized in tandem repeat arrays in most eukaryotes and transcribed by RNA polymerase III, expression of 5S rRNA genes is under epigenetic control. To unveil mechanisms of transcriptional regulation, we obtained here in depth sequence information on 5S rRNA genes from the Arabidopsis thaliana genome and identified differential enrichment in epigenetic marks between the three 5S rDNA loci situated on chromosomes 3, 4 and 5. We reveal the chromosome 5 locus as the major source of an atypical, long 5S rRNA transcript characteristic of an open chromatin structure. 5S rRNA genes from this locus translocated in the Landsberg erecta ecotype as shown by linkage mapping and chromosome-specific FISH analysis. These variations in 5S rDNA locus organization cause changes in the spatial arrangement of chromosomes in the nucleus. Furthermore, 5S rRNA gene arrangements are highly dynamic with alterations in chromosomal positions through translocations in certain mutants of the RNA-directed DNA methylation pathway and important copy number variations among ecotypes. Finally, variations in 5S rRNA gene sequence, chromatin organization and transcripts indicate differential usage of 5S rDNA loci in distinct ecotypes. We suggest that both the usage of existing and new 5S rDNA loci resulting from translocations may impact neighboring chromatin organization.

Intra-Genomic Heterogeneity in 16S rRNA Genes in Strictly Anaerobic Clinical Isolates from Periodontal Abscesses.

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Chen, Jiazhen; Miao, Xinyu; Xu, Meng; He, Junlin; Xie, Yi; Wu, Xingwen; Chen, Gang; Yu, Liying; Zhang, Wenhong

2015-01-01

Members of the genera Prevotella, Veillonella and Fusobacterium are the predominant culturable obligate anaerobic bacteria isolated from periodontal abscesses. When determining the cumulative number of clinical anaerobic isolates from periodontal abscesses, ambiguous or overlapping signals were frequently encountered in 16S rRNA gene sequencing chromatograms, resulting in ambiguous identifications. With the exception of the genus Veillonella, the high intra-chromosomal heterogeneity of rrs genes has not been reported. The 16S rRNA genes of 138 clinical, strictly anaerobic isolates and one reference strain were directly sequenced, and the chromatograms were carefully examined. Gene cloning was performed for 22 typical isolates with doublet sequencing signals for the 16S rRNA genes, and four copies of the rrs-ITS genes of 9 Prevotella intermedia isolates were separately amplified by PCR, sequenced and compared. Five conserved housekeeping genes, hsp60, recA, dnaJ, gyrB1 and rpoB from 89 clinical isolates of Prevotella were also amplified by PCR and sequenced for identification and phylogenetic analysis along with 18 Prevotella reference strains. Heterogeneity of 16S rRNA genes was apparent in clinical, strictly anaerobic oral bacteria, particularly in the genera Prevotella and Veillonella. One hundred out of 138 anaerobic strains (72%) had intragenomic nucleotide polymorphisms (SNPs) in multiple locations, and 13 strains (9.4%) had intragenomic insertions or deletions in the 16S rRNA gene. In the genera Prevotella and Veillonella, 75% (67/89) and 100% (19/19) of the strains had SNPs in the 16S rRNA gene, respectively. Gene cloning and separate amplifications of four copies of the rrs-ITS genes confirmed that 2 to 4 heterogeneous 16S rRNA copies existed. Sequence alignment of five housekeeping genes revealed that intra-species nucleotide similarities were very high in the genera Prevotella, ranging from 94.3-100%. However, the inter-species similarities were

Maternally Expressed Gene 3, an imprinted non-coding RNA gene, is associated with meningioma pathogenesis and progression

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Zhang, Xun; Gejman, Roger; Mahta, Ali; Zhong, Ying; Rice, Kimberley A.; Zhou, Yunli; Cheunsuchon, Pornsuk; Louis, David N.; Klibanski, Anne

2010-01-01

Meningiomas are common tumors, representing 15-25% of all central nervous system tumors. NF2 gene inactivation on chromosome 22 has been shown as an early event in tumorigenesis; however, few factors underlying tumor growth and progression have been identified. Chromosomal abnormalities of 14q32 are often associated with meningioma pathogenesis and progression; therefore it has been proposed that an as yet unidentified tumor suppressor is present at this locus. MEG3 is an imprinted gene located at 14q32 that encodes a non-coding RNA with an anti-proliferative function. We found that MEG3 mRNA is highly expressed in normal arachnoidal cells. However, MEG3 is not expressed in the majority of human meningiomas or the human meningioma cell lines IOMM-Lee and CH157-MN. There is a strong association between loss of MEG3 expression and tumor grade. Allelic loss at the MEG3 locus is also observed in meningiomas, with increasing prevalence in higher grade tumors. In addition, there is an increase in CpG methylation within the promoter and the imprinting control region of MEG3 gene in meningiomas. Functionally, MEG3 suppresses DNA synthesis in both IOMM-Lee and CH157-MN cells by approximately 60% in BrdU incorporation assays. Colony-forming efficiency assays show that MEG3 inhibits colony formation in CH157-MN cells by approximately 80%. Furthermore, MEG3 stimulates p53-mediated transactivation in these cell lines. Therefore, these data are consistent with the hypothesis that MEG3, which encodes a non-coding RNA, may be a tumor suppressor gene at chromosome 14q32 involved in meningioma progression via a novel mechanism. PMID:20179190

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

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Giuseppe Fiume

2016-11-01

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

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

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

2016-11-07

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

Selection of reference genes is critical for miRNA expression analysis in human cardiac tissue. A focus on atrial fibrillation.

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Masè, Michela; Grasso, Margherita; Avogaro, Laura; D'Amato, Elvira; Tessarolo, Francesco; Graffigna, Angelo; Denti, Michela Alessandra; Ravelli, Flavia

2017-01-24

MicroRNAs (miRNAs) are emerging as key regulators of complex biological processes in several cardiovascular diseases, including atrial fibrillation (AF). Reverse transcription-quantitative polymerase chain reaction is a powerful technique to quantitatively assess miRNA expression profile, but reliable results depend on proper data normalization by suitable reference genes. Despite the increasing number of studies assessing miRNAs in cardiac disease, no consensus on the best reference genes has been reached. This work aims to assess reference genes stability in human cardiac tissue with a focus on AF investigation. We evaluated the stability of five reference genes (U6, SNORD48, SNORD44, miR-16, and 5S) in atrial tissue samples from eighteen cardiac-surgery patients in sinus rhythm and AF. Stability was quantified by combining BestKeeper, delta-C q , GeNorm, and NormFinder statistical tools. All methods assessed SNORD48 as the best and U6 as the worst reference gene. Applications of different normalization strategies significantly impacted miRNA expression profiles in the study population. Our results point out the necessity of a consensus on data normalization in AF studies to avoid the emergence of divergent biological conclusions.

An intronic microRNA silences genes that are functionally antagonistic to its host gene.

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Barik, Sailen

2008-09-01

MicroRNAs (miRNAs) are short noncoding RNAs that down-regulate gene expression by silencing specific target mRNAs. While many miRNAs are transcribed from their own genes, nearly half map within introns of 'host' genes, the significance of which remains unclear. We report that transcriptional activation of apoptosis-associated tyrosine kinase (AATK), essential for neuronal differentiation, also generates miR-338 from an AATK gene intron that silences a family of mRNAs whose protein products are negative regulators of neuronal differentiation. We conclude that an intronic miRNA, transcribed together with the host gene mRNA, may serve the interest of its host gene by silencing a cohort of genes that are functionally antagonistic to the host gene itself.

Rapid Genome-wide Recruitment of RNA Polymerase II Drives Transcription, Splicing, and Translation Events during T Cell Responses

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Kathrin Davari

2017-04-01

Full Text Available Summary: Activation of immune cells results in rapid functional changes, but how such fast changes are accomplished remains enigmatic. By combining time courses of 4sU-seq, RNA-seq, ribosome profiling (RP, and RNA polymerase II (RNA Pol II ChIP-seq during T cell activation, we illustrate genome-wide temporal dynamics for ∼10,000 genes. This approach reveals not only immediate-early and posttranscriptionally regulated genes but also coupled changes in transcription and translation for >90% of genes. Recruitment, rather than release of paused RNA Pol II, primarily mediates transcriptional changes. This coincides with a genome-wide temporary slowdown in cotranscriptional splicing, even for polyadenylated mRNAs that are localized at the chromatin. Subsequent splicing optimization correlates with increasing Ser-2 phosphorylation of the RNA Pol II carboxy-terminal domain (CTD and activation of the positive transcription elongation factor (pTEFb. Thus, rapid de novo recruitment of RNA Pol II dictates the course of events during T cell activation, particularly transcription, splicing, and consequently translation. : Davari et al. visualize global changes in RNA Pol II binding, transcription, splicing, and translation. T cells change their functional program by rapid de novo recruitment of RNA Pol II and coupled changes in transcription and translation. This coincides with fluctuations in RNA Pol II phosphorylation and a temporary reduction in cotranscriptional splicing. Keywords: RNA Pol II, cotranscriptional splicing, T cell activation, ribosome profiling, 4sU, H3K36, Ser-5 RNA Pol II, Ser-2 RNA Pol II, immune response, immediate-early genes

miRNA-mediated functional changes through co-regulating function related genes.

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Jie He

Full Text Available BACKGROUND: MicroRNAs play important roles in various biological processes involving fairly complex mechanism. Analysis of genome-wide miRNA microarray demonstrate that a single miRNA can regulate hundreds of genes, but the regulative extent on most individual genes is surprisingly mild so that it is difficult to understand how a miRNA provokes detectable functional changes with such mild regulation. RESULTS: To explore the internal mechanism of miRNA-mediated regulation, we re-analyzed the data collected from genome-wide miRNA microarray with bioinformatics assay, and found that the transfection of miR-181b and miR-34a in Hela and HCT-116 tumor cells regulated large numbers of genes, among which, the genes related to cell growth and cell death demonstrated high Enrichment scores, suggesting that these miRNAs may be important in cell growth and cell death. MiR-181b induced changes in protein expression of most genes that were seemingly related to enhancing cell growth and decreasing cell death, while miR-34a mediated contrary changes of gene expression. Cell growth assays further confirmed this finding. In further study on miR-20b-mediated osteogenesis in hMSCs, miR-20b was found to enhance osteogenesis by activating BMPs/Runx2 signaling pathway in several stages by co-repressing of PPARγ, Bambi and Crim1. CONCLUSIONS: With its multi-target characteristics, miR-181b, miR-34a and miR-20b provoked detectable functional changes by co-regulating functionally-related gene groups or several genes in the same signaling pathway, and thus mild regulation from individual miRNA targeting genes could have contributed to an additive effect. This might also be one of the modes of miRNA-mediated gene regulation.

Identification of Phosphoglycerate Kinase 1 (PGK1 as a reference gene for quantitative gene expression measurements in human blood RNA

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Unger Elizabeth R

2011-09-01

Full Text Available Abstract Background Blood is a convenient sample and increasingly used for quantitative gene expression measurements with a variety of diseases including chronic fatigue syndrome (CFS. Quantitative gene expression measurements require normalization of target genes to reference genes that are stable and independent from variables being tested in the experiment. Because there are no genes that are useful for all situations, reference gene selection is an essential step to any quantitative reverse transcription-PCR protocol. Many publications have described appropriate genes for a wide variety of tissues and experimental conditions, however, reference genes that may be suitable for the analysis of CFS, or human blood RNA derived from whole blood as well as isolated peripheral blood mononuclear cells (PBMCs, have not been described. Findings Literature review and analyses of our unpublished microarray data were used to narrow down the pool of candidate reference genes to six. We assayed whole blood RNA from Tempus tubes and cell preparation tube (CPT-collected PBMC RNA from 46 subjects, and used the geNorm and NormFinder algorithms to select the most stable reference genes. Phosphoglycerate kinase 1 (PGK1 was one of the optimal normalization genes for both whole blood and PBMC RNA, however, additional genes differed for the two sample types; Ribosomal protein large, P0 (RPLP0 for PBMC RNA and Peptidylprolyl isomerase B (PPIB for whole blood RNA. We also show that the use of a single reference gene is sufficient for normalization when the most stable candidates are used. Conclusions We have identified PGK1 as a stable reference gene for use with whole blood RNA and RNA derived from PBMC. When stable genes are selected it is possible to use a single gene for normalization rather than two or three. Optimal normalization will improve the ability of results from PBMC RNA to be compared with those from whole blood RNA and potentially allows comparison of

Genes misregulated in C. elegans deficient in Dicer, RDE-4, or RDE-1 are enriched for innate immunity genes.

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Welker, Noah C; Habig, Jeffrey W; Bass, Brenda L

2007-07-01

We describe the first microarray analysis of a whole animal containing a mutation in the Dicer gene. We used adult Caenorhabditis elegans and, to distinguish among different roles of Dicer, we also performed microarray analyses of animals with mutations in rde-4 and rde-1, which are involved in silencing by siRNA, but not miRNA. Surprisingly, we find that the X chromosome is greatly enriched for genes regulated by Dicer. Comparison of all three microarray data sets indicates the majority of Dicer-regulated genes are not dependent on RDE-4 or RDE-1, including the X-linked genes. However, all three data sets are enriched in genes important for innate immunity and, specifically, show increased expression of innate immunity genes.

Systematic identification of novel, essential host genes affecting bromovirus RNA replication.

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Brandi L Gancarz

Full Text Available Positive-strand RNA virus replication involves viral proteins and cellular proteins at nearly every replication step. Brome mosaic virus (BMV is a well-established model for dissecting virus-host interactions and is one of very few viruses whose RNA replication, gene expression and encapsidation have been reproduced in the yeast Saccharomyces cerevisiae. Previously, our laboratory identified ∼100 non-essential host genes whose loss inhibited or enhanced BMV replication at least 3-fold. However, our isolation of additional BMV-modulating host genes by classical genetics and other results underscore that genes essential for cell growth also contribute to BMV RNA replication at a frequency that may be greater than that of non-essential genes. To systematically identify novel, essential host genes affecting BMV RNA replication, we tested a collection of ∼900 yeast strains, each with a single essential gene promoter replaced by a doxycycline-repressible promoter, allowing repression of gene expression by adding doxycycline to the growth medium. Using this strain array of ∼81% of essential yeast genes, we identified 24 essential host genes whose depleted expression reproducibly inhibited or enhanced BMV RNA replication. Relevant host genes are involved in ribosome biosynthesis, cell cycle regulation and protein homeostasis, among other cellular processes. BMV 2a(Pol levels were significantly increased in strains depleted for a heat shock protein (HSF1 or proteasome components (PRE1 and RPT6, suggesting these genes may affect BMV RNA replication by directly or indirectly modulating 2a(Pol localization, post-translational modification or interacting partners. Investigating the diverse functions of these newly identified essential host genes should advance our understanding of BMV-host interactions and normal cellular pathways, and suggest new modes of virus control.

Neighborhood of 16S rRNA nucleotides U788/U789 in the 30S ribosomal subunit determined by site-directed crosslinking.

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Mundus, D; Wollenzien, P

1998-01-01

Site-specific photo crosslinking has been used to investigate the RNA neighborhood of 16S rRNA positions U788/ U789 in Escherichia coli 30S subunits. For these studies, site-specific psoralen (SSP) which contains a sulfhydryl group on a 17 A side chain was first added to nucleotides U788/U789 using a complementary guide DNA by annealing and phototransfer. Modified RNA was purified from the DNA and unmodified RNA. For some experiments, the SSP, which normally crosslinks at an 8 A distance, was...

Dysregulation of RNA Mediated Gene Expression in Motor Neuron Diseases.

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Gonçalves, Inês do Carmo G; Rehorst, Wiebke A; Kye, Min Jeong

2016-01-01

Recent findings indicate an important role for RNA-mediated gene expression in motor neuron diseases, including ALS (amyotrophic lateral sclerosis) and SMA (spinal muscular atrophy). ALS, also known as Lou Gehrig's disease, is an adult-onset progressive neurodegenerative disorder, whereby SMA or "children's Lou Gehrig's disease" is considered a pediatric neurodevelopmental disorder. Despite the difference in genetic causes, both ALS and SMA share common phenotypes; dysfunction/loss of motor neurons that eventually leads to muscle weakness and atrophy. With advanced techniques in molecular genetics and cell biology, current data suggest that these two distinct motor neuron diseases share more than phenotypes; ALS and SMA have similar cellular pathological mechanisms including mitochondrial dysfunction, oxidative stress and dysregulation in RNA-mediated gene expression. Here, we will discuss the current findings on these two diseases with specific focus on RNA-mediated gene regulation including miRNA expression, pre-mRNA processing and RNA binding proteins.

Permuted tRNA genes of Cyanidioschyzon merolae, the origin of the tRNA molecule and the root of the Eukarya domain.

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Di Giulio, Massimo

2008-08-07

An evolutionary analysis is conducted on the permuted tRNA genes of Cyanidioschyzon merolae, in which the 5' half of the tRNA molecule is codified at the 3' end of the gene and its 3' half is codified at the 5' end. This analysis has shown that permuted genes cannot be considered as derived traits but seem to possess characteristics that suggest they are ancestral traits, i.e. they originated when tRNA molecule genes originated for the first time. In particular, if the hypothesis that permuted genes are a derived trait were true, then we should not have been able to observe that the most frequent class of permuted genes is that of the anticodon loop type, for the simple reason that this class would derive by random permutation from a class of non-permuted tRNA genes, which instead is the rarest. This would not explain the high frequency with which permuted tRNA genes with perfectly separate 5' and 3' halves were observed. Clearly the mechanism that produced this class of permuted genes would envisage the existence, in an advanced stage of evolution, of minigenes codifying for the 5' and 3' halves of tRNAs which were assembled in a permuted way at the origin of the tRNA molecule, thus producing a high frequency of permuted genes of the class here referred. Therefore, this evidence supports the hypothesis that the genes of the tRNA molecule were assembled by minigenes codifying for hairpin-like RNA molecules, as suggested by one model for the origin of tRNA [Di Giulio, M., 1992. On the origin of the transfer RNA molecule. J. Theor. Biol. 159, 199-214; Di Giulio, M., 1999. The non-monophyletic origin of tRNA molecule. J. Theor. Biol. 197, 403-414]. Moreover, the late assembly of the permuted genes of C. merolae, as well as their ancestrality, strengthens the hypothesis of the polyphyletic origins of these genes. Finally, on the basis of the uniqueness and the ancestrality of these permuted genes, I suggest that the root of the Eukarya domain is in the super

Origin of sphinx, a young chimeric RNA gene in Drosophila melanogaster

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Wang, Wen; Brunet, Frédéric G.; Nevo, Eviatar; Long, Manyuan

2002-01-01

Non-protein-coding RNA genes play an important role in various biological processes. How new RNA genes originated and whether this process is controlled by similar evolutionary mechanisms for the origin of protein-coding genes remains unclear. A young chimeric RNA gene that we term sphinx (spx) provides the first insight into the early stage of evolution of RNA genes. spx originated as an insertion of a retroposed sequence of the ATP synthase chain F gene at the cytological region 60DB since the divergence of Drosophila melanogaster from its sibling species 2–3 million years ago. This retrosequence, which is located at 102F on the fourth chromosome, recruited a nearby exon and intron, thereby evolving a chimeric gene structure. This molecular process suggests that the mechanism of exon shuffling, which can generate protein-coding genes, also plays a role in the origin of RNA genes. The subsequent evolutionary process of spx has been associated with a high nucleotide substitution rate, possibly driven by a continuous positive Darwinian selection for a novel function, as is shown in its sex- and development-specific alternative splicing. To test whether spx has adapted to different environments, we investigated its population genetic structure in the unique “Evolution Canyon” in Israel, revealing a similar haplotype structure in spx, and thus similar evolutionary forces operating on spx between environments. PMID:11904380

Identification of genes regulating migration and invasion using a new model of metastatic prostate cancer

International Nuclear Information System (INIS)

Banyard, Jacqueline; Chung, Ivy; Migliozzi, Matthew; Phan, Derek T; Wilson, Arianne M; Zetter, Bruce R; Bielenberg, Diane R

2014-01-01

Understanding the complex, multistep process of metastasis remains a major challenge in cancer research. Metastasis models can reveal insights in tumor development and progression and provide tools to test new intervention strategies. To develop a new cancer metastasis model, we used DU145 human prostate cancer cells and performed repeated rounds of orthotopic prostate injection and selection of subsequent lymph node metastases. Tumor growth, metastasis, cell migration and invasion were analyzed. Microarray analysis was used to identify cell migration- and cancer-related genes correlating with metastasis. Selected genes were silenced using siRNA, and their roles in cell migration and invasion were determined in transwell migration and Matrigel invasion assays. Our in vivo cycling strategy created cell lines with dramatically increased tumorigenesis and increased ability to colonize lymph nodes (DU145LN1-LN4). Prostate tumor xenografts displayed increased vascularization, enlarged podoplanin-positive lymphatic vessels and invasive margins. Microarray analysis revealed gene expression profiles that correlated with metastatic potential. Using gene network analysis we selected 3 significantly upregulated cell movement and cancer related genes for further analysis: EPCAM (epithelial cell adhesion molecule), ITGB4 (integrin β4) and PLAU (urokinase-type plasminogen activator (uPA)). These genes all showed increased protein expression in the more metastatic DU145-LN4 cells compared to the parental DU145. SiRNA knockdown of EpCAM, integrin-β4 or uPA all significantly reduced cell migration in DU145-LN4 cells. In contrast, only uPA siRNA inhibited cell invasion into Matrigel. This role of uPA in cell invasion was confirmed using the uPA inhibitors, amiloride and UK122. Our approach has identified genes required for the migration and invasion of metastatic tumor cells, and we propose that our new in vivo model system will be a powerful tool to interrogate the metastatic

Amniotic fluid RNA gene expression profiling provides insights into the phenotype of Turner syndrome.

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Massingham, Lauren J; Johnson, Kirby L; Scholl, Thomas M; Slonim, Donna K; Wick, Heather C; Bianchi, Diana W

2014-09-01

Turner syndrome is a sex chromosome aneuploidy with characteristic malformations. Amniotic fluid, a complex biological material, could contribute to the understanding of Turner syndrome pathogenesis. In this pilot study, global gene expression analysis of cell-free RNA in amniotic fluid supernatant was utilized to identify specific genes/organ systems that may play a role in Turner syndrome pathophysiology. Cell-free RNA from amniotic fluid of five mid-trimester Turner syndrome fetuses and five euploid female fetuses matched for gestational age was extracted, amplified, and hybridized onto Affymetrix(®) U133 Plus 2.0 arrays. Significantly differentially regulated genes were identified using paired t tests. Biological interpretation was performed using Ingenuity Pathway Analysis and BioGPS gene expression atlas. There were 470 statistically significantly differentially expressed genes identified. They were widely distributed across the genome. XIST was significantly down-regulated (p Turner syndrome transcriptome from other aneuploidies we previously studied. Manual curation of the differentially expressed gene list identified genes of possible pathologic significance, including NFATC3, IGFBP5, and LDLR. Transcriptomic differences in the amniotic fluid of Turner syndrome fetuses are due to genome-wide dysregulation. The hematologic/immune system differences may play a role in early-onset autoimmune dysfunction. Other genes identified with possible pathologic significance are associated with cardiac and skeletal systems, which are known to be affected in females with Turner syndrome. The discovery-driven approach described here may be useful in elucidating novel mechanisms of disease in Turner syndrome.

The translation initiation factor eIF4E regulates the sex-specific expression of the master switch gene Sxl in Drosophila melanogaster.

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Patricia L Graham

2011-07-01

Full Text Available In female fruit flies, Sex-lethal (Sxl turns off the X chromosome dosage compensation system by a mechanism involving a combination of alternative splicing and translational repression of the male specific lethal-2 (msl-2 mRNA. A genetic screen identified the translation initiation factor eif4e as a gene that acts together with Sxl to repress expression of the Msl-2 protein. However, eif4e is not required for Sxl mediated repression of msl-2 mRNA translation. Instead, eif4e functions as a co-factor in Sxl-dependent female-specific alternative splicing of msl-2 and also Sxl pre-mRNAs. Like other factors required for Sxl regulation of splicing, eif4e shows maternal-effect female-lethal interactions with Sxl. This female lethality can be enhanced by mutations in other co-factors that promote female-specific splicing and is caused by a failure to properly activate the Sxl-positive autoregulatory feedback loop in early embryos. In this feedback loop Sxl proteins promote their own synthesis by directing the female-specific alternative splicing of Sxl-Pm pre-mRNAs. Analysis of pre-mRNA splicing when eif4e activity is compromised demonstrates that Sxl-dependent female-specific splicing of both Sxl-Pm and msl-2 pre-mRNAs requires eif4e activity. Consistent with a direct involvement in Sxl-dependent alternative splicing, eIF4E is associated with unspliced Sxl-Pm pre-mRNAs and is found in complexes that contain early acting splicing factors--the U1/U2 snRNP protein Sans-fils (Snf, the U1 snRNP protein U1-70k, U2AF38, U2AF50, and the Wilms' Tumor 1 Associated Protein Fl(2d--that have been directly implicated in Sxl splicing regulation.

Glucocorticoids selectively inhibit the transcription of the interleukin 1β gene and decrease the stability of interleukin 1β mRNA

International Nuclear Information System (INIS)

Lee, S.W.; Tsou, A.P.; Chan, H.; Thomas, J.; Petrie, K.; Eugui, E.M.; Allison, A.C.

1988-01-01

Transcription of the interleukin 1β (IL-1β) gene was studied by mRNA hybridization with a cDNA probe in the human promonocytic cell line U-937. Phorbol ester and lipopolysaccharide increased the steady-state level of Il-1β mRNA. Glucocorticoids markedly decreased IL-1β mRNA levels by two mechanisms. Transcription of the IL-1 gene was inhibited, as shown by in vitro transcription assays with nuclei isolated from glucocorticoid-treated cells. Moreover, kinetic analyses and pulse-labeling of mRNAs showed that glucocorticoids selectively decrease the stability of IL-1β mRNA, without affecting the stability of β-actin and FOS mRNAs. Inhibition of the formation and effects IL-1 is a mechanism by which glucocorticoids can exert antiinflammatory and immunosuppressive effects

Genome-Wide Analysis of the RNA Helicase Gene Family in Gossypium raimondii

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Jie Chen

2014-03-01

Full Text Available The RNA helicases, which help to unwind stable RNA duplexes, and have important roles in RNA metabolism, belong to a class of motor proteins that play important roles in plant development and responses to stress. Although this family of genes has been the subject of systematic investigation in Arabidopsis, rice, and tomato, it has not yet been characterized in cotton. In this study, we identified 161 putative RNA helicase genes in the genome of the diploid cotton species Gossypium raimondii. We classified these genes into three subfamilies, based on the presence of either a DEAD-box (51 genes, DEAH-box (52 genes, or DExD/H-box (58 genes in their coding regions. Chromosome location analysis showed that the genes that encode RNA helicases are distributed across all 13 chromosomes of G. raimondii. Syntenic analysis revealed that 62 of the 161 G. raimondii helicase genes (38.5% are within the identified syntenic blocks. Sixty-six (40.99% helicase genes from G. raimondii have one or several putative orthologs in tomato. Additionally, GrDEADs have more conserved gene structures and more simple domains than GrDEAHs and GrDExD/Hs. Transcriptome sequencing data demonstrated that many of these helicases, especially GrDEADs, are highly expressed at the fiber initiation stage and in mature leaves. To our knowledge, this is the first report of a genome-wide analysis of the RNA helicase gene family in cotton.

Predicting gene regulatory networks of soybean nodulation from RNA-Seq transcriptome data.

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Zhu, Mingzhu; Dahmen, Jeremy L; Stacey, Gary; Cheng, Jianlin

2013-09-22

High-throughput RNA sequencing (RNA-Seq) is a revolutionary technique to study the transcriptome of a cell under various conditions at a systems level. Despite the wide application of RNA-Seq techniques to generate experimental data in the last few years, few computational methods are available to analyze this huge amount of transcription data. The computational methods for constructing gene regulatory networks from RNA-Seq expression data of hundreds or even thousands of genes are particularly lacking and urgently needed. We developed an automated bioinformatics method to predict gene regulatory networks from the quantitative expression values of differentially expressed genes based on RNA-Seq transcriptome data of a cell in different stages and conditions, integrating transcriptional, genomic and gene function data. We applied the method to the RNA-Seq transcriptome data generated for soybean root hair cells in three different development stages of nodulation after rhizobium infection. The method predicted a soybean nodulation-related gene regulatory network consisting of 10 regulatory modules common for all three stages, and 24, 49 and 70 modules separately for the first, second and third stage, each containing both a group of co-expressed genes and several transcription factors collaboratively controlling their expression under different conditions. 8 of 10 common regulatory modules were validated by at least two kinds of validations, such as independent DNA binding motif analysis, gene function enrichment test, and previous experimental data in the literature. We developed a computational method to reliably reconstruct gene regulatory networks from RNA-Seq transcriptome data. The method can generate valuable hypotheses for interpreting biological data and designing biological experiments such as ChIP-Seq, RNA interference, and yeast two hybrid experiments.

Dendrimers as Carriers for siRNA Delivery and Gene Silencing: A Review

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Jiangyu Wu

2013-01-01

Full Text Available RNA interference (RNAi was first literaturally reported in 1998 and has become rapidly a promising tool for therapeutic applications in gene therapy. In a typical RNAi process, small interfering RNAs (siRNA are used to specifically downregulate the expression of the targeted gene, known as the term “gene silencing.” One key point for successful gene silencing is to employ a safe and efficient siRNA delivery system. In this context, dendrimers are emerging as potential nonviral vectors to deliver siRNA for RNAi purpose. Dendrimers have attracted intense interest since their emanating research in the 1980s and are extensively studied as efficient DNA delivery vectors in gene transfer applications, due to their unique features based on the well-defined and multivalent structures. Knowing that DNA and RNA possess a similar structure in terms of nucleic acid framework and the electronegative nature, one can also use the excellent DNA delivery properties of dendrimers to develop effective siRNA delivery systems. In this review, the development of dendrimer-based siRNA delivery vectors is summarized, focusing on the vector features (siRNA delivery efficiency, cytotoxicity, etc. of different types of dendrimers and the related investigations on structure-activity relationship to promote safe and efficient siRNA delivery system.

Dendrimers as Carriers for siRNA Delivery and Gene Silencing: A Review

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Huang, Weizhe; He, Ziying

2013-01-01

RNA interference (RNAi) was first literaturally reported in 1998 and has become rapidly a promising tool for therapeutic applications in gene therapy. In a typical RNAi process, small interfering RNAs (siRNA) are used to specifically downregulate the expression of the targeted gene, known as the term “gene silencing.” One key point for successful gene silencing is to employ a safe and efficient siRNA delivery system. In this context, dendrimers are emerging as potential nonviral vectors to deliver siRNA for RNAi purpose. Dendrimers have attracted intense interest since their emanating research in the 1980s and are extensively studied as efficient DNA delivery vectors in gene transfer applications, due to their unique features based on the well-defined and multivalent structures. Knowing that DNA and RNA possess a similar structure in terms of nucleic acid framework and the electronegative nature, one can also use the excellent DNA delivery properties of dendrimers to develop effective siRNA delivery systems. In this review, the development of dendrimer-based siRNA delivery vectors is summarized, focusing on the vector features (siRNA delivery efficiency, cytotoxicity, etc.) of different types of dendrimers and the related investigations on structure-activity relationship to promote safe and efficient siRNA delivery system. PMID:24288498

Elevation of D4 dopamine receptor mRNA in postmortem schizophrenic brain.

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Stefanis, N C; Bresnick, J N; Kerwin, R W; Schofield, W N; McAllister, G

1998-01-01

The D4 dopamine (DA) receptor has been proposed to be a target for the development of a novel antipsychotic drug based on its pharmacological and distribution profile. There is much interest in whether D4 DA receptor levels are altered in schizophrenia, but the lack of an available receptor subtype-specific radioligand made this difficult to quantitate. In this study, we examined whether D4 mRNA levels are altered in different brain regions of schizophrenics compared to controls. Ribonuclease protection assays were carried out on total RNA samples isolated postmortem from frontal cortex and caudate brain regions of schizophrenics and matched controls. 32P-labelled RNA probes to the D4 DA receptor and to the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (G3PDH), were hybridised with the RNA samples, digested with ribonucleases to remove unhybridised probe, and separated on 6% sequencing gels. Densitometer analysis on the subsequent autoradiogams was used to calculate the relative optical density of D4 mRNA compared to G3PDH mRNA. Statistical analysis of the data revealed a 3-fold higher level (P<0.011) of D4 mRNA in the frontal cortex of schizophrenics compared to controls. No increase was seen in caudate. D4 receptors could play a role in mediating dopaminergic activity in frontal cortex, an activity which may be malfunctioning in schizophrenia.

[Investigation into the relationship between mitochondrial 12 S rRNA gene, tRNA gene and cytochrome oxidase Ⅱ gene variations and the risk of noise-induced hearing loss].

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Jiao, J; Gu, G Z; Chen, G S; Li, Y H; Zhang, H L; Yang, Q Y; Xu, X R; Zhou, W H; Wu, H; He, L H; Zheng, Y X; Yu, S F

2017-01-06

Objective: To explore the relationship between mitochondrial 12 S rRNA gene variation, tRNA gene variation and cytochrome oxidase Ⅱ gene point mutations and the risk of noise-induced hearing loss (NIHL). Methods: A nested case-control study was performed that followed a cohort of 7 445 noise-exposed workers in a steel factory in Henan province, China, from January 1, 2006 to December 31, 2015. Subjects whose average hearing threshold was more than 40 dB(A) in high frequency were defined as the case group, and subjects whose average hearing threshold was less than 35 dB(A) in high frequency and less than 25 dB (A) in speech frequency were defined as the control group. Subjects was recruited into the case group ( n =286) and the control group ( n= 286) according to gender, age, job category and time of exposure to noise, and a 1∶1 case-control study was carried out. We genotyped eight single nucleotide polymorphisms in the mitochondrial 12 S rRNA gene, the mitochondrial tRNA gene and the mitochondrial cytochrome oxidase Ⅱ gene using SNPscan high-throughput genotyping technology from the recruited subjects. The relationship between polymorphic sites and NIHL, adjusted for covariates, was analyzed using conditional logistic regression analysis, as were the subgroup data. Results: The average age of the recruited subjects was (40.3±8.1) years and the length of service exposure to noise was (18.6±8.9) years. The range of noise exposed levels and cumulative noise exposure (CNE) was 80.1- 93.4 dB (A) and 86.8- 107.9 dB (A) · year, respectively. For workers exposed to noise at a CNE level<98 dB (A) · year, smokers showed an increased risk of NIHL of 1.88 (1.16-3.05) compared with non-smokers; for workers exposed to noise at a CNE level ≥98 dB(A) · year, smokers showed an increased risk of NIHL of 2.53 (1.49- 4.30) compared with non-smokers. For workers exposed to noise at a CNE level<98 dB (A) · year, the results of univariate analysis and multifactor analysis

RNA amplification for successful gene profiling analysis

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Wang Ena

2005-07-01

Full Text Available Abstract The study of clinical samples is often limited by the amount of material available to study. While proteins cannot be multiplied in their natural form, DNA and RNA can be amplified from small specimens and used for high-throughput analyses. Therefore, genetic studies offer the best opportunity to screen for novel insights of human pathology when little material is available. Precise estimates of DNA copy numbers in a given specimen are necessary. However, most studies investigate static variables such as the genetic background of patients or mutations within pathological specimens without a need to assess proportionality of expression among different genes throughout the genome. Comparative genomic hybridization of DNA samples represents a crude exception to this rule since genomic amplification or deletion is compared among different specimens directly. For gene expression analysis, however, it is critical to accurately estimate the proportional expression of distinct RNA transcripts since such proportions directly govern cell function by modulating protein expression. Furthermore, comparative estimates of relative RNA expression at different time points portray the response of cells to environmental stimuli, indirectly informing about broader biological events affecting a particular tissue in physiological or pathological conditions. This cognitive reaction of cells is similar to the detection of electroencephalographic patterns which inform about the status of the brain in response to external stimuli. As our need to understand human pathophysiology at the global level increases, the development and refinement of technologies for high fidelity messenger RNA amplification have become the focus of increasing interest during the past decade. The need to increase the abundance of RNA has been met not only for gene specific amplification, but, most importantly for global transcriptome wide, unbiased amplification. Now gene

Intrinsic challenges in ancient microbiome reconstruction using 16S rRNA gene amplification.

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Ziesemer, Kirsten A; Mann, Allison E; Sankaranarayanan, Krithivasan; Schroeder, Hannes; Ozga, Andrew T; Brandt, Bernd W; Zaura, Egija; Waters-Rist, Andrea; Hoogland, Menno; Salazar-García, Domingo C; Aldenderfer, Mark; Speller, Camilla; Hendy, Jessica; Weston, Darlene A; MacDonald, Sandy J; Thomas, Gavin H; Collins, Matthew J; Lewis, Cecil M; Hofman, Corinne; Warinner, Christina

2015-11-13

To date, characterization of ancient oral (dental calculus) and gut (coprolite) microbiota has been primarily accomplished through a metataxonomic approach involving targeted amplification of one or more variable regions in the 16S rRNA gene. Specifically, the V3 region (E. coli 341-534) of this gene has been suggested as an excellent candidate for ancient DNA amplification and microbial community reconstruction. However, in practice this metataxonomic approach often produces highly skewed taxonomic frequency data. In this study, we use non-targeted (shotgun metagenomics) sequencing methods to better understand skewed microbial profiles observed in four ancient dental calculus specimens previously analyzed by amplicon sequencing. Through comparisons of microbial taxonomic counts from paired amplicon (V3 U341F/534R) and shotgun sequencing datasets, we demonstrate that extensive length polymorphisms in the V3 region are a consistent and major cause of differential amplification leading to taxonomic bias in ancient microbiome reconstructions based on amplicon sequencing. We conclude that systematic amplification bias confounds attempts to accurately reconstruct microbiome taxonomic profiles from 16S rRNA V3 amplicon data generated using universal primers. Because in silico analysis indicates that alternative 16S rRNA hypervariable regions will present similar challenges, we advocate for the use of a shotgun metagenomics approach in ancient microbiome reconstructions.

Methylation of 10 miRNA genes in clear cell renal cell carcinoma and their diagnostic value

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V. I. Loginov

2017-01-01

Full Text Available Introduction. Clear cell renal cell carcinoma (ccRCC is characterized by the high (30–40 % of cases frequency of lethal outcomes which at metastasis reaches 90 %. Lack of efficient diagnostics at early stages of a disease indicates the need of searching on new ccRCC markers.Objective: for definition of methylation role of some tumor suppressor microRNA (miRNA genes in ccRCC pathogenesis and progression and marker identification for ccRCC diagnostics and metastasis predictions.Materials and methods. The alterations of methylation status of 10 miRNA genes were determined by methylation specific polymerase chain reaction in tumor DNA samples and matched histologically unchanged tissues from 70 patients with ccRCC, as well as in DNA samples of kidney tissues from 19 post-mortal individuals without cancer history. Methylation of MIR MIR-107, -130b and -148a genes in ccRCC was studied for the first time.Results. It was shown that 8 miRNA genes (MIR-9-1/3, -34b/c, -124a-1/2/3, -129-2, -130b were methylated in ccRCC tumors with significantly higher frequency than in the matched histologically unchanged kidney tissues. It was established the association of methylation of 4 miRNA genes (MIR-107, -124a-3, -129-2, -130b with ccRCC progression (stage, tumor size, differentiation grade, including metastasis in the lymph nodes or distant organs, revealed for MIR-107 and -129-2. The association of MIR-107 and -130b methylation with progression of ccRCC is shown for the first time. Potential marker systems are made for ccRCC diagnostics using tumor biopsy; according to the ROC analysis, systems from 4 and 5 genes (MIR-9-1, -4b/c, -124a-3, -129-2/with addition of MIR-130b are characterized by high clinical sensitivity of 90 % and specificity of 94 % (area under ROC curve 0.93 and 0.94. Conclusion. The received results will form the basis of noninvasive ccRCC diagnostics further development. To conclude, it is shown the association of methylation of 9

[Clinical Significance of ID4 Gene Mehtylation in Demethylation-Treated MDS Cell Line and 2 MDS Patients].

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Kang, Hui-Yuan; Wang, Xin-Rong; Gao, Li; Wang, Wei; Li, Mian-Yang; Wang, Li-Li; Wang, Cheng-Bin; Yu, Li

2015-04-01

To evaluate significance of ID4 gene mehtylation in demethylating myelodysplastic syndrome(MDS) cell Line MUTZ1 and 2 patients with MDS. The methylation-specific PCR (MS-PCR) and reverse transcription-PCR (RT-PCR) were applied to identify the methylation status and gene expression of ID4 gene in MDS cell line MUTZ1, a patient with aplastic anemia(AA) and a donor with normal bone marrow (NBM). RT-PCR was applied to detect the ID4 gene expression status in MUTZ1 cell line treated with decitabine at 3 different concentrations. Then bisulfite sequencing PCR (BSP) was applied to detect ID4 gene methylation status in 2 MDS parients treated with decitabine. The MDS cell line MUTZ-1 displayed a complete methylation of ID4 gene promoter with little mRNA expression. Inversely, bone marrow of an AA patient and NBM showed complete unmethylation of this gene with intensity mRNA expression. With the increase of decitabine concentration, ID4 gene mRNA expression was more and more increased. After decitabine treatment, ID4 gene methylation-positive frequencies of both the 2 MDS patients were much more decreased than that of the first treatment. So, ID4 gene mRNA expression inhibited by promoter hypemethylation could be recovered by using demethylation medicine. ID4 as a new potential anti-oncogene suggests that its methylation may become a marker for selection and assessment of therapeutic schedules in patients with MDS.

MicroRNA and gene signature of severe cutaneous drug ...

African Journals Online (AJOL)

Purpose: To build a microRNA and gene signature of severe cutaneous adverse drug reactions (SCAR), including Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN). Methods: MicroRNA expression profiles were downloaded from miRNA expression profile of patients' skin suffering from TEN using an ...

Identifying miRNA and gene modules of colon cancer associated with pathological stage by weighted gene co-expression network analysis

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Zhou X

2018-05-01

Full Text Available Xian-guo Zhou,1,2,* Xiao-liang Huang,1,2,* Si-yuan Liang,1–3 Shao-mei Tang,1,2 Si-kao Wu,1,2 Tong-tong Huang,1,2 Zeng-nan Mo,1,2,4 Qiu-yan Wang1,2,5 1Center for Genomic and Personalized Medicine, Guangxi Medical University, Nanning, Guangxi Zhuang Autonomous Region, People’s Republic of China; 2Guangxi Key Laboratory for Genomic and Personalized Medicine, Guangxi Collaborative Innovation Center for Genomic and Personalized Medicine, Nanning, Guangxi Zhuang Autonomous Region, People’s Republic of China; 3Department of Colorectal Surgery, First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi Zhuang Autonomous Region, People’s Republic of China; 4Department of Urology and Nephrology, The First Affiliated Hospital of Guangxi, Medical University, Nanning, Guangxi Zhuang Autonomous Region, People’s Republic of China; 5Guangxi Colleges and Universities Key Laboratory of Biological Molecular Medicine Research, Guangxi Medical University, Nanning, Guangxi Zhuang Autonomous Region, People’s Republic of China *These authors contributed equally to this work Introduction: Colorectal cancer (CRC is the fourth most common cause of cancer-related mortality worldwide. The tumor, node, metastasis (TNM stage remains the standard for CRC prognostication. Identification of meaningful microRNA (miRNA and gene modules or representative biomarkers related to the pathological stage of colon cancer helps to predict prognosis and reveal the mechanisms behind cancer progression.Materials and methods: We applied a systems biology approach by combining differential expression analysis and weighted gene co-expression network analysis (WGCNA to detect the pathological stage-related miRNA and gene modules and construct a miRNA–gene network. The Cancer Genome Atlas (TCGA colon adenocarcinoma (CAC RNA-sequencing data and miRNA-sequencing data were subjected to WGCNA analysis, and the GSE29623, GSE35602 and GSE39396 were utilized to validate and

Fragmentation of the large subunit ribosomal RNA gene in oyster mitochondrial genomes

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Milbury Coren A

2010-09-01

Full Text Available Abstract Background Discontinuous genes have been observed in bacteria, archaea, and eukaryotic nuclei, mitochondria and chloroplasts. Gene discontinuity occurs in multiple forms: the two most frequent forms result from introns that are spliced out of the RNA and the resulting exons are spliced together to form a single transcript, and fragmented gene transcripts that are not covalently attached post-transcriptionally. Within the past few years, fragmented ribosomal RNA (rRNA genes have been discovered in bilateral metazoan mitochondria, all within a group of related oysters. Results In this study, we have characterized this fragmentation with comparative analysis and experimentation. We present secondary structures, modeled using comparative sequence analysis of the discontinuous mitochondrial large subunit rRNA genes of the cupped oysters C. virginica, C. gigas, and C. hongkongensis. Comparative structure models for the large subunit rRNA in each of the three oyster species are generally similar to those for other bilateral metazoans. We also used RT-PCR and analyzed ESTs to determine if the two fragmented LSU rRNAs are spliced together. The two segments are transcribed separately, and not spliced together although they still form functional rRNAs and ribosomes. Conclusions Although many examples of discontinuous ribosomal genes have been documented in bacteria and archaea, as well as the nuclei, chloroplasts, and mitochondria of eukaryotes, oysters are some of the first characterized examples of fragmented bilateral animal mitochondrial rRNA genes. The secondary structures of the oyster LSU rRNA fragments have been predicted on the basis of previous comparative metazoan mitochondrial LSU rRNA structure models.

Negative regulation of neuromedin U mRNA expression in the rat pars tuberalis by melatonin.

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Sayaka Aizawa

Full Text Available The pars tuberalis (PT is part of the anterior pituitary gland surrounding the median eminence as a thin cell layer. The characteristics of PT differ from those of the pars distalis (PD, such as cell composition and gene expression, suggesting that the PT has a unique physiological function compared to the PD. Because the PT highly expresses melatonin receptor type 1, it is considered a mediator of seasonal and/or circadian signals of melatonin. Expression of neuromedin U (NMU that is known to regulate energy balance has been previously reported in the rat PT; however, the regulatory mechanism of NMU mRNA expression and secretion in the PT are still obscure. In this study, we examined both the diurnal change of NMU mRNA expression in the rat PT and the effects of melatonin on NMU in vivo. In situ hybridization and quantitative PCR analysis of laser microdissected PT samples revealed that NMU mRNA expression in the PT has diurnal variation that is high during the light phase and low during the dark phase. Furthermore, melatonin administration significantly suppressed NMU mRNA expression in the PT in vivo. On the other hand, 48 h fasting did not have an effect on PT-NMU mRNA expression, and the diurnal change of NMU mRNA expression was maintained. We also found the highest expression of neuromedin U receptor type 2 (NMUR2 mRNA in the third ventricle ependymal cell layer, followed by the arcuate nucleus and the spinal cord. These results suggest that NMU mRNA expression in the PT is downregulated by melatonin during the dark phase and shows diurnal change. Considering that NMU mRNA in the PT showed the highest expression level in the brain, PT-NMU may act on NMUR2 in the brain, especially in the third ventricle ependymal cell layer, with a circadian rhythm.

RNA interference and retinoblastoma-related genes are required for repression of endogenous siRNA targets in Caenorhabditis elegans.

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Grishok, Alla; Hoersch, Sebastian; Sharp, Phillip A

2008-12-23

In Caenorhabditis elegans, a vast number of endogenous short RNAs corresponding to thousands of genes have been discovered recently. This finding suggests that these short interfering RNAs (siRNAs) may contribute to regulation of many developmental and other signaling pathways in addition to silencing viruses and transposons. Here, we present a microarray analysis of gene expression in RNA interference (RNAi)-related mutants rde-4, zfp-1, and alg-1 and the retinoblastoma (Rb) mutant lin-35. We found that a component of Dicer complex RDE-4 and a chromatin-related zinc finger protein ZFP-1, not implicated in endogenous RNAi, regulate overlapping sets of genes. Notably, genes a) up-regulated in the rde-4 and zfp-1 mutants and b) up-regulated in the lin-35(Rb) mutant, but not the down-regulated genes are highly represented in the set of genes with corresponding endogenous siRNAs (endo-siRNAs). Our study suggests that endogenous siRNAs cooperate with chromatin factors, either C. elegans ortholog of acute lymphoblastic leukemia-1 (ALL-1)-fused gene from chromosome 10 (AF10), ZFP-1, or tumor suppressor Rb, to regulate overlapping sets of genes and predicts a large role for RNAi-based chromatin silencing in control of gene expression in C. elegans.

About miRNAs, miRNA seeds, target genes and target pathways.

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Kehl, Tim; Backes, Christina; Kern, Fabian; Fehlmann, Tobias; Ludwig, Nicole; Meese, Eckart; Lenhof, Hans-Peter; Keller, Andreas

2017-12-05

miRNAs are typically repressing gene expression by binding to the 3' UTR, leading to degradation of the mRNA. This process is dominated by the eight-base seed region of the miRNA. Further, miRNAs are known not only to target genes but also to target significant parts of pathways. A logical line of thoughts is: miRNAs with similar (seed) sequence target similar sets of genes and thus similar sets of pathways. By calculating similarity scores for all 3.25 million pairs of 2,550 human miRNAs, we found that this pattern frequently holds, while we also observed exceptions. Respective results were obtained for both, predicted target genes as well as experimentally validated targets. We note that miRNAs target gene set similarity follows a bimodal distribution, pointing at a set of 282 miRNAs that seems to target genes with very high specificity. Further, we discuss miRNAs with different (seed) sequences that nonetheless regulate similar gene sets or pathways. Most intriguingly, we found miRNA pairs that regulate different gene sets but similar pathways such as miR-6886-5p and miR-3529-5p. These are jointly targeting different parts of the MAPK signaling cascade. The main goal of this study is to provide a general overview on the results, to highlight a selection of relevant results on miRNAs, miRNA seeds, target genes and target pathways and to raise awareness for artifacts in respective comparisons. The full set of information that allows to infer detailed results on each miRNA has been included in miRPathDB, the miRNA target pathway database (https://mpd.bioinf.uni-sb.de).

RNA-seq reveals more consistent reference genes for gene expression studies in human non-melanoma skin cancers

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Van L.T. Hoang

2017-08-01

Full Text Available Identification of appropriate reference genes (RGs is critical to accurate data interpretation in quantitative real-time PCR (qPCR experiments. In this study, we have utilised next generation RNA sequencing (RNA-seq to analyse the transcriptome of a panel of non-melanoma skin cancer lesions, identifying genes that are consistently expressed across all samples. Genes encoding ribosomal proteins were amongst the most stable in this dataset. Validation of this RNA-seq data was examined using qPCR to confirm the suitability of a set of highly stable genes for use as qPCR RGs. These genes will provide a valuable resource for the normalisation of qPCR data for the analysis of non-melanoma skin cancer.

Folate deficiency facilitates recruitment of upstream binding factor to hot spots of DNA double-strand breaks of rRNA genes and promotes its transcription.

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Xie, Qiu; Li, Caihua; Song, Xiaozhen; Wu, Lihua; Jiang, Qian; Qiu, Zhiyong; Cao, Haiyan; Yu, Kaihui; Wan, Chunlei; Li, Jianting; Yang, Feng; Huang, Zebing; Niu, Bo; Jiang, Zhengwen; Zhang, Ting

2017-03-17

The biogenesis of ribosomes in vivo is an essential process for cellular functions. Transcription of ribosomal RNA (rRNA) genes is the rate-limiting step in ribosome biogenesis controlled by environmental conditions. Here, we investigated the role of folate antagonist on changes of DNA double-strand breaks (DSBs) landscape in mouse embryonic stem cells. A significant DSB enhancement was detected in the genome of these cells and a large majority of these DSBs were found in rRNA genes. Furthermore, spontaneous DSBs in cells under folate deficiency conditions were located exclusively within the rRNA gene units, representing a H3K4me1 hallmark. Enrichment H3K4me1 at the hot spots of DSB regions enhanced the recruitment of upstream binding factor (UBF) to rRNA genes, resulting in the increment of rRNA genes transcription. Supplement of folate resulted in a restored UBF binding across DNA breakage sites of rRNA genes, and normal rRNA gene transcription. In samples from neural tube defects (NTDs) with low folate level, up-regulation of rRNA gene transcription was observed, along with aberrant UBF level. Our results present a new view by which alterations in folate levels affects DNA breakage through epigenetic control leading to the regulation of rRNA gene transcription during the early stage of development. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Substrate-assisted mechanism of RNP disruption by the spliceosomal Brr2 RNA helicase

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Theuser, Matthias; Höbartner, Claudia; Wahl, Markus C.; Santos, Karine F.

2016-01-01

The Brr2 RNA helicase disrupts the U4/U6 di-small nuclear RNA–protein complex (di-snRNP) during spliceosome activation via ATP-driven translocation on the U4 snRNA strand. However, it is unclear how bound proteins influence U4/U6 unwinding, which regions of the U4/U6 duplex the helicase actively unwinds, and whether U4/U6 components are released as individual molecules or as subcomplexes. Here, we set up a recombinant Brr2-mediated U4/U6 di-snRNP disruption system, showing that sequential addition of the U4/U6 proteins small nuclear ribonucleoprotein-associated protein 1 (Snu13), pre-mRNA processing factor 31 (Prp31), and Prp3 to U4/U6 di-snRNA leads to a stepwise decrease of Brr2-mediated U4/U6 unwinding, but that unwinding is largely restored by a Brr2 cofactor, the C-terminal Jab1/MPN domain of the Prp8 protein. Brr2-mediated U4/U6 unwinding was strongly inhibited by mutations in U4/U6 di-snRNAs that diminish the ability of U6 snRNA to adopt an alternative conformation but leave the number and kind of U4/U6 base pairs unchanged. Irrespective of the presence of the cofactor, the helicase segregated a Prp3-Prp31-Snu13-U4/U6 RNP into an intact Prp31-Snu13-U4 snRNA particle, free Prp3, and free U6 snRNA. Together, these observations suggest that Brr2 translocates only a limited distance on the U4 snRNA strand and does not actively release RNA-bound proteins. Unwinding is then completed by the partially displaced U6 snRNA adopting an alternative conformation, which leads to dismantling of the Prp3-binding site on U4/U6 di-snRNA but leaves the Prp31- and Snu13-binding sites on U4 snRNA unaffected. In this fashion, Brr2 can activate the spliceosome by stripping U6 snRNA of all precatalytic binding partners, while minimizing logistic requirements for U4/U6 di-snRNP reassembly after splicing. PMID:27354531

AGO6 functions in RNA-mediated transcriptional gene silencing in shoot and root meristems in Arabidopsis thaliana.

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Changho Eun

Full Text Available RNA-directed DNA methylation (RdDM is a small interfering RNA (siRNA-mediated epigenetic modification that contributes to transposon silencing in plants. RdDM requires a complex transcriptional machinery that includes specialized RNA polymerases, named Pol IV and Pol V, as well as chromatin remodelling proteins, transcription factors, RNA binding proteins, and other plant-specific proteins whose functions are not yet clarified. In Arabidopsis thaliana, DICER-LIKE3 and members of the ARGONAUTE4 group of ARGONAUTE (AGO proteins are involved, respectively, in generating and using 24-nt siRNAs that trigger methylation and transcriptional gene silencing of homologous promoter sequences. AGO4 is the main AGO protein implicated in the RdDM pathway. Here we report the identification of the related AGO6 in a forward genetic screen for mutants defective in RdDM and transcriptional gene silencing in shoot and root apical meristems in Arabidopsis thaliana. The identification of AGO6, and not AGO4, in our screen is consistent with the primary expression of AGO6 in shoot and root growing points.

Alteration of the SETBP1 gene and splicing pathway genes SF3B1, U2AF1, and SRSF2 in childhood acute myeloid leukemia.

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Choi, Hyun-Woo; Kim, Hye-Ran; Baek, Hee-Jo; Kook, Hoon; Cho, Duck; Shin, Jong-Hee; Suh, Soon-Pal; Ryang, Dong-Wook; Shin, Myung-Geun

2015-01-01

Recurrent somatic SET-binding protein 1 (SETBP1) and splicing pathway gene mutations have recently been found in atypical chronic myeloid leukemia and other hematologic malignancies. These mutations have been comprehensively analyzed in adult AML, but not in childhood AML. We investigated possible alteration of the SETBP1, splicing factor 3B subunit 1 (SF3B1), U2 small nuclear RNA auxiliary factor 1 (U2AF1), and serine/arginine-rich splicing factor 2 (SRSF2) genes in childhood AML. Cytogenetic and molecular analyses were performed to reveal chromosomal and genetic alterations. Sequence alterations in the SETBP1, SF3B1, U2AF1, and SRSF2 genes were examined by using direct sequencing in a cohort of 53 childhood AML patients. Childhood AML patients did not harbor any recurrent SETBP1 gene mutations, although our study did identify a synonymous mutation in one patient. None of the previously reported aberrations in the mutational hotspot of SF3B1, U2AF1, and SRSF2 were identified in any of the 53 patients. Alterations of the SETBP1 gene or SF3B1, U2AF1, and SRSF2 genes are not common genetic events in childhood AML, implying that the mutations are unlikely to exert a driver effect in myeloid leukemogenesis during childhood.

An RNA-seq transcriptome analysis of histone modifiers and RNA silencing genes in soybean during floral initiation process.

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Lim Chee Liew

Full Text Available Epigenetics has been recognised to play vital roles in many plant developmental processes, including floral initiation through the epigenetic regulation of gene expression. The histone modifying proteins that mediate these modifications involve the SET domain-containing histone methyltransferases, JmjC domain-containing demethylase, acetylases and deacetylases. In addition, RNA interference (RNAi-associated genes are also involved in epigenetic regulation via RNA-directed DNA methylation and post-transcriptional gene silencing. Soybean, a major crop legume, requires a short day to induce flowering. How histone modifications regulate the plant response to external cues that initiate flowering is still largely unknown. Here, we used RNA-seq to address the dynamics of transcripts that are potentially involved in the epigenetic programming and RNAi mediated gene silencing during the floral initiation of soybean. Soybean is a paleopolyploid that has been subjected to at least two rounds of whole genome duplication events. We report that the expanded genomic repertoire of histone modifiers and RNA silencing genes in soybean includes 14 histone acetyltransferases, 24 histone deacetylases, 47 histone methyltransferases, 15 protein arginine methyltransferases, 24 JmjC domain-containing demethylases and 47 RNAi-associated genes. To investigate the role of these histone modifiers and RNA silencing genes during floral initiation, we compared the transcriptional dynamics of the leaf and shoot apical meristem at different time points after a short-day treatment. Our data reveal that the extensive activation of genes that are usually involved in the epigenetic programming and RNAi gene silencing in the soybean shoot apical meristem are reprogrammed for floral development following an exposure to inductive conditions.

An RNA editing/dsRNA binding-independent gene regulatory mechanism of ADARs and its clinical implication in cancer.

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Qi, Lihua; Song, Yangyang; Chan, Tim Hon Man; Yang, Henry; Lin, Chi Ho; Tay, Daryl Jin Tai; Hong, HuiQi; Tang, Sze Jing; Tan, Kar Tong; Huang, Xi Xiao; Lin, Jaymie Siqi; Ng, Vanessa Hui En; Maury, Julien Jean Pierre; Tenen, Daniel G; Chen, Leilei

2017-10-13

Adenosine-to-inosine (A-to-I) RNA editing, catalyzed by Adenosine DeAminases acting on double-stranded RNA(dsRNA) (ADAR), occurs predominantly in the 3' untranslated regions (3'UTRs) of spliced mRNA. Here we uncover an unanticipated link between ADARs (ADAR1 and ADAR2) and the expression of target genes undergoing extensive 3'UTR editing. Using METTL7A (Methyltransferase Like 7A), a novel tumor suppressor gene with multiple editing sites at its 3'UTR, we demonstrate that its expression could be repressed by ADARs beyond their RNA editing and double-stranded RNA (dsRNA) binding functions. ADARs interact with Dicer to augment the processing of pre-miR-27a to mature miR-27a. Consequently, mature miR-27a targets the METTL7A 3'UTR to repress its expression level. In sum, our study unveils that the extensive 3'UTR editing of METTL7A is merely a footprint of ADAR binding, and there are a subset of target genes that are equivalently regulated by ADAR1 and ADAR2 through their non-canonical RNA editing and dsRNA binding-independent functions, albeit maybe less common. The functional significance of ADARs is much more diverse than previously appreciated and this gene regulatory function of ADARs is most likely to be of high biological importance beyond the best-studied editing function. This non-editing side of ADARs opens another door to target cancer. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Temporal aspects of DNA and RNA synthesis during human immunodeficiency virus infection: Evidence for differential gene expression

International Nuclear Information System (INIS)

Kim, Sunyoung; Baltimore, D.; Byrn, R.; Groopman, J.

1989-01-01

The kinetics of retroviral DNA and RNA synthesis are parameters vital to understanding viral growth, especially for human immunodeficiency virus (HIV), which encodes several of its own regulatory genes. The authors have established a single-cycle growth condition for HIV in H9 cells, a human CD4 + lymphocyte line. The full-length viral linear DNA is first detectable by 4 h postinfection. During a one-step growth of HIV, amounts of viral DNA gradually increase until 8 to 12 h postinfection and then decrease. The copy number of unintegrated viral DNA is not extraordinarily high even at its peak. Most strikingly, there is a temporal program of RNA accumulation: the earliest RNA is greatly enriched in the 2-kilobase subgenomic mRNA species, while the level of 9.2-kilobase RNA which is both genomic RNA and mRNA remains low until after 24 h of infection. Virus production begins at about 24 h postinfection. Thus, viral DNA synthesis is as rapid as for other retroviruses, but viral RNA synthesis involves temporal alteration in the species that accumulate, presumably as a consequence of viral regulatory genes

MicroRNA gene polymorphisms and environmental factors increase patient susceptibility to hepatocellular carcinoma.

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Yin-Hung Chu

Full Text Available BACKGROUND: Micro RNAs (miRNAs are small RNA fragments that naturally exist in the human body. Through various physiological mechanisms, miRNAs can generate different functions for regulating RNA protein levels and balancing abnormalities. Abnormal miRNA expression has been reported to be highly related to several diseases and cancers. Single-nucleotide polymorphisms (SNPs in miRNAs have been reported to increase patient susceptibility and affect patient prognosis and survival. We adopted a case-control research design to verify the relationship between miRNAs and hepatocellular carcinoma. METHODOLOGY/PRINCIPAL FINDINGS: A total of 525 subjects, including 377 controls and 188 hepatocellular carcinoma patients, were selected. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP and real-time PCR were used to analyze miRNA146a (rs2910164, miRNA149 (rs2292832, miRNA196 (rs11614913, and miRNA499 (rs3746444 genetic polymorphisms between the control group and the case group. The results indicate that people who carry the rs3746444 CT or CC genotypes may have a significantly increased susceptibility to hepatocellular carcinoma (adjusted odds ratio [AOR] = 2.84, 95% confidence interval [CI] = 1.88-4.30. In addition, when combined with environmental risk factors, such as smoking and alcohol consumption, interaction effects were observed between gene polymorphisms and environmental factors (odds ratio [OR] = 4.69, 95% CI = 2.52-8.70; AOR = 3.38, 95% CI = 1.68-6.80. CONCLUSIONS: These results suggest that a significant association exists between miRNA499 SNPs and hepatocellular carcinoma. Gene-environment interactions of miRNA499 polymorphisms, smoking, and alcohol consumption might alter hepatocellular carcinoma susceptibility.

Inhibition of HIV transmission in human cervicovaginal explants and humanized mice using CD4 aptamer-siRNA chimeras

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Wheeler, Lee Adam; Trifonova, Radiana; Vrbanac, Vladimir; Basar, Emre; McKernan, Shannon; Xu, Zhan; Seung, Edward; Deruaz, Maud; Dudek, Tim; Einarsson, Jon Ivar; Yang, Linda; Allen, Todd M.; Luster, Andrew D.; Tager, Andrew M.; Dykxhoorn, Derek M.; Lieberman, Judy

2011-01-01

The continued spread of the HIV epidemic underscores the need to interrupt transmission. One attractive strategy is a topical vaginal microbicide. Sexual transmission of herpes simplex virus type 2 (HSV-2) in mice can be inhibited by intravaginal siRNA application. To overcome the challenges of knocking down gene expression in immune cells susceptible to HIV infection, we used chimeric RNAs composed of an aptamer fused to an siRNA for targeted gene knockdown in cells bearing an aptamer-binding receptor. Here, we showed that CD4 aptamer-siRNA chimeras (CD4-AsiCs) specifically suppress gene expression in CD4+ T cells and macrophages in vitro, in polarized cervicovaginal tissue explants, and in the female genital tract of humanized mice. CD4-AsiCs do not activate lymphocytes or stimulate innate immunity. CD4-AsiCs that knock down HIV genes and/or CCR5 inhibited HIV infection in vitro and in tissue explants. When applied intravaginally to humanized mice, CD4-AsiCs protected against HIV vaginal transmission. Thus, CD4-AsiCs could be used as the active ingredient of a microbicide to prevent HIV sexual transmission. PMID:21576818

An albumin-mediated cholesterol design-based strategy for tuning siRNA pharmacokinetics and gene silencing.

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Bienk, Konrad; Hvam, Michael Lykke; Pakula, Malgorzata Maria; Dagnæs-Hansen, Frederik; Wengel, Jesper; Malle, Birgitte Mølholm; Kragh-Hansen, Ulrich; Cameron, Jason; Bukrinski, Jens Thostrup; Howard, Kenneth A

2016-06-28

Major challenges for the clinical translation of small interfering RNA (siRNA) include overcoming the poor plasma half-life, site-specific delivery and modulation of gene silencing. In this work, we exploit the intrinsic transport properties of human serum albumin to tune the blood circulatory half-life, hepatic accumulation and gene silencing; based on the number of siRNA cholesteryl modifications. We demonstrate by a gel shift assay a strong and specific affinity of recombinant human serum albumin (rHSA) towards cholesteryl-modified siRNA (Kd>1×10(-7)M) dependent on number of modifications. The rHSA/siRNA complex exhibited reduced nuclease degradation and reduced induction of TNF-α production by human peripheral blood mononuclear cells. The increased solubility of heavily cholesteryl modified siRNA in the presence of rHSA facilitated duplex annealing and consequent interaction that allowed in vivo studies using multiple cholesteryl modifications. A structural-activity-based screen of in vitro EGFP-silencing was used to select optimal siRNA designs containing cholesteryl modifications within the sense strand that were used for in vivo studies. We demonstrate plasma half-life extension in NMRI mice from t1/2 12min (naked) to t1/2 45min (single cholesteryl) and t1/2 71min (double cholesteryl) using fluorescent live bioimaging. The biodistribution showed increased accumulation in the liver for the double cholesteryl modified siRNA that correlated with an increase in hepatic Factor VII gene silencing of 28% (rHSA/siRNA) compared to 4% (naked siRNA) 6days post-injection. This work presents a novel albumin-mediated cholesteryl design-based strategy for tuning pharmacokinetics and systemic gene silencing. Copyright © 2016 Elsevier B.V. All rights reserved.

Evaluation of tools for highly variable gene discovery from single-cell RNA-seq data.

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Yip, Shun H; Sham, Pak Chung; Wang, Junwen

2018-02-21

Traditional RNA sequencing (RNA-seq) allows the detection of gene expression variations between two or more cell populations through differentially expressed gene (DEG) analysis. However, genes that contribute to cell-to-cell differences are not discoverable with RNA-seq because RNA-seq samples are obtained from a mixture of cells. Single-cell RNA-seq (scRNA-seq) allows the detection of gene expression in each cell. With scRNA-seq, highly variable gene (HVG) discovery allows the detection of genes that contribute strongly to cell-to-cell variation within a homogeneous cell population, such as a population of embryonic stem cells. This analysis is implemented in many software packages. In this study, we compare seven HVG methods from six software packages, including BASiCS, Brennecke, scLVM, scran, scVEGs and Seurat. Our results demonstrate that reproducibility in HVG analysis requires a larger sample size than DEG analysis. Discrepancies between methods and potential issues in these tools are discussed and recommendations are made.

Post-transcriptional bursting in genes regulated by small RNA molecules

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Rodrigo, Guillermo

2018-03-01

Gene expression programs in living cells are highly dynamic due to spatiotemporal molecular signaling and inherent biochemical stochasticity. Here we study a mechanism based on molecule-to-molecule variability at the RNA level for the generation of bursts of protein production, which can lead to heterogeneity in a cell population. We develop a mathematical framework to show numerically and analytically that genes regulated post transcriptionally by small RNA molecules can exhibit such bursts due to different states of translation activity (on or off), mostly revealed in a regime of few molecules. We exploit this framework to compare transcriptional and post-transcriptional bursting and also to illustrate how to tune the resulting protein distribution with additional post-transcriptional regulations. Moreover, because RNA-RNA interactions are predictable with an energy model, we define the kinetic constants of on-off switching as functions of the two characteristic free-energy differences of the system, activation and formation, with a nonequilibrium scheme. Overall, post-transcriptional bursting represents a distinctive principle linking gene regulation to gene expression noise, which highlights the importance of the RNA layer beyond the simple information transfer paradigm and significantly contributes to the understanding of the intracellular processes from a first-principles perspective.

Photochemistry of U(BH/sub 4/)/sub 4/ and U(BD/sub 4/)/sub 4/

Energy Technology Data Exchange (ETDEWEB)

Paine, R T; Schonberg, P R; Light, R W [New Mexico Univ., Albuquerque (USA). Dept. of Chemistry; Danen, W C; Freund, S M

1979-01-01

U(BH/sub 4/)/sub 4/ and U(BD/sub 4/)/sub 4/ are observed to undergo complex degradation reactions promoted by broadband UV radiation. The primary products of these reactions appear to be U(BH/sub 4/)/sub 3/, B/sub 2/H/sub 6/, H/sub 2/, U(BD/sub 4/)/sub 3/, B/sub 2/D/sub 6/ and D/sub 2/. Further, U(BD/sub 4/)/sub 4/ undergoes a related decomposition reaction under the influence of CO/sub 2/ laser irradiation at 924.97 cm/sup -1/.

Identification of putative noncoding RNA genes in the Burkholderia cenocepacia J2315 genome

DEFF Research Database (Denmark)

Coenye, T.; Drevinek, P.; Mahenthiralingam, E.

2007-01-01

Noncoding RNA (ncRNA) genes are not involved in the production of mRNA and proteins, but produce transcripts that function directly as structural or regulatory RNAs. In the present study, the presence of ncRNA genes in the genome of Burkholderia cenocepacia J2315 was evaluated by combining...

Impact of MicroRNA Levels, Target-Site Complementarity, and Cooperativity on Competing Endogenous RNA-Regulated Gene Expression.

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Denzler, Rémy; McGeary, Sean E; Title, Alexandra C; Agarwal, Vikram; Bartel, David P; Stoffel, Markus

2016-11-03

Expression changes of competing endogenous RNAs (ceRNAs) have been proposed to influence microRNA (miRNA) activity and thereby regulate other transcripts containing miRNA-binding sites. Here, we find that although miRNA levels define the extent of repression, they have little effect on the magnitude of the ceRNA expression change required to observe derepression. Canonical 6-nt sites, which typically mediate modest repression, can nonetheless compete for miRNA binding, with potency ∼20% of that observed for canonical 8-nt sites. In aggregate, low-affinity/background sites also contribute to competition. Sites with extensive additional complementarity can appear as more potent, but only because they induce miRNA degradation. Cooperative binding of proximal sites for the same or different miRNAs does increase potency. These results provide quantitative insights into the stoichiometric relationship between miRNAs and target abundance, target-site spacing, and affinity requirements for ceRNA-mediated gene regulation, and the unusual circumstances in which ceRNA-mediated gene regulation might be observed. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Histone and ribosomal RNA repetitive gene clusters of the boll weevil are linked in a tandem array.

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Roehrdanz, R; Heilmann, L; Senechal, P; Sears, S; Evenson, P

2010-08-01

Histones are the major protein component of chromatin structure. The histone family is made up of a quintet of proteins, four core histones (H2A, H2B, H3 & H4) and the linker histones (H1). Spacers are found between the coding regions. Among insects this quintet of genes is usually clustered and the clusters are tandemly repeated. Ribosomal DNA contains a cluster of the rRNA sequences 18S, 5.8S and 28S. The rRNA genes are separated by the spacers ITS1, ITS2 and IGS. This cluster is also tandemly repeated. We found that the ribosomal RNA repeat unit of at least two species of Anthonomine weevils, Anthonomus grandis and Anthonomus texanus (Coleoptera: Curculionidae), is interspersed with a block containing the histone gene quintet. The histone genes are situated between the rRNA 18S and 28S genes in what is known as the intergenic spacer region (IGS). The complete reiterated Anthonomus grandis histone-ribosomal sequence is 16,248 bp.

Dendrosomal curcumin nanoformulation downregulates pluripotency genes via miR-145 activation in U87MG glioblastoma cells

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Tahmasebi Mirgani M

2014-01-01

Full Text Available Maryam Tahmasebi Mirgani,1 Benedetta Isacchi,2 Majid Sadeghizadeh,1,* Fabio Marra,3 Anna Rita Bilia,2,* Seyed Javad Mowla,1 Farhood Najafi,4 Esmael Babaei51Department of Genetics, Tarbiat Modares University, Tehran, Iran; 2Department of Chemistry, University of Florence, Sesto Fiorentino, Italy; 3Department of Experimental and Clinical Medicine, University of Florence, Italy; 4Department of Resin and Additives, Institute for Color Science and Technology, Tehran, Iran; 5Department of Biology, University of Tabriz, Tabriz, Iran*These authors contributed equally to this workAbstract: Glioblastoma is an invasive tumor of the central nervous system. Tumor recurrence resulting from ineffective current treatments, mainly due to the blood–brain barrier, highlights the need for innovative therapeutic alternatives. The recent availability of nanotechnology represents a novel targeted strategy in cancer therapy. Natural products have received considerable attention for cancer therapy because of general lower side effects. Curcumin is a new candidate for anticancer treatment, but its low bioavailability and water solubility represent the main disadvantages of its use. Here, curcumin was efficiently encapsulated in a nontoxic nanocarrier, termed dendrosome, to overcome these problems. Dendrosomal curcumin was prepared as 142 nm spherical structures with constant physical and chemical stability. The inhibitory role of dendrosomal curcumin on the proliferation of U87MG cells, a cellular model of glioblastoma, was evaluated by considering master genes of pluripotency and regulatory miRNA (microribonucleic acid. Methylthiazol tetrazolium assay and flow cytometry were used to detect the antiproliferative effects of dendrosomal curcumin. Annexin-V-FLUOS and caspase assay were used to quantify apoptosis. Real-time polymerase chain reaction was used to analyze the expression of OCT4 (octamer binding protein 4 gene variants (OCT4A, OCT4B, and OCT4B1, SOX-2 (SRY

Endogenous MCM7 microRNA cluster as a novel platform to multiplex small interfering and nucleolar RNAs for combinational HIV-1 gene therapy.

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Chung, Janet; Zhang, Jane; Li, Haitang; Ouellet, Dominique L; DiGiusto, David L; Rossi, John J

2012-11-01

Combinational therapy with small RNA inhibitory agents against multiple viral targets allows efficient inhibition of viral production by controlling gene expression at critical time points. Here we explore combinations of different classes of therapeutic anti-HIV-1 RNAs expressed from within the context of an intronic MCM7 (minichromosome maintenance complex component-7) platform that naturally harbors 3 microRNAs (miRNAs). We replaced the endogenous miRNAs with anti-HIV small RNAs, including small interfering RNAs (siRNAs) targeting HIV-1 tat and rev messages that function to induce post-transcriptional gene silencing by the RNA interference pathway, a nucleolar-localizing RNA ribozyme that targets the conserved U5 region of HIV-1 transcripts for degradation, and finally nucleolar trans-activation response (TAR) and Rev-binding element (RBE) RNA decoys designed to sequester HIV-1 Tat and Rev proteins inside the nucleolus. We demonstrate the versatility of the MCM7 platform in expressing and efficiently processing the siRNAs as miRNA mimics along with nucleolar small RNAs. Furthermore, three of the combinatorial constructs tested potently suppressed viral replication during a 1-month HIV challenge, with greater than 5-log inhibition compared with untransduced, HIV-1-infected CEM T lymphocytes. One of the most effective constructs contains an anti-HIV siRNA combined with a nucleolar-localizing U5 ribozyme and TAR decoy. This represents the first efficacious example of combining Drosha-processed siRNAs with small nucleolar ribonucleoprotein (snoRNP)-processed nucleolar RNA chimeras from a single intron platform for effective inhibition of viral replication. Moreover, we demonstrated enrichment/selection for cells expressing levels of the antiviral RNAs that provide optimal inhibition under the selective pressure of HIV. The combinations of si/snoRNAs represent a new paradigm for combinatorial RNA-based gene therapy applications.

Analysis of the siRNA-Mediated Gene Silencing Process Targeting Three Homologous Genes Controlling Soybean Seed Oil Quality.

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Lu, Sha; Yin, Xiaoyan; Spollen, William; Zhang, Ning; Xu, Dong; Schoelz, James; Bilyeu, Kristin; Zhang, Zhanyuan J

2015-01-01

In the past decade, RNA silencing has gained significant attention because of its success in genomic scale research and also in the genetic improvement of crop plants. However, little is known about the molecular basis of siRNA processing in association with its target transcript. To reveal this process for improving hpRNA-mediated gene silencing in crop plants, the soybean GmFAD3 gene family was chosen as a test model. We analyzed RNAi mutant soybean lines in which three members of the GmFAD3 gene family were silenced. The silencing levels of FAD3A, FAD3B and FAD3C were correlated with the degrees of sequence homology between the inverted repeat of hpRNA and the GmFAD3 transcripts in the RNAi lines. Strikingly, transgenes in two of the three RNAi lines were heavily methylated, leading to a dramatic reduction of hpRNA-derived siRNAs. Small RNAs corresponding to the loop portion of the hairpin transcript were detected while much lower levels of siRNAs were found outside of the target region. siRNAs generated from the 318-bp inverted repeat were found to be diced much more frequently at stem sequences close to the loop and associated with the inferred cleavage sites on the target transcripts, manifesting "hot spots". The top candidate hpRNA-derived siRNA share certain sequence features with mature miRNA. This is the first comprehensive and detailed study revealing the siRNA-mediated gene silencing mechanism in crop plants using gene family GmFAD3 as a test model.

High throughput 16S rRNA gene amplicon sequencing

DEFF Research Database (Denmark)

Nierychlo, Marta; Larsen, Poul; Jørgensen, Mads Koustrup

S rRNA gene amplicon sequencing has been developed over the past few years and is now ready to use for more comprehensive studies related to plant operation and optimization thanks to short analysis time, low cost, high throughput, and high taxonomic resolution. In this study we show how 16S r......RNA gene amplicon sequencing can be used to reveal factors of importance for the operation of full-scale nutrient removal plants related to settling problems and floc properties. Using optimized DNA extraction protocols, indexed primers and our in-house Illumina platform, we prepared multiple samples...... be correlated to the presence of the species that are regarded as “strong” and “weak” floc formers. In conclusion, 16S rRNA gene amplicon sequencing provides a high throughput approach for a rapid and cheap community profiling of activated sludge that in combination with multivariate statistics can be used...

Conditional RNA interference achieved by Oct-1 POU/rtTA fusion protein activator and a modified TRE-mouse U6 promoter

International Nuclear Information System (INIS)

Fei Zhaoliang; Chen Zheng; Wang Zhugang; Fei Jian

2007-01-01

RNA interference (RNAi) is a powerful technique and is widely used to down-regulate expression of specific genes in cultured cells and in vivo. In this paper, we report our development of a new tetracycline-inducible RNAi expression using a modified TRE-mouse U6 promoter in which the distal sequence element (DSE) was replaced by the tetracycline-responsive element (TRE). The modified TRE-mouse U6 promoter can be activated by a Tet-on version tetracycline-regulated artificial activator rTetOct which was constructed by fusing the rtTA DNA binding domain with the Oct-1 POU activation domain. This rTetOct/TRE-U6 system was successfully applied to conditionally and reversibly down-regulate the expression of endogenous p53 gene in MCF7 cells, and the expression of β-defensin gene (mBin1b) either transiently expressed in COS7 cells or stably expressed in CHO cells

Evaluating whole transcriptome amplification for gene profiling experiments using RNA-Seq.

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Faherty, Sheena L; Campbell, C Ryan; Larsen, Peter A; Yoder, Anne D

2015-07-30

RNA-Seq has enabled high-throughput gene expression profiling to provide insight into the functional link between genotype and phenotype. Low quantities of starting RNA can be a severe hindrance for studies that aim to utilize RNA-Seq. To mitigate this bottleneck, whole transcriptome amplification (WTA) technologies have been developed to generate sufficient sequencing targets from minute amounts of RNA. Successful WTA requires accurate replication of transcript abundance without the loss or distortion of specific mRNAs. Here, we test the efficacy of NuGEN's Ovation RNA-Seq V2 system, which uses linear isothermal amplification with a unique chimeric primer for amplification, using white adipose tissue from standard laboratory rats (Rattus norvegicus). Our goal was to investigate potential biological artifacts introduced through WTA approaches by establishing comparisons between matched raw and amplified RNA libraries derived from biological replicates. We found that 93% of expressed genes were identical between all unamplified versus matched amplified comparisons, also finding that gene density is similar across all comparisons. Our sequencing experiment and downstream bioinformatic analyses using the Tuxedo analysis pipeline resulted in the assembly of 25,543 high-quality transcripts. Libraries constructed from raw RNA and WTA samples averaged 15,298 and 15,253 expressed genes, respectively. Although significant differentially expressed genes (P < 0.05) were identified in all matched samples, each of these represents less than 0.15% of all shared genes for each comparison. Transcriptome amplification is efficient at maintaining relative transcript frequencies with no significant bias when using this NuGEN linear isothermal amplification kit under ideal laboratory conditions as presented in this study. This methodology has broad applications, from clinical and diagnostic, to field-based studies when sample acquisition, or sample preservation, methods prove

Regulation of bacterial photosynthesis genes by the small noncoding RNA PcrZ.

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Mank, Nils N; Berghoff, Bork A; Hermanns, Yannick N; Klug, Gabriele

2012-10-02

The small RNA PcrZ (photosynthesis control RNA Z) of the facultative phototrophic bacterium Rhodobacter sphaeroides is induced upon a drop of oxygen tension with similar kinetics to those of genes for components of photosynthetic complexes. High expression of PcrZ depends on PrrA, the response regulator of the PrrB/PrrA two-component system with a central role in redox regulation in R. sphaeroides. In addition the FnrL protein, an activator of some photosynthesis genes at low oxygen tension, is involved in redox-dependent expression of this small (s)RNA. Overexpression of full-length PcrZ in R. sphaeroides affects expression of a small subset of genes, most of them with a function in photosynthesis. Some mRNAs from the photosynthetic gene cluster were predicted to be putative PcrZ targets and results from an in vivo reporter system support these predictions. Our data reveal a negative effect of PcrZ on expression of its target mRNAs. Thus, PcrZ counteracts the redox-dependent induction of photosynthesis genes, which is mediated by protein regulators. Because PrrA directly activates photosynthesis genes and at the same time PcrZ, which negatively affects photosynthesis gene expression, this is one of the rare cases of an incoherent feed-forward loop including an sRNA. Our data identified PcrZ as a trans acting sRNA with a direct regulatory function in formation of photosynthetic complexes and provide a model for the control of photosynthesis gene expression by a regulatory network consisting of proteins and a small noncoding RNA.

Duplication and Loss of Function of Genes Encoding RNA Polymerase III Subunit C4 Causes Hybrid Incompatibility in Rice

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Giao Ngoc Nguyen

2017-08-01

Full Text Available Reproductive barriers are commonly observed in both animals and plants, in which they maintain species integrity and contribute to speciation. This report shows that a combination of loss-of-function alleles at two duplicated loci, DUPLICATED GAMETOPHYTIC STERILITY 1 (DGS1 on chromosome 4 and DGS2 on chromosome 7, causes pollen sterility in hybrid progeny derived from an interspecific cross between cultivated rice, Oryza sativa, and an Asian annual wild rice, O. nivara. Male gametes carrying the DGS1 allele from O. nivara (DGS1-nivaras and the DGS2 allele from O. sativa (DGS2-T65s were sterile, but female gametes carrying the same genotype were fertile. We isolated the causal gene, which encodes a protein homologous to DNA-dependent RNA polymerase (RNAP III subunit C4 (RPC4. RPC4 facilitates the transcription of 5S rRNAs and tRNAs. The loss-of-function alleles at DGS1-nivaras and DGS2-T65s were caused by weak or nonexpression of RPC4 and an absence of RPC4, respectively. Phylogenetic analysis demonstrated that gene duplication of RPC4 at DGS1 and DGS2 was a recent event that occurred after divergence of the ancestral population of Oryza from other Poaceae or during diversification of AA-genome species.

Deciphering the transcriptional circuitry of microRNA genes expressed during human monocytic differentiation

KAUST Repository

Schmeier, Sebastian; MacPherson, Cameron R; Essack, Magbubah; Kaur, Mandeep; Schaefer, Ulf; Suzuki, Harukazu; Hayashizaki, Yoshihide; Bajic, Vladimir B.

2009-01-01

Background: Macrophages are immune cells involved in various biological processes including host defence, homeostasis, differentiation, and organogenesis. Disruption of macrophage biology has been linked to increased pathogen infection, inflammation and malignant diseases. Differential gene expression observed in monocytic differentiation is primarily regulated by interacting transcription factors (TFs). Current research suggests that microRNAs (miRNAs) degrade and repress translation of mRNA, but also may target genes involved in differentiation. We focus on getting insights into the transcriptional circuitry regulating miRNA genes expressed during monocytic differentiation. Results: We computationally analysed the transcriptional circuitry of miRNA genes during monocytic differentiation using in vitro time-course expression data for TFs and miRNAs. A set of TF?miRNA associations was derived from predicted TF binding sites in promoter regions of miRNA genes. Time-lagged expression correlation analysis was utilised to evaluate the TF?miRNA associations. Our analysis identified 12 TFs that potentially play a central role in regulating miRNAs throughout the differentiation process. Six of these 12 TFs (ATF2, E2F3, HOXA4, NFE2L1, SP3, and YY1) have not previously been described to be important for monocytic differentiation. The remaining six TFs are CEBPB, CREB1, ELK1, NFE2L2, RUNX1, and USF2. For several miRNAs (miR-21, miR-155, miR-424, and miR-17-92), we show how their inferred transcriptional regulation impacts monocytic differentiation. Conclusions: The study demonstrates that miRNAs and their transcriptional regulatory control are integral molecular mechanisms during differentiation. Furthermore, it is the first study to decipher on a large-scale, how miRNAs are controlled by TFs during human monocytic differentiation. Subsequently, we have identified 12 candidate key controllers of miRNAs during this differentiation process. 2009 Schmeier et al; licensee Bio

Deciphering the transcriptional circuitry of microRNA genes expressed during human monocytic differentiation

KAUST Repository

Schmeier, Sebastian

2009-12-10

Background: Macrophages are immune cells involved in various biological processes including host defence, homeostasis, differentiation, and organogenesis. Disruption of macrophage biology has been linked to increased pathogen infection, inflammation and malignant diseases. Differential gene expression observed in monocytic differentiation is primarily regulated by interacting transcription factors (TFs). Current research suggests that microRNAs (miRNAs) degrade and repress translation of mRNA, but also may target genes involved in differentiation. We focus on getting insights into the transcriptional circuitry regulating miRNA genes expressed during monocytic differentiation. Results: We computationally analysed the transcriptional circuitry of miRNA genes during monocytic differentiation using in vitro time-course expression data for TFs and miRNAs. A set of TF?miRNA associations was derived from predicted TF binding sites in promoter regions of miRNA genes. Time-lagged expression correlation analysis was utilised to evaluate the TF?miRNA associations. Our analysis identified 12 TFs that potentially play a central role in regulating miRNAs throughout the differentiation process. Six of these 12 TFs (ATF2, E2F3, HOXA4, NFE2L1, SP3, and YY1) have not previously been described to be important for monocytic differentiation. The remaining six TFs are CEBPB, CREB1, ELK1, NFE2L2, RUNX1, and USF2. For several miRNAs (miR-21, miR-155, miR-424, and miR-17-92), we show how their inferred transcriptional regulation impacts monocytic differentiation. Conclusions: The study demonstrates that miRNAs and their transcriptional regulatory control are integral molecular mechanisms during differentiation. Furthermore, it is the first study to decipher on a large-scale, how miRNAs are controlled by TFs during human monocytic differentiation. Subsequently, we have identified 12 candidate key controllers of miRNAs during this differentiation process. 2009 Schmeier et al; licensee Bio

Involvement of DNA topoisomerase I in transcription of human ribosomal RNA genes

International Nuclear Information System (INIS)

Zhang, H.; Wang, J.C.; Liu, L.F.

1988-01-01

Treatment of HeLa cells with a DNA topoisomerase I-specific inhibitor, camptothecin, results in rapid cessation of the synthesis of the 45S rRNA precursor. The inhibition of rRNA synthesis is reversible following drug removal and correlates with the presence of camptothecin-trapped topoisomerase I-DNA abortive complexes, which can be detected as topoisomerase I-linked DNA breaks upon lysis with sodium dodecyl sulfate. These breaks were found to be concentrated within the transcribed region of human rRNA genes. No such sites can be detected in the inactive human rRNA genes in mouse-human hybrid cells, suggesting a preferential association of topoisomerase I with actively transcribed genes. The distribution of RNA polymerase molecules along the transcription unit of human rRNA genes in camptothecin-treated HeLa cells, as assayed by nuclear run-on transcription, shows a graded decrease of the RNA polymerase density toward the 3' end of the transcription unit; the density is minimally affected near the 5' start of the transcription unit. These results suggest that DNA topoisomerase I is normally involved in the elongation step of transcription, especially when the transcripts are long, and that camptothecin interferes with this role

Low-level lasers alter mRNA levels from traditional reference genes used in breast cancer cells

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Teixeira, A. F.; Canuto, K. S.; Rodrigues, J. A.; Fonseca, A. S.; Mencalha, A. L.

2017-07-01

Cancer is among the leading causes of mortality worldwide, increasing the importance of treatment development. Low-level lasers are used in several diseases, but some concerns remains on cancers. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) is a technique used to understand cellular behavior through quantification of mRNA levels. Output data from target genes are commonly relative to a reference that cannot vary according to treatment. This study evaluated reference genes levels from MDA-MB-231 cells exposed to red or infrared lasers at different fluences. Cultures were exposed to red and infrared lasers, incubated (4 h, 37 °C), total RNA was extracted and cDNA synthesis was performed to evaluate mRNA levels from ACTB, GUSB and TRFC genes by RT-qPCR. Specific amplification was verified by melting curves and agarose gel electrophoresis. RefFinder enabled data analysis by geNorm, NormFinder and BestKeeper. Specific amplifications were obtained and, although mRNA levels from ACTB, GUSB or TRFC genes presented no significant variation through traditional statistical analysis, Excel-based tools revealed that the use of these reference genes are dependent of laser characteristics. Our data showed that exposure to low-level red and infrared lasers at different fluences alter the mRNA levels from ACTB, GUSB and TRFC in MDA-MB-231 cells.

SLAM-seq defines direct gene-regulatory functions of the BRD4-MYC axis.

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Muhar, Matthias; Ebert, Anja; Neumann, Tobias; Umkehrer, Christian; Jude, Julian; Wieshofer, Corinna; Rescheneder, Philipp; Lipp, Jesse J; Herzog, Veronika A; Reichholf, Brian; Cisneros, David A; Hoffmann, Thomas; Schlapansky, Moritz F; Bhat, Pooja; von Haeseler, Arndt; Köcher, Thomas; Obenauf, Anna C; Popow, Johannes; Ameres, Stefan L; Zuber, Johannes

2018-05-18

Defining direct targets of transcription factors and regulatory pathways is key to understanding their roles in physiology and disease. We combined SLAM-seq [thiol(SH)-linked alkylation for the metabolic sequencing of RNA], a method for direct quantification of newly synthesized messenger RNAs (mRNAs), with pharmacological and chemical-genetic perturbation in order to define regulatory functions of two transcriptional hubs in cancer, BRD4 and MYC, and to interrogate direct responses to BET bromodomain inhibitors (BETis). We found that BRD4 acts as general coactivator of RNA polymerase II-dependent transcription, which is broadly repressed upon high-dose BETi treatment. At doses triggering selective effects in leukemia, BETis deregulate a small set of hypersensitive targets including MYC. In contrast to BRD4, MYC primarily acts as a selective transcriptional activator controlling metabolic processes such as ribosome biogenesis and de novo purine synthesis. Our study establishes a simple and scalable strategy to identify direct transcriptional targets of any gene or pathway. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Computational prediction and experimental validation of Ciona intestinalis microRNA genes

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Pasquinelli Amy E

2007-11-01

Full Text Available Abstract Background This study reports the first collection of validated microRNA genes in the sea squirt, Ciona intestinalis. MicroRNAs are processed from hairpin precursors to ~22 nucleotide RNAs that base pair to target mRNAs and inhibit expression. As a member of the subphylum Urochordata (Tunicata whose larval form has a notochord, the sea squirt is situated at the emergence of vertebrates, and therefore may provide information about the evolution of molecular regulators of early development. Results In this study, computational methods were used to predict 14 microRNA gene families in Ciona intestinalis. The microRNA prediction algorithm utilizes configurable microRNA sequence conservation and stem-loop specificity parameters, grouping by miRNA family, and phylogenetic conservation to the related species, Ciona savignyi. The expression for 8, out of 9 attempted, of the putative microRNAs in the adult tissue of Ciona intestinalis was validated by Northern blot analyses. Additionally, a target prediction algorithm was implemented, which identified a high confidence list of 240 potential target genes. Over half of the predicted targets can be grouped into the gene ontology categories of metabolism, transport, regulation of transcription, and cell signaling. Conclusion The computational techniques implemented in this study can be applied to other organisms and serve to increase the understanding of the origins of non-coding RNAs, embryological and cellular developmental pathways, and the mechanisms for microRNA-controlled gene regulatory networks.

Differential impact of the HEN1 homolog HENN-1 on 21U and 26G RNAs in the germline of Caenorhabditis elegans.

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Leonie M Kamminga

Full Text Available RNA interference (RNAi-related pathways affect gene activity by sequence-specific recruitment of Ago proteins to mRNA target molecules. The sequence specificity of this process stems from small RNA (sRNA co-factors bound by the Ago protein. Stability of sRNA molecules in some pathways is in part regulated by Hen1-mediated methylation of their 3' ends. Here we describe the effects of the Caenorhabditis elegans HEN1 RNA-methyl-transferase homolog, HENN-1, on the different RNAi pathways in this nematode. We reveal differential effects of HENN-1 on the two pathways that are known to employ methylated sRNA molecules: the 26G and 21U pathways. Surprisingly, in the germline, stability of 21U RNAs, the C. elegans piRNAs, is only mildly affected by loss of methylation; and introduction of artificial 21U target RNA does not further destabilize non-methylated 21U RNAs. In contrast, most 26G RNAs display reduced stability and respond to loss of HENN-1 by displaying increased 3'-uridylation frequencies. Within the 26G RNA class, we find that specifically ERGO-1-bound 26G RNAs are modified by HENN-1, while ALG-3/ALG-4-bound 26G RNAs are not. Global gene expression analysis of henn-1 mutants reveals mild effects, including down-regulation of many germline-expressed genes. Our data suggest that, apart from direct effects of reduced 26G RNA levels of henn-1 on gene expression, most effects on global gene expression are indirect. These studies further refine our understanding of endogenous RNAi in C. elegans and the roles for Hen1 like enzymes in these pathways.

β-glucuronidase use as a single internal control gene may confound analysis in FMR1 mRNA toxicity studies.

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Kraan, Claudine M; Cornish, Kim M; Bui, Quang M; Li, Xin; Slater, Howard R; Godler, David E

2018-01-01

Relationships between Fragile X Mental Retardation 1 (FMR1) mRNA levels in blood and intragenic FMR1 CGG triplet expansions support the pathogenic role of RNA gain of function toxicity in premutation (PM: 55-199 CGGs) related disorders. Real-time PCR (RT-PCR) studies reporting these findings normalised FMR1 mRNA level to a single internal control gene called β-glucuronidase (GUS). This study evaluated FMR1 mRNA-CGG correlations in 33 PM and 33 age- and IQ-matched control females using three normalisation strategies in peripheral blood mononuclear cells (PBMCs): (i) GUS as a single internal control; (ii) the mean of GUS, Eukaryotic Translation Initiation Factor 4A2 (EIF4A2) and succinate dehydrogenase complex flavoprotein subunit A (SDHA); and (iii) the mean of EIF4A2 and SDHA (with no contribution from GUS). GUS mRNA levels normalised to the mean of EIF4A2 and SDHA mRNA levels and EIF4A2/SDHA ratio were also evaluated. FMR1mRNA level normalised to the mean of EIF4A2 and SDHA mRNA levels, with no contribution from GUS, showed the most significant correlation with CGG size and the greatest difference between PM and control groups (p = 10-11). Only 15% of FMR1 mRNA PM results exceeded the maximum control value when normalised to GUS, compared with over 42% when normalised to the mean of EIF4A2 and SDHA mRNA levels. Neither GUS mRNA level normalised to the mean RNA levels of EIF4A2 and SDHA, nor to the EIF4A2/SDHA ratio were correlated with CGG size. However, greater variability in GUS mRNA levels were observed for both PM and control females across the full range of CGG repeat as compared to the EIF4A2/SDHA ratio. In conclusion, normalisation with multiple control genes, excluding GUS, can improve assessment of the biological significance of FMR1 mRNA-CGG size relationships.

Cross disease analysis of co-functional microRNA pairs on a reconstructed network of disease-gene-microRNA tripartite.

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Peng, Hui; Lan, Chaowang; Zheng, Yi; Hutvagner, Gyorgy; Tao, Dacheng; Li, Jinyan

2017-03-24

MicroRNAs always function cooperatively in their regulation of gene expression. Dysfunctions of these co-functional microRNAs can play significant roles in disease development. We are interested in those multi-disease associated co-functional microRNAs that regulate their common dysfunctional target genes cooperatively in the development of multiple diseases. The research is potentially useful for human disease studies at the transcriptional level and for the study of multi-purpose microRNA therapeutics. We designed a computational method to detect multi-disease associated co-functional microRNA pairs and conducted cross disease analysis on a reconstructed disease-gene-microRNA (DGR) tripartite network. The construction of the DGR tripartite network is by the integration of newly predicted disease-microRNA associations with those relationships of diseases, microRNAs and genes maintained by existing databases. The prediction method uses a set of reliable negative samples of disease-microRNA association and a pre-computed kernel matrix instead of kernel functions. From this reconstructed DGR tripartite network, multi-disease associated co-functional microRNA pairs are detected together with their common dysfunctional target genes and ranked by a novel scoring method. We also conducted proof-of-concept case studies on cancer-related co-functional microRNA pairs as well as on non-cancer disease-related microRNA pairs. With the prioritization of the co-functional microRNAs that relate to a series of diseases, we found that the co-function phenomenon is not unusual. We also confirmed that the regulation of the microRNAs for the development of cancers is more complex and have more unique properties than those of non-cancer diseases.

Polymorphisms in miRNA genes and their involvement in autoimmune diseases susceptibility.

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Latini, Andrea; Ciccacci, Cinzia; Novelli, Giuseppe; Borgiani, Paola

2017-08-01

MicroRNAs (miRNAs) are small non-coding RNA molecules that negatively regulate the expression of multiple protein-encoding genes at the post-transcriptional level. MicroRNAs are involved in different pathways, such as cellular proliferation and differentiation, signal transduction and inflammation, and play crucial roles in the development of several diseases, such as cancer, diabetes, and cardiovascular diseases. They have recently been recognized to play a role also in the pathogenesis of autoimmune diseases. Although the majority of studies are focused on miRNA expression profiles investigation, a growing number of studies have been investigating the role of polymorphisms in miRNA genes in the autoimmune diseases development. Indeed, polymorphisms affecting the miRNA genes can modify the set of targets they regulate or the maturation efficiency. This review is aimed to give an overview about the available studies that have investigated the association of miRNA gene polymorphisms with the susceptibility to various autoimmune diseases and to their clinical phenotypes.

Phorbol esters induce interleukin 2 mRNA in sensitive but not in resistant EL4 cells

International Nuclear Information System (INIS)

Harrison, J.R.; Lynch, K.R.; Sando, J.J.

1986-01-01

Phorbol ester (PE) sensitive EL4 cells are growth-inhibited and produce interleukin 2 (IL2) when treated with PE. Resistant EL4 cells lack both responses. To determine whether the defect in resistant cells occurs pre or post-transcriptionally, an assay for IL2 mRNA was developed using a synthetic oligonucleotide to mouse IL2 as a probe. Total RNA (15 μg) from cells +/- PE was electrophoresed, blotted onto a cationic nylon membrane, and probed with radiolabeled oligomer. This probe hybridized to a 1.1 kb band in RNA from PE-treated sensitive cells. This RNA was detectable within 3h of PE administration, was clearly visible by 6h, and peaked by 9 to 12h. No bands hybridizing with the IL2 probe were detected in RNA isolated from unstimulated cells or from resistant EL4 cells at any time following PE stimulation. Since levels of the protooncogene c-myc have been shown to decrease in a number of cell lines during differentiation and growth inhibition, total RNA from EL4 cells was probed with a nick-translated plasmid containing the protein coding region of the c-myc gene. In PE sensitive cells, levels of c-myc RNA are markedly reduced by 3h. In a pilot experiment with resistant cells, c-myc levels appeared to remain constant. These results demonstrate that PE induced IL2 mRNA in PE sensitive but not resistant EL4 cells. Sensitive and resistant EL4 cell lines provide a useful model for the investigation of the regulation of gene expression by PE

[Selection and construction of cell line stably expressing survivin gene in lower level through eukaryotic plasmid vector of shRNA].

Science.gov (United States)

Wang, Wen-Xia; Sun, Shan-Zhen; Song, Ying

2008-06-01

To construct a short hairpin RNA(shRNA) interference expression plasmid vector of survivin gene, transfect tongue squamous cell carcinoma line Tca8113 which expressed survivin gene in a high level, and choose the cells whose survivin gene were suppressed significantly. Two pairs of oligonucleotide sequences specific for survivin gene were designed and synthesized, and cloned into pSilencer-2.1U6-neo plasmid. The recombinant plasmids (named PS1 and PS2) were amplified in Ecoli. DH5alpha was identified by restriction digestion, PCR and sequencing. The vectors were transfected into Tca8113 cells with lipofectamine 2000. After selection with G418, the stable cell clones were attained. Survivn expression was assayed with real-time quantitative PCR and Western blotting. SAS8.0 software package was used for Student t test. Two vectors were constructed successfully and stable cell clones with PS1 or PS2 plasmid were obtained. As compared with those of control, survivin expression of transfected cell with PS1 or PS2 in mRNA level was significantly suppressed (P<0.05). In protein level, only those of transfected cell with PS2 was significantly suppressed (P<0.01). The shRNA interference expression plasmid vectors of survivin gene are successfully constructed, and Tca8113 cells which express survivin gene in a stable lower level are attained, which enable us to carry out further research on gene therapy of oral squamous cell carcinoma. Supported by National Natural Science Foundation of China (Grant No.30572056).

Molecular characterization of 5S ribosomal RNA genes and transcripts in the protozoan parasite Leishmania major.

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Moreno-Campos, Rodrigo; Florencio-Martínez, Luis E; Nepomuceno-Mejía, Tomás; Rojas-Sánchez, Saúl; Vélez-Ramírez, Daniel E; Padilla-Mejía, Norma E; Figueroa-Angulo, Elisa; Manning-Cela, Rebeca; Martínez-Calvillo, Santiago

2016-12-01

Eukaryotic 5S rRNA, synthesized by RNA polymerase III (Pol III), is an essential component of the large ribosomal subunit. Most organisms contain hundreds of 5S rRNA genes organized into tandem arrays. However, the genome of the protozoan parasite Leishmania major contains only 11 copies of the 5S rRNA gene, which are interspersed and associated with other Pol III-transcribed genes. Here we report that, in general, the number and order of the 5S rRNA genes is conserved between different species of Leishmania. While in most organisms 5S rRNA genes are normally associated with the nucleolus, combined fluorescent in situ hybridization and indirect immunofluorescence experiments showed that 5S rRNA genes are mainly located at the nuclear periphery in L. major. Similarly, the tandemly repeated 5S rRNA genes in Trypanosoma cruzi are dispersed throughout the nucleus. In contrast, 5S rRNA transcripts in L. major were localized within the nucleolus, and scattered throughout the cytoplasm, where mature ribosomes are located. Unlike other rRNA species, stable antisense RNA complementary to 5S rRNA is not detected in L. major.

The rde-1 gene, RNA interference, and transposon silencing in C. elegans.

Science.gov (United States)

Tabara, H; Sarkissian, M; Kelly, W G; Fleenor, J; Grishok, A; Timmons, L; Fire, A; Mello, C C

1999-10-15

Double-stranded (ds) RNA can induce sequence-specific inhibition of gene function in several organisms. However, both the mechanism and the physiological role of the interference process remain mysterious. In order to study the interference process, we have selected C. elegans mutants resistant to dsRNA-mediated interference (RNAi). Two loci, rde-1 and rde-4, are defined by mutants strongly resistant to RNAi but with no obvious defects in growth or development. We show that rde-1 is a member of the piwi/sting/argonaute/zwille/eIF2C gene family conserved from plants to vertebrates. Interestingly, several, but not all, RNAi-deficient strains exhibit mobilization of the endogenous transposons. We discuss implications for the mechanism of RNAi and the possibility that one natural function of RNAi is transposon silencing.

Analysis of the siRNA-Mediated Gene Silencing Process Targeting Three Homologous Genes Controlling Soybean Seed Oil Quality.

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Sha Lu

Full Text Available In the past decade, RNA silencing has gained significant attention because of its success in genomic scale research and also in the genetic improvement of crop plants. However, little is known about the molecular basis of siRNA processing in association with its target transcript. To reveal this process for improving hpRNA-mediated gene silencing in crop plants, the soybean GmFAD3 gene family was chosen as a test model. We analyzed RNAi mutant soybean lines in which three members of the GmFAD3 gene family were silenced. The silencing levels of FAD3A, FAD3B and FAD3C were correlated with the degrees of sequence homology between the inverted repeat of hpRNA and the GmFAD3 transcripts in the RNAi lines. Strikingly, transgenes in two of the three RNAi lines were heavily methylated, leading to a dramatic reduction of hpRNA-derived siRNAs. Small RNAs corresponding to the loop portion of the hairpin transcript were detected while much lower levels of siRNAs were found outside of the target region. siRNAs generated from the 318-bp inverted repeat were found to be diced much more frequently at stem sequences close to the loop and associated with the inferred cleavage sites on the target transcripts, manifesting "hot spots". The top candidate hpRNA-derived siRNA share certain sequence features with mature miRNA. This is the first comprehensive and detailed study revealing the siRNA-mediated gene silencing mechanism in crop plants using gene family GmFAD3 as a test model.

Promoter-wide hypermethylation of the ribosomal RNA gene promoter in the suicide brain.

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Patrick O McGowan

Full Text Available BACKGROUND: Alterations in gene expression in the suicide brain have been reported and for several genes DNA methylation as an epigenetic regulator is thought to play a role. rRNA genes, that encode ribosomal RNA, are the backbone of the protein synthesis machinery and levels of rRNA gene promoter methylation determine rRNA transcription. METHODOLOGY/PRINCIPAL FINDINGS: We test here by sodium bisulfite mapping of the rRNA promoter and quantitative real-time PCR of rRNA expression the hypothesis that epigenetic differences in critical loci in the brain are involved in the pathophysiology of suicide. Suicide subjects in this study were selected for a history of early childhood neglect/abuse, which is associated with decreased hippocampal volume and cognitive impairments. rRNA was significantly hypermethylated throughout the promoter and 5' regulatory region in the brain of suicide subjects, consistent with reduced rRNA expression in the hippocampus. This difference in rRNA methylation was not evident in the cerebellum and occurred in the absence of genome-wide changes in methylation, as assessed by nearest neighbor. CONCLUSIONS/SIGNIFICANCE: This is the first study to show aberrant regulation of the protein synthesis machinery in the suicide brain. The data implicate the epigenetic modulation of rRNA in the pathophysiology of suicide.

RNA preparation and characterization for gene expression studies

DEFF Research Database (Denmark)

Stangegaard, Michael

2009-01-01

Much information can be obtained from knowledge of the relative expression level of each gene in the transcriptome. With the current advances in technology as little as a single cell is required as starting material for gene expression experiments. The mRNA from a single cell may be linearly...

Use of tiling array data and RNA secondary structure predictions to identify noncoding RNA genes

DEFF Research Database (Denmark)

Weile, Christian; Gardner, Paul P; Hedegaard, Mads M

2007-01-01

neuroblastoma cell line SK-N-AS. Using this strategy, we identify thousands of human candidate RNA genes. To further verify the expression of these genes, we focused on candidate genes that had a stable hairpin structures or a high level of covariance. Using northern blotting, we verify the expression of 2 out...

Synergistic Effect of Auto-Activation and Small RNA Regulation on Gene Expression

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Xiong, Li-Ping; Ma, Yu-Qiang; Tang, Lei-Han

2010-09-01

Auto-activation and small ribonucleic acid (RNA)-mediated regulation are two important mechanisms in controlling gene expression. We study the synergistic effect of these two regulations on gene expression. It is found that under this combinatorial regulation, gene expression exhibits bistable behaviors at the transition regime, while each of these two regulations, if working solely, only leads to monostability. Within the stochastic framework, the base pairing strength between sRNA and mRNA plays an important role in controlling the transition time between on and off states. The noise strength of protein number in the off state approaches 1 and is smaller than that in the on state. The noise strength also depends on which parameters, the feedback strength or the synthesis rate of small RNA, are tuned in switching the gene expression on and off. Our findings may provide a new insight into gene-regulation mechanism and can be applied in synthetic biology.

Synergistic Effect of Auto-Activation and Small RNA Regulation on Gene Expression

International Nuclear Information System (INIS)

Li-Ping, Xiong; Yu-Qiang, Ma; Lei-Han, Tang

2010-01-01

Auto-activation and small ribonucleic acid (RNA)-mediated regulation are two important mechanisms in controlling gene expression. We study the synergistic effect of these two regulations on gene expression. It is found that under this combinatorial regulation, gene expression exhibits bistable behaviors at the transition regime, while each of these two regulations, if working solely, only leads to monostability. Within the stochastic framework, the base pairing strength between sRNA and mRNA plays an important role in controlling the transition time between on and off states. The noise strength of protein number in the off state approaches 1 and is smaller than that in the on state. The noise strength also depends on which parameters, the feedback strength or the synthesis rate of small RNA, are tuned in switching the gene expression on and off. Our findings may provide a new insight into gene-regulation mechanism and can be applied in synthetic biology

International interlaboratory study comparing single organism 16S rRNA gene sequencing data: Beyond consensus sequence comparisons

Science.gov (United States)

Olson, Nathan D.; Lund, Steven P.; Zook, Justin M.; Rojas-Cornejo, Fabiola; Beck, Brian; Foy, Carole; Huggett, Jim; Whale, Alexandra S.; Sui, Zhiwei; Baoutina, Anna; Dobeson, Michael; Partis, Lina; Morrow, Jayne B.

2015-01-01

This study presents the results from an interlaboratory sequencing study for which we developed a novel high-resolution method for comparing data from different sequencing platforms for a multi-copy, paralogous gene. The combination of PCR amplification and 16S ribosomal RNA gene (16S rRNA) sequencing has revolutionized bacteriology by enabling rapid identification, frequently without the need for culture. To assess variability between laboratories in sequencing 16S rRNA, six laboratories sequenced the gene encoding the 16S rRNA from Escherichia coli O157:H7 strain EDL933 and Listeria monocytogenes serovar 4b strain NCTC11994. Participants performed sequencing methods and protocols available in their laboratories: Sanger sequencing, Roche 454 pyrosequencing®, or Ion Torrent PGM®. The sequencing data were evaluated on three levels: (1) identity of biologically conserved position, (2) ratio of 16S rRNA gene copies featuring identified variants, and (3) the collection of variant combinations in a set of 16S rRNA gene copies. The same set of biologically conserved positions was identified for each sequencing method. Analytical methods using Bayesian and maximum likelihood statistics were developed to estimate variant copy ratios, which describe the ratio of nucleotides at each identified biologically variable position, as well as the likely set of variant combinations present in 16S rRNA gene copies. Our results indicate that estimated variant copy ratios at biologically variable positions were only reproducible for high throughput sequencing methods. Furthermore, the likely variant combination set was only reproducible with increased sequencing depth and longer read lengths. We also demonstrate novel methods for evaluating variable positions when comparing multi-copy gene sequence data from multiple laboratories generated using multiple sequencing technologies. PMID:27077030

International interlaboratory study comparing single organism 16S rRNA gene sequencing data: Beyond consensus sequence comparisons

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Nathan D. Olson

2015-03-01

Full Text Available This study presents the results from an interlaboratory sequencing study for which we developed a novel high-resolution method for comparing data from different sequencing platforms for a multi-copy, paralogous gene. The combination of PCR amplification and 16S ribosomal RNA gene (16S rRNA sequencing has revolutionized bacteriology by enabling rapid identification, frequently without the need for culture. To assess variability between laboratories in sequencing 16S rRNA, six laboratories sequenced the gene encoding the 16S rRNA from Escherichia coli O157:H7 strain EDL933 and Listeria monocytogenes serovar 4b strain NCTC11994. Participants performed sequencing methods and protocols available in their laboratories: Sanger sequencing, Roche 454 pyrosequencing®, or Ion Torrent PGM®. The sequencing data were evaluated on three levels: (1 identity of biologically conserved position, (2 ratio of 16S rRNA gene copies featuring identified variants, and (3 the collection of variant combinations in a set of 16S rRNA gene copies. The same set of biologically conserved positions was identified for each sequencing method. Analytical methods using Bayesian and maximum likelihood statistics were developed to estimate variant copy ratios, which describe the ratio of nucleotides at each identified biologically variable position, as well as the likely set of variant combinations present in 16S rRNA gene copies. Our results indicate that estimated variant copy ratios at biologically variable positions were only reproducible for high throughput sequencing methods. Furthermore, the likely variant combination set was only reproducible with increased sequencing depth and longer read lengths. We also demonstrate novel methods for evaluating variable positions when comparing multi-copy gene sequence data from multiple laboratories generated using multiple sequencing technologies.

Analysis of gene expression in normal and neoplastic human testis: new roles of RNA

DEFF Research Database (Denmark)

Novotny, G W; Nielsen, J E; Sonne, Si Brask

2007-01-01

Large-scale methods for analysing gene expression, such as microarrays, have yielded a wealth of information about gene expression at the mRNA level. However, expression of alternative transcripts, together with the presence of a wide range of largely undescribed RNA transcripts combined with reg......Large-scale methods for analysing gene expression, such as microarrays, have yielded a wealth of information about gene expression at the mRNA level. However, expression of alternative transcripts, together with the presence of a wide range of largely undescribed RNA transcripts combined...

Synthesis of 4-thiouridine, 6-thioinosine, and 6-thioguanosine 3',5'-O-bisphosphates as donor molecules for RNA ligation and their application to the synthesis of photoactivatable TMG-capped U1 snRNA fragments.

Science.gov (United States)

Kadokura, M; Wada, T; Seio, K; Sekine, M

2000-08-25

4-Thiouridine, 6-thioguanosine, and 6-thioinosine 3',5'-bisphosphates (9, 20, and 28) were synthesized in good yields by considerably improved methods. In the former two compounds, uridine and 2-N-phenylacetylguanosine were converted via transient O-trimethylsilylation to the corresponding 4- and 6-O-benzenesulfonyl intermediates (2 and 13), which, in turn, were allowed to react with 2-cyanoethanethiol in the presence of N-methylpyrrolidine to give 4-thiouridine (3) and 2-N-phenylacetyl-6-thioguanosine derivatives (14), respectively. In situ dimethoxytritylation of these thionucleoside derivatives gave the 5'-masked products 4 and 15 in high overall yields from 1 and 11. 6-S-(2-Cyanoethyl)-5'-O-(4,4'-dimethoxytrityl)-6-thioinosine (23) was synthesized via substitution of the 5'-O-tritylated 6-chloropurine riboside derivative 22 with 2-cyanoethanethiol. These S-(2-cyanoethyl)thionucleosides were converted to the 2'-O-(tert-butyldimethylsilyl)ribonucleoside 3'-phosphoramidite derivatives 7, 18, and 26 or 3',5'-bisphosphate derivatives 8, 19, and 27. Treatment of 8, 19, and 27 with DBU gave thionucleoside 3',5'-bisphosphate derivatives 9, 20, and 28, which were found to be substrates of T4 RNA ligase. These thionucleoside 3',5'-bisphosphates were examined as donors for ligation with m3(2,2,7) G5'pppAmUmA, i.e., the 5'-terminal tetranucleotide fragment of U1 snRNA, The 4-thiouridine 3',5'-bisphosphate derivative 9 was found to serve as the most active substrate of T4 RNA ligase with a reaction efficiency of 96%.

Yeast RNA viruses as indicators of exosome activity: human exosome hCsl4p participates in RNA degradation in Saccharomyces cerevisiae'.

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Ramírez-Garrastacho, Manuel; Esteban, Rosa

2011-12-01

The exosome is an evolutionarily conserved 10-mer complex involved in RNA metabolism, located in both the nucleus and the cytoplasm. The cytoplasmic exosome plays an important role in mRNA turnover through its 3'→5' exonucleolytic activity. The superkiller (SKI) phenotype of yeast was originally identified as an increase of killer toxin production due to elevated levels of the L-A double-stranded RNA (dsRNA) Totivirus and its satellite toxin-encoding M dsRNA. Most SKI genes were later shown to be either components of the exosome or modulators of its activity. Variations in the amount of Totivirus are, thus, good indicators of yeast exosome activity, and can be used to analyse its components. Furthermore, if exosome proteins of higher eukaryotes were functional in S. cerevisiae, these viruses would provide a simple tool to analyse their function. In this work, we have found that hCSL4, the human orthologue of SKI4 in the yeast exosome, rescues the null phenotype of the deletion mutant. hCsl4p shares with Ski4p conserved S1 RNA-binding domains, but lacks the N-terminal third of Ski4p. Nevertheless, it interacts with the Dis3p exonuclease of yeast exosome, and partially complements the superkiller phenotype of ski4-1 mutation. The elimination of the N-terminal third of Ski4p does not affect its activity, indicating that it is dispensable for RNA degradation. We have also identified the point mutation G152E in hCSL4, equivalent to the ski4-1 mutation G253E, which impairs the activity of the protein, thus validating our approach of using yeast RNA virus to analyse the exosome of higher eukaryotes. Copyright © 2011 John Wiley & Sons, Ltd.

Prevalence of 16S rRNA methylase genes among β-lactamase ...

African Journals Online (AJOL)

Background: Co production of 16S rRNA methylases gene and β-Lactamase gene among Enterobacteriaceae isolates conferring resistance to both therapeutic options has serious implications for clinicians worldwide. Methods: To study co existence of 16S rRNA methylases (armA, rmtA, rmtB, rmtC, rmtD, and npmA) and ...

Direct Regulation of tRNA and 5S rRNA Gene Transcription by Polo-like Kinase 1

NARCIS (Netherlands)

Fairley, Jennifer A.; Mitchell, Louise E.; Berg, Tracy; Kenneth, Niall S.; von Schubert, Conrad; Sillje, Herman H. W.; Medema, Rene H.; Nigg, Erich A.; White, Robert J.

2012-01-01

Polo-like kinase Plk1 controls numerous aspects of cell-cycle progression. We show that it associates with tRNA and 5S rRNA genes and regulates their transcription by RNA polymerase Ill (pol Ill) through direct binding and phosphorylation of transcription factor Brit During interphase, Plk1 promotes

A Caenorhabditis elegans RNA polymerase II gene, ama-1 IV, and nearby essential genes.

Science.gov (United States)

Rogalski, T M; Riddle, D L

1988-01-01

The amanitin-binding subunit of RNA polymerase II in Caenorhabditis elegans is encoded by the ama-1 gene, located approximately 0.05 map unit to the right of dpy-13 IV. Using the amanitin-resistant ama-1(m118) strain as a parent, we have isolated amanitin-sensitive mutants that carry recessive-lethal ama-1 alleles. Of the six ethyl methanesulfonate-induced mutants examined, two are arrested late in embryogenesis. One of these is a large deficiency, mDf9, but the second may be a novel point mutation. The four other mutants are hypomorphs, and presumably produce altered RNA polymerase II enzymes with some residual function. Two of these mutants develop into sterile adults at 20 degrees but are arrested as larvae at 25 degrees, and two others are fertile at 20 degrees and sterile at 25 degrees. Temperature-shift experiments performed with the adult sterile mutant, ama-1(m118m238ts), have revealed a temperature-sensitive period that begins late in gonadogenesis and is centered around the initiation of egg-laying. Postembryonic development at 25 degrees is slowed by 30%. By contrast, the amanitin-resistant allele of ama-1 has very little effect on developmental rate or fertility. We have identified 15 essential genes in an interval of 4.5 map units surrounding ama-1, as well as four gamma-ray-induced deficiencies and two duplications that include the ama-1 gene. The larger duplication, mDp1, may include the entire left arm of chromosome IV, and it recombines with the normal homologue at a low frequency. The smallest deficiency, mDf10, complements all but three identified genes: let-278, dpy-13 and ama-1, which define an interval of only 0.1 map unit. The terminal phenotype of mDf10 homozygotes is developmental arrest during the first larval stage, suggesting that there is sufficient maternal RNA polymerase II to complete embryonic development.

Equilibrium electrode U(4)-U and redox U(4)-U(3) potentials in molten alkali metal chlorides medium

Energy Technology Data Exchange (ETDEWEB)

Smirnov, M V; Kudyakov, V Ya; Komarov, V E; Salyulev, A B [AN SSSR, Sverdlovsk. Inst. Ehlektrokhimii

1979-02-01

Conditional standard electrode potentials of uranium are determined for diluted solutions of its tetrachloride in alkali metal chloride melts (LiCl, NaCl, NaCl-KCl, KCl, RbCl and CsCl) when using U(4) ion activity coefficient values experimentally found by the tensimetric method. These potentials shift to the electronegative side at the temperature decrease and alkali cation radius increase rsub(Msup(+)) according to the empiric ratio E*U(4)-U= -3.06+6.87x10/sup -4/ T-(1.67-10/sup -4/T-0.44) 1/rsub(Msup(+)) +-0.01. The temperature dependences of formal conditional redox potentials of the U(4)-U(3) system for above melted chlorides are estimated. The E*U(4)-U(3) value also becomes more electronegative in the series LiCl, NaCl, NaCl-KCl, KCl, RbCl and CsCl. This alternation is satisfactorily described by the empiric expression E*U(4)-U(3)= -1.74+1.74x10/sup -4/T-(0.71x10/sup -4/T-0.20) 1rsub(Msup(+)) +-0.05. The calculated values Eu*(4)-U(3) are compared with those directly measured for the NaCl-KCl equimolar mixture and 3LiCl-2KCl eutectic mixture. A satisfactory confirmity has been observed.

Equilibrium electrode U(4)-U and redox U(4)-U(3) potentials in molten alkali metal chlorides medium

International Nuclear Information System (INIS)

Smirnov, M.V.; Kudyakov, V.Ya.; Komarov, V.E.; Salyulev, A.B.

1979-01-01

Conditional standard electrode potentials of uranium are determined for diluted solutions of its tetrachloride in alkali metal chloride melts (LiCl, NaCl, NaCl-KCl, KCl, RbCl and CsCl) when using U(4) ion activity coefficient values experimentally found by the tensimetric method. These potentials shift to the electronegative side at the temperature decrease and alkali cation radius increase rsub(Msup(+)) according to the empiric ratio E*U(4)-U= -3.06+6.87x10 -4 T-(1.67-10 -4 T-0.44) 1/rsub(Msup(+)) +-0.01. The temperature dependences of formal conditional redox potentials of the U(4)-U(3) system for above melted chlorides are estimated. The E*U(4)-U(3) value also becomes more electronegative in the series LiCl, NaCl, NaCl-KCl, KCl, RbCl and CsCl. This alternation is satisfactorily described by the empiric expression E*U(4)-U(3)= -1.74+1.74x10 -4 T-(0.71x10 -4 T-0.20) 1rsub(Msup(+)) +-0.05. The calculated values Eu*(4)-U(3) are compared with those directly measured for the NaCl-KCl equimolar mixture and 3LiCl-2KCl eutectic mixture. A satisfactory confirmity has been observed

Gene Ranking of RNA-Seq Data via Discriminant Non-Negative Matrix Factorization.

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Jia, Zhilong; Zhang, Xiang; Guan, Naiyang; Bo, Xiaochen; Barnes, Michael R; Luo, Zhigang

2015-01-01

RNA-sequencing is rapidly becoming the method of choice for studying the full complexity of transcriptomes, however with increasing dimensionality, accurate gene ranking is becoming increasingly challenging. This paper proposes an accurate and sensitive gene ranking method that implements discriminant non-negative matrix factorization (DNMF) for RNA-seq data. To the best of our knowledge, this is the first work to explore the utility of DNMF for gene ranking. When incorporating Fisher's discriminant criteria and setting the reduced dimension as two, DNMF learns two factors to approximate the original gene expression data, abstracting the up-regulated or down-regulated metagene by using the sample label information. The first factor denotes all the genes' weights of two metagenes as the additive combination of all genes, while the second learned factor represents the expression values of two metagenes. In the gene ranking stage, all the genes are ranked as a descending sequence according to the differential values of the metagene weights. Leveraging the nature of NMF and Fisher's criterion, DNMF can robustly boost the gene ranking performance. The Area Under the Curve analysis of differential expression analysis on two benchmarking tests of four RNA-seq data sets with similar phenotypes showed that our proposed DNMF-based gene ranking method outperforms other widely used methods. Moreover, the Gene Set Enrichment Analysis also showed DNMF outweighs others. DNMF is also computationally efficient, substantially outperforming all other benchmarked methods. Consequently, we suggest DNMF is an effective method for the analysis of differential gene expression and gene ranking for RNA-seq data.

A mitochondrial tRNA(His) gene mutation causing pigmentary retinopathy and neurosensorial deafness.

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Crimi, M; Galbiati, S; Perini, M P; Bordoni, A; Malferrari, G; Sciacco, M; Biunno, I; Strazzer, S; Moggio, M; Bresolin, N; Comi, G P

2003-04-08

We have identified a heteroplasmic G to A mutation at position 12,183 of the mitochondrial transfer RNA Histidine (tRNA(His)) gene in three related patients. These phenotypes varied according to mutation heteroplasmy: one had severe pigmentary retinopathy, neurosensorial deafness, testicular dysfunction, muscle hypotrophy, and ataxia; the other two had only retinal and inner ear involvement. The mutation is in a highly conserved region of the T(psi)C stem of the tRNA(His) gene and may alter secondary structure formation. This is the first described pathogenic, maternally inherited mutation of the mitochondrial tRNA(His) gene.

GENE-counter: a computational pipeline for the analysis of RNA-Seq data for gene expression differences.

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Cumbie, Jason S; Kimbrel, Jeffrey A; Di, Yanming; Schafer, Daniel W; Wilhelm, Larry J; Fox, Samuel E; Sullivan, Christopher M; Curzon, Aron D; Carrington, James C; Mockler, Todd C; Chang, Jeff H

2011-01-01

GENE-counter is a complete Perl-based computational pipeline for analyzing RNA-Sequencing (RNA-Seq) data for differential gene expression. In addition to its use in studying transcriptomes of eukaryotic model organisms, GENE-counter is applicable for prokaryotes and non-model organisms without an available genome reference sequence. For alignments, GENE-counter is configured for CASHX, Bowtie, and BWA, but an end user can use any Sequence Alignment/Map (SAM)-compliant program of preference. To analyze data for differential gene expression, GENE-counter can be run with any one of three statistics packages that are based on variations of the negative binomial distribution. The default method is a new and simple statistical test we developed based on an over-parameterized version of the negative binomial distribution. GENE-counter also includes three different methods for assessing differentially expressed features for enriched gene ontology (GO) terms. Results are transparent and data are systematically stored in a MySQL relational database to facilitate additional analyses as well as quality assessment. We used next generation sequencing to generate a small-scale RNA-Seq dataset derived from the heavily studied defense response of Arabidopsis thaliana and used GENE-counter to process the data. Collectively, the support from analysis of microarrays as well as the observed and substantial overlap in results from each of the three statistics packages demonstrates that GENE-counter is well suited for handling the unique characteristics of small sample sizes and high variability in gene counts.

GENE-counter: a computational pipeline for the analysis of RNA-Seq data for gene expression differences.

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Jason S Cumbie

Full Text Available GENE-counter is a complete Perl-based computational pipeline for analyzing RNA-Sequencing (RNA-Seq data for differential gene expression. In addition to its use in studying transcriptomes of eukaryotic model organisms, GENE-counter is applicable for prokaryotes and non-model organisms without an available genome reference sequence. For alignments, GENE-counter is configured for CASHX, Bowtie, and BWA, but an end user can use any Sequence Alignment/Map (SAM-compliant program of preference. To analyze data for differential gene expression, GENE-counter can be run with any one of three statistics packages that are based on variations of the negative binomial distribution. The default method is a new and simple statistical test we developed based on an over-parameterized version of the negative binomial distribution. GENE-counter also includes three different methods for assessing differentially expressed features for enriched gene ontology (GO terms. Results are transparent and data are systematically stored in a MySQL relational database to facilitate additional analyses as well as quality assessment. We used next generation sequencing to generate a small-scale RNA-Seq dataset derived from the heavily studied defense response of Arabidopsis thaliana and used GENE-counter to process the data. Collectively, the support from analysis of microarrays as well as the observed and substantial overlap in results from each of the three statistics packages demonstrates that GENE-counter is well suited for handling the unique characteristics of small sample sizes and high variability in gene counts.

Punctual mutations in 23S rRNA gene of clarithromycin-resistant Helicobacter pylori in Colombian populations.

Science.gov (United States)

Matta, Andrés Jenuer; Zambrano, Diana Carolina; Pazos, Alvaro Jairo

2018-04-14

To characterize punctual mutations in 23S rRNA gene of clarithromycin-resistant Helicobacter pylori ( H. pylori ) and determine their association with therapeutic failure. PCR products of 23S rRNA gene V domain of 74 H. pylori isolates; 34 resistant to clarithromycin (29 from a low-risk gastric cancer (GC) population: Tumaco-Colombia, and 5 from a high-risk population: Tuquerres-Colombia) and 40 from a susceptible population (28 from Tumaco and 12 from Túquerres) were sequenced using capillary electrophoresis. The concordance between mutations of V domain 23S rRNA gene of H. pylori and therapeutic failure was determined using the Kappa coefficient and McNemar's test was performed to determine the relationship between H. pylori mutations and clarithromycin resistance. 23S rRNA gene from H. pylori was amplified in 56/74 isolates, of which 25 were resistant to clarithromycin (20 from Tumaco and 5 from Túquerres, respectively). In 17 resistant isolates (13 from Tumaco and 4 from Túquerres) the following mutations were found: A1593T1, A1653G2, C1770T, C1954T1, and G1827C in isolates from Tumaco, and A2144G from Túquerres. The mutations T2183C, A2144G and C2196T in H. pylori isolates resistant to clarithromycin from Colombia are reported for the first time. No association between the H. pylori mutations and in vitro clarithromycin resistance was found. However, therapeutic failure of eradication treatment was associated with mutations of 23S rRNA gene in clarithromycin-resistant H. pylori ( κ = 0.71). The therapeutic failure of eradication treatment in the two populations from Colombia was associated with mutations of the 23S rRNA gene in clarithromycin-resistant H. pylori .

Multilevel Regulation of Bacterial Gene Expression with the Combined STAR and Antisense RNA System.

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Lee, Young Je; Kim, Soo-Jung; Moon, Tae Seok

2018-03-16

Synthetic small RNA regulators have emerged as a versatile tool to predictably control bacterial gene expression. Owing to their simple design principles, small size, and highly orthogonal behavior, these engineered genetic parts have been incorporated into genetic circuits. However, efforts to achieve more sophisticated cellular functions using RNA regulators have been hindered by our limited ability to integrate different RNA regulators into complex circuits. Here, we present a combined RNA regulatory system in Escherichia coli that uses small transcription activating RNA (STAR) and antisense RNA (asRNA) to activate or deactivate target gene expression in a programmable manner. Specifically, we demonstrated that the activated target output by the STAR system can be deactivated by expressing two different types of asRNAs: one binds to and sequesters the STAR regulator, affecting the transcription process, while the other binds to the target mRNA, affecting the translation process. We improved deactivation efficiencies (up to 96%) by optimizing each type of asRNA and then integrating the two optimized asRNAs into a single circuit. Furthermore, we demonstrated that the combined STAR and asRNA system can control gene expression in a reversible way and can regulate expression of a gene in the genome. Lastly, we constructed and simultaneously tested two A AND NOT B logic gates in the same cell to show sophisticated multigene regulation by the combined system. Our approach establishes a methodology for integrating multiple RNA regulators to rationally control multiple genes.

Evolutionary acquisition of promoter-associated non-coding RNA (pancRNA) repertoires diversifies species-dependent gene activation mechanisms in mammals

OpenAIRE

Uesaka, Masahiro; Agata, Kiyokazu; Oishi, Takao; Nakashima, Kinichi; Imamura, Takuya

2017-01-01

Background Recent transcriptome analyses have shown that long non-coding RNAs (ncRNAs) play extensive roles in transcriptional regulation. In particular, we have reported that promoter-associated ncRNAs (pancRNAs) activate the partner gene expression via local epigenetic changes. Results Here, we identify thousands of genes under pancRNA-mediated transcriptional activation in five mammalian species in common. In the mouse, 1) pancRNA-partnered genes confined their expression pattern to certai...

High-resolution microscopy of active ribosomal genes and key members of the rRNA processing machinery inside nucleolus-like bodies of fully-grown mouse oocytes.

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Shishova, Kseniya V; Khodarovich, Yuriy M; Lavrentyeva, Elena A; Zatsepina, Olga V

2015-10-01

Nucleolus-like bodies (NLBs) of fully-grown (germinal vesicle, GV) mammalian oocytes are traditionally considered as morphologically distinct entities, which, unlike normal nucleoli, contain transcribed ribosomal genes (rDNA) solely at their surface. In the current study, we for the first time showed that active ribosomal genes are present not only on the surface but also inside NLBs of the NSN-type oocytes. The "internal" rRNA synthesis was evidenced by cytoplasmic microinjections of BrUTP as precursor and by fluorescence in situ hybridization with a probe to the short-lived 5'ETS segment of the 47S pre-rRNA. We further showed that in the NLB mass of NSN-oocytes, distribution of active rDNA, RNA polymerase I (UBF) and rRNA processing (fibrillarin) protein factors, U3 snoRNA, pre-rRNAs and 18S/28S rRNAs is remarkably similar to that in somatic nucleoli capable to make pre-ribosomes. Overall, these observations support the occurrence of rDNA transcription, rRNA processing and pre-ribosome assembly in the NSN-type NLBs and so that their functional similarity to normal nucleoli. Unlike the NSN-type NLBs, the NLBs of more mature SN-oocytes do not contain transcribed rRNA genes, U3 snoRNA, pre-rRNAs, 18S and 28S rRNAs. These results favor the idea that in a process of transformation of NSN-oocytes to SN-oocytes, NLBs cease to produce pre-ribosomes and, moreover, lose their rRNAs. We also concluded that a denaturing fixative 70% ethanol used in the study to fix oocytes could be more appropriate for light microscopy analysis of nucleolar RNAs and proteins in mammalian fully-grown oocytes than a commonly used cross-linking aldehyde fixative, formalin. Copyright © 2015 Elsevier Inc. All rights reserved.

Characterization of Hydrocortisone Biometabolites and 18S rRNA Gene in Chlamydomonas reinhardtii Cultures

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Seyed Bagher Mosavi-Azam

2008-10-01

Full Text Available A unicellular microalga, Chlamydomonas reinhardtii, was isolated from rice paddy-field soil and water samples and used in the biotransformation of hydrocortisone (1. This strain has not been previously tested for steroid bioconversion. Fermentation was carried out in BG-11 medium supplemented with 0.05% substrate at 25ºC for 14 days of incubation. The products obtained were chromatographically purified and characterized using spectroscopic methods. 11b,17b-Dihydroxyandrost-4-en-3-one (2, 11b-hydroxyandrost-4-en-3,17-dione (3, 11b,17a,20b,21-tetrahydroxypregn-4-en-3-one (4 and prednisolone (5 were the main products of the bioconversion. The observed bioreaction features were the side chain degradation of the substrate to give compounds 2 and 3 and the 20-ketone reduction and 1,2-dehydrogenation affording compounds 4 and 5, respectively. A time course study showed the accumulation of product 2 from the second day of the fermentation and of compounds 3, 4 and 5 from the third day. All the metabolites reached their maximum concentration in seven days. Microalgal 18S rRNA gene was also amplified by PCR. PCR products were sequenced to confirm their authenticity as 18S rRNA gene of microalgae. The result of PCR blasted with other sequenced microalgae in NCBI showed 100% homology to the 18S small subunit rRNA of two Chlamydomonas reinhardtii spp.

Characterization of hydrocortisone biometabolites and 18S rRNA gene in Chlamydomonas reinhardtii cultures.

Science.gov (United States)

Ghasemi, Younes; Rasoul-Amini, Sara; Morowvat, Mohammad Hossein; Raee, Mohammad Javad; Ghoshoon, Mohammad Bagher; Nouri, Fatemeh; Negintaji, Narges; Parvizi, Rezvan; Mosavi-Azam, Seyed Bagher

2008-10-31

A unicellular microalga, Chlamydomonas reinhardtii, was isolated from rice paddy-field soil and water samples and used in the biotransformation of hydrocortisone (1). This strain has not been previously tested for steroid bioconversion. Fermentation was carried out in BG-11 medium supplemented with 0.05% substrate at 25 degrees C for 14 days of incubation. The products obtained were chromatographically purified and characterized using spectroscopic methods. 11b,17 beta-Dihydroxyandrost-4-en-3-one (2), 11 beta-hydroxyandrost-4-en-3,17-dione (3), 11 beta,17 alpha,20 beta,21-tetrahydroxypregn-4-en-3-one (4) and prednisolone (5) were the main products of the bioconversion. The observed bioreaction features were the side chain degradation of the substrate to give compounds 2 and 3 and the 20-ketone reduction and 1,2-dehydrogenation affording compounds 4 and 5, respectively. A time course study showed the accumulation of product 2 from the second day of the fermentation and of compounds 3, 4 and 5 from the third day. All the metabolites reached their maximum concentration in seven days. Microalgal 18S rRNA gene was also amplified by PCR. PCR products were sequenced to confirm their authenticity as 18S rRNA gene of microalgae. The result of PCR blasted with other sequenced microalgae in NCBI showed 100% homology to the 18S small subunit rRNA of two Chlamydomonas reinhardtii spp.

miRNA genes of an invasive vector mosquito, Aedes albopictus.

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Jinbao Gu

Full Text Available Aedes albopictus, a vector of Dengue and Chikungunya viruses, is a robust invasive species in both tropical and temperate environments. MicroRNAs (miRNAs regulate gene expression and biological processes including embryonic development, innate immunity and infection. While a number of miRNAs have been discovered in some mosquitoes, no comprehensive effort has been made to characterize them from different developmental stages from a single species. Systematic analysis of miRNAs in Ae. albopictus will improve our understanding of its basic biology and inform novel strategies to prevent virus transmission. Between 10-14 million Illumina sequencing reads per sample were obtained from embryos, larvae, pupae, adult males, sugar-fed and blood-fed adult females. A total of 119 miRNA genes represented by 215 miRNA or miRNA star (miRNA* sequences were identified, 15 of which are novel. Eleven, two, and two of the newly-discovered miRNA genes appear specific to Aedes, Culicinae, and Culicidae, respectively. A number of miRNAs accumulate predominantly in one or two developmental stages and the large number that showed differences in abundance following a blood meal likely are important in blood-induced mosquito biology. Gene Ontology (GO analysis of the targets of all Ae. albopictus miRNAs provides a useful starting point for the study of their functions in mosquitoes. This study is the first systematic analysis of miRNAs based on deep-sequencing of small RNA samples of all developmental stages of a mosquito species. A number of miRNAs are related to specific physiological states, most notably, pre- and post-blood feeding. The distribution of lineage-specific miRNAs is consistent with mosquito phylogeny and the presence of a number of Aedes-specific miRNAs likely reflects the divergence between the Aedes and Culex genera.

[Mechanism of reaction catalyzed by RNA-ligase from bacteriophage T4].

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Zagrebel'nyĭ, S N; Zernov, Iu P

1987-01-01

The dissociation constants of the complexes of RNA-ligase with acceptors, donors and the adenylylated donor A(5')ppAp have been determined on the basis of the inhibition of ATP-pyrophosphate exchange reaction. The dissociation constants of the complexes of the enzyme with "poor" acceptors (oligouridilates) have been shown to be slightly different from those with "good" acceptors (oligoadenylates). The dependence of the reaction velocity of the formation of ligation products on the concentration of acceptors (pA)4, (pU)4 and the adenylylated donor A(5)ppAp has been studied. On the basis of the data obtained the conclusion about the random addition mechanism has been drawn. The reaction takes place in the steady-state conditions in the case of (pA)4 and in the equilibrium conditions--in the case of (pU)4.

Distinct roles for RDE-1 and RDE-4 during RNA interference in Caenorhabditis elegans.

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Parrish, S; Fire, A

2001-10-01

RNA interference (RNAi) is a cellular defense mechanism that uses double-stranded RNA (dsRNA) as a sequence-specific trigger to guide the degradation of homologous single-stranded RNAs. RNAi is a multistep process involving several proteins and at least one type of RNA intermediate, a population of small 21-25 nt RNAs (called siRNAs) that are initially derived from cleavage of the dsRNA trigger. Genetic screens in Caenorhabditis elegans have identified numerous mutations that cause partial or complete loss of RNAi. In this work, we analyzed cleavage of injected dsRNA to produce the initial siRNA population in animals mutant for rde-1 and rde-4, two genes that are essential for RNAi but that are not required for organismal viability or fertility. Our results suggest distinct roles for RDE-1 and RDE-4 in the interference process. Although null mutants lacking rde-1 show no phenotypic response to dsRNA, the amount of siRNAs generated from an injected dsRNA trigger was comparable to that of wild-type. By contrast, mutations in rde-4 substantially reduced the population of siRNAs derived from an injected dsRNA trigger. Injection of chemically synthesized 24- or 25-nt siRNAs could circumvent RNAi resistance in rde-4 mutants, whereas no bypass was observed in rde-1 mutants. These results support a model in which RDE-4 is involved before or during production of siRNAs, whereas RDE-1 acts after the siRNAs have been formed.

BRAKER1: Unsupervised RNA-Seq-Based Genome Annotation with GeneMark-ET and AUGUSTUS.

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Hoff, Katharina J; Lange, Simone; Lomsadze, Alexandre; Borodovsky, Mark; Stanke, Mario

2016-03-01

Gene finding in eukaryotic genomes is notoriously difficult to automate. The task is to design a work flow with a minimal set of tools that would reach state-of-the-art performance across a wide range of species. GeneMark-ET is a gene prediction tool that incorporates RNA-Seq data into unsupervised training and subsequently generates ab initio gene predictions. AUGUSTUS is a gene finder that usually requires supervised training and uses information from RNA-Seq reads in the prediction step. Complementary strengths of GeneMark-ET and AUGUSTUS provided motivation for designing a new combined tool for automatic gene prediction. We present BRAKER1, a pipeline for unsupervised RNA-Seq-based genome annotation that combines the advantages of GeneMark-ET and AUGUSTUS. As input, BRAKER1 requires a genome assembly file and a file in bam-format with spliced alignments of RNA-Seq reads to the genome. First, GeneMark-ET performs iterative training and generates initial gene structures. Second, AUGUSTUS uses predicted genes for training and then integrates RNA-Seq read information into final gene predictions. In our experiments, we observed that BRAKER1 was more accurate than MAKER2 when it is using RNA-Seq as sole source for training and prediction. BRAKER1 does not require pre-trained parameters or a separate expert-prepared training step. BRAKER1 is available for download at http://bioinf.uni-greifswald.de/bioinf/braker/ and http://exon.gatech.edu/GeneMark/ katharina.hoff@uni-greifswald.de or borodovsky@gatech.edu Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Human Immunodeficiency Virus-Type 1 LTR DNA contains an intrinsic gene producing antisense RNA and protein products

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Hsiao Chiu-Bin

2006-11-01

Full Text Available Abstract Background While viruses have long been shown to capitalize on their limited genomic size by utilizing both strands of DNA or complementary DNA/RNA intermediates to code for viral proteins, it has been assumed that human retroviruses have all their major proteins translated only from the plus or sense strand of RNA, despite their requirement for a dsDNA proviral intermediate. Several studies, however, have suggested the presence of antisense transcription for both HIV-1 and HTLV-1. More recently an antisense transcript responsible for the HTLV-1 bZIP factor (HBZ protein has been described. In this study we investigated the possibility of an antisense gene contained within the human immunodeficiency virus type 1 (HIV-1 long terminal repeat (LTR. Results Inspection of published sequences revealed a potential transcription initiator element (INR situated downstream of, and in reverse orientation to, the usual HIV-1 promoter and transcription start site. This antisense initiator (HIVaINR suggested the possibility of an antisense gene responsible for RNA and protein production. We show that antisense transcripts are generated, in vitro and in vivo, originating from the TAR DNA of the HIV-1 LTR. To test the possibility that protein(s could be translated from this novel HIV-1 antisense RNA, recombinant HIV antisense gene-FLAG vectors were designed. Recombinant protein(s were produced and isolated utilizing carboxy-terminal FLAG epitope (DYKDDDDK sequences. In addition, affinity-purified antisera to an internal peptide derived from the HIV antisense protein (HAP sequences identified HAPs from HIV+ human peripheral blood lymphocytes. Conclusion HIV-1 contains an antisense gene in the U3-R regions of the LTR responsible for both an antisense RNA transcript and proteins. This antisense transcript has tremendous potential for intrinsic RNA regulation because of its overlap with the beginning of all HIV-1 sense RNA transcripts by 25 nucleotides. The

MicroRNA genes and their target 3'-untranslated regions are infrequently somatically mutated in ovarian cancers.

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Georgina L Ryland

Full Text Available MicroRNAs are key regulators of gene expression and have been shown to have altered expression in a variety of cancer types, including epithelial ovarian cancer. MiRNA function is most often achieved through binding to the 3'-untranslated region of the target protein coding gene. Mutation screening using massively-parallel sequencing of 712 miRNA genes in 86 ovarian cancer cases identified only 5 mutated miRNA genes, each in a different case. One mutation was located in the mature miRNA, and three mutations were predicted to alter the secondary structure of the miRNA transcript. Screening of the 3'-untranslated region of 18 candidate cancer genes identified one mutation in each of AKT2, EGFR, ERRB2 and CTNNB1. The functional effect of these mutations is unclear, as expression data available for AKT2 and EGFR showed no increase in gene transcript. Mutations in miRNA genes and 3'-untranslated regions are thus uncommon in ovarian cancer.

Packaging of Mason-Pfizer monkey virus (MPMV) genomic RNA depends upon conserved long-range interactions (LRIs) between U5 and gag sequences.

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Kalloush, Rawan M; Vivet-Boudou, Valérie; Ali, Lizna M; Mustafa, Farah; Marquet, Roland; Rizvi, Tahir A

2016-06-01

MPMV has great potential for development as a vector for gene therapy. In this respect, precisely defining the sequences and structural motifs that are important for dimerization and packaging of its genomic RNA (gRNA) are of utmost importance. A distinguishing feature of the MPMV gRNA packaging signal is two phylogenetically conserved long-range interactions (LRIs) between U5 and gag complementary sequences, LRI-I and LRI-II. To test their biological significance in the MPMV life cycle, we introduced mutations into these structural motifs and tested their effects on MPMV gRNA packaging and propagation. Furthermore, we probed the structure of key mutants using SHAPE (selective 2'hydroxyl acylation analyzed by primer extension). Disrupting base-pairing of the LRIs affected gRNA packaging and propagation, demonstrating their significance to the MPMV life cycle. A double mutant restoring a heterologous LRI-I was fully functional, whereas a similar LRI-II mutant failed to restore gRNA packaging and propagation. These results demonstrate that while LRI-I acts at the structural level, maintaining base-pairing is not sufficient for LRI-II function. In addition, in vitro RNA dimerization assays indicated that the loss of RNA packaging in LRI mutants could not be attributed to the defects in dimerization. Our findings suggest that U5-gag LRIs play an important architectural role in maintaining the structure of the 5' region of the MPMV gRNA, expanding the crucial role of LRIs to the nonlentiviral group of retroviruses. © 2016 Kalloush et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

How many 5S rRNA genes and pseudogenes are there in ''Aspergillus nidulans''?

International Nuclear Information System (INIS)

Pelczar, P.; Fiett, J.; Bartnik, E.

1994-01-01

We have estimated the number of 5S rRNA genes in ''Aspergillus nidulans'' using two-dimensional agarose gel electrophoresis and hybridization to appropriate probes, representing the 5'-halves, the 3'-halves of the 5S rRNA sequence and a sequence found at the 3'-end of all known. ''A. nidulans'' pseudogenes (block C). We have found 23 5S rRNA genes, 15 pseudogenes consisting of the 5'-half of the 5S rRNA sequence (of which 3 are flanked by block C) and 12 copies of block C which do not seem to be in the vicinity of 5S rRNA sequences. This number of genes is much lower than our earlier estimates, and makes our previously analyzed sample of 9 sequenced genes and 3 pseudogenes much more representative. (author). 7 refs, 1 fig

Integration of the Pokeweed miRNA and mRNA Transcriptomes Reveals Targeting of Jasmonic Acid-Responsive Genes

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Kira C. M. Neller

2018-05-01

Full Text Available The American pokeweed plant, Phytolacca americana, displays broad-spectrum resistance to plant viruses and is a heavy metal hyperaccumulator. However, little is known about the regulation of biotic and abiotic stress responses in this non-model plant. To investigate the control of miRNAs in gene expression, we sequenced the small RNA transcriptome of pokeweed treated with jasmonic acid (JA, a hormone that mediates pathogen defense and stress tolerance. We predicted 145 miRNAs responsive to JA, most of which were unique to pokeweed. These miRNAs were low in abundance and condition-specific, with discrete expression change. Integration of paired mRNA-Seq expression data enabled us to identify correlated, novel JA-responsive targets that mediate hormone biosynthesis, signal transduction, and pathogen defense. The expression of approximately half the pairs was positively correlated, an uncommon finding that we functionally validated by mRNA cleavage. Importantly, we report that a pokeweed-specific miRNA targets the transcript of OPR3, novel evidence that a miRNA regulates a JA biosynthesis enzyme. This first large-scale small RNA study of a Phytolaccaceae family member shows that miRNA-mediated control is a significant component of the JA response, associated with widespread changes in expression of genes required for stress adaptation.

Promoter Analysis Reveals Globally Differential Regulation of Human Long Non-Coding RNA and Protein-Coding Genes

KAUST Repository

Alam, Tanvir

2014-10-02

Transcriptional regulation of protein-coding genes is increasingly well-understood on a global scale, yet no comparable information exists for long non-coding RNA (lncRNA) genes, which were recently recognized to be as numerous as protein-coding genes in mammalian genomes. We performed a genome-wide comparative analysis of the promoters of human lncRNA and protein-coding genes, finding global differences in specific genetic and epigenetic features relevant to transcriptional regulation. These two groups of genes are hence subject to separate transcriptional regulatory programs, including distinct transcription factor (TF) proteins that significantly favor lncRNA, rather than coding-gene, promoters. We report a specific signature of promoter-proximal transcriptional regulation of lncRNA genes, including several distinct transcription factor binding sites (TFBS). Experimental DNase I hypersensitive site profiles are consistent with active configurations of these lncRNA TFBS sets in diverse human cell types. TFBS ChIP-seq datasets confirm the binding events that we predicted using computational approaches for a subset of factors. For several TFs known to be directly regulated by lncRNAs, we find that their putative TFBSs are enriched at lncRNA promoters, suggesting that the TFs and the lncRNAs may participate in a bidirectional feedback loop regulatory network. Accordingly, cells may be able to modulate lncRNA expression levels independently of mRNA levels via distinct regulatory pathways. Our results also raise the possibility that, given the historical reliance on protein-coding gene catalogs to define the chromatin states of active promoters, a revision of these chromatin signature profiles to incorporate expressed lncRNA genes is warranted in the future.

Bioinformatics analysis of RNA-seq data revealed critical genes in colon adenocarcinoma.

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Xi, W-D; Liu, Y-J; Sun, X-B; Shan, J; Yi, L; Zhang, T-T

2017-07-01

RNA-seq data of colon adenocarcinoma (COAD) were analyzed with bioinformatics tools to discover critical genes in the disease. Relevant small molecule drugs, transcription factors (TFs) and microRNAs (miRNAs) were also investigated. RNA-seq data of COAD were downloaded from The Cancer Genome Atlas (TCGA). Differential analysis was performed with package edgeR. False positive discovery (FDR) 1 were set as the cut-offs to screen out differentially expressed genes (DEGs). Gene coexpression network was constructed with package Ebcoexpress. GO enrichment analysis was performed for the DEGs in the gene coexpression network with DAVID. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was also performed for the genes with KOBASS 2.0. Modules were identified with MCODE of Cytoscape. Relevant small molecules drugs were predicted by Connectivity map. Relevant miRNAs and TFs were searched by WebGestalt. A total of 457 DEGs, including 255 up-regulated and 202 down-regulated genes, were identified from 437 COAD and 39 control samples. A gene coexpression network was constructed containing 40 DEGs and 101 edges. The genes were mainly associated with collagen fibril organization, extracellular matrix organization and translation. Two modules were identified from the gene coexpression network, which were implicated in muscle contraction and extracellular matrix organization, respectively. Several critical genes were disclosed, such as MYH11, COL5A2 and ribosomal proteins. Nine relevant small molecule drugs were identified, such as scriptaid and STOCK1N-35874. Accordingly, a total of 17 TFs and 10 miRNAs related to COAD were acquired, such as ETS2, NFAT, AP4, miR-124A, MiR-9, miR-96 and let-7. Several critical genes and relevant drugs, TFs and miRNAs were revealed in COAD. These findings could advance the understanding of the disease and benefit therapy development.

Complete genome sequence of Fer-de-Lance Virus reveals a novel gene in reptilian Paramyxoviruses

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Kurath, G.; Batts, W.N.; Ahne, W.; Winton, J.R.

2004-01-01

The complete RNA genome sequence of the archetype reptilian paramyxovirus, Fer-de-Lance virus (FDLV), has been determined. The genome is 15,378 nucleotides in length and consists of seven nonoverlapping genes in the order 3??? N-U-P-M-F-HN-L 5???, coding for the nucleocapsid, unknown, phospho-, matrix, fusion, hemagglutinin-neuraminidase, and large polymerase proteins, respectively. The gene junctions contain highly conserved transcription start and stop signal sequences and tri-nucleotide intergenic regions similar to those of other Paramyxoviridae. The FDLV P gene expression strategy is like that of rubulaviruses, which express the accessory V protein from the primary transcript and edit a portion of the mRNA to encode P and I proteins. There is also an overlapping open reading frame potentially encoding a small basic protein in the P gene. The gene designated U (unknown), encodes a deduced protein of 19.4 kDa that has no counterpart in other paramyxoviruses and has no similarity with sequences in the National Center for Biotechnology Information database. Active transcription of the U gene in infected cells was demonstrated by Northern blot analysis, and bicistronic N-U mRNA was also evident. The genomes of two other snake paramyxovirus genotypes were also found to have U genes, with 11 to 16% nucleotide divergence from the FDLV U gene. Pairwise comparisons of amino acid identities and phylogenetic analyses of all deduced FDLV protein sequences with homologous sequences from other Paramyxoviridae indicate that FDLV represents a new genus within the subfamily Paramyxovirinae. We suggest the name Ferlavirus for the new genus, with FDLV as the type species.

The distribution, diversity, and importance of 16S rRNA gene introns in the order Thermoproteales.

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Jay, Zackary J; Inskeep, William P

2015-07-09

Intron sequences are common in 16S rRNA genes of specific thermophilic lineages of Archaea, specifically the Thermoproteales (phylum Crenarchaeota). Environmental sequencing (16S rRNA gene and metagenome) from geothermal habitats in Yellowstone National Park (YNP) has expanded the available datasets for investigating 16S rRNA gene introns. The objectives of this study were to characterize and curate archaeal 16S rRNA gene introns from high-temperature habitats, evaluate the conservation and distribution of archaeal 16S rRNA introns in geothermal systems, and determine which "universal" archaeal 16S rRNA gene primers are impacted by the presence of intron sequences. Several new introns were identified and their insertion loci were constrained to thirteen locations across the 16S rRNA gene. Many of these introns encode homing endonucleases, although some introns were short or partial sequences. Pyrobaculum, Thermoproteus, and Caldivirga 16S rRNA genes contained the most abundant and diverse intron sequences. Phylogenetic analysis of introns revealed that sequences within the same locus are distributed biogeographically. The most diverse set of introns were observed in a high-temperature, circumneutral (pH 6) sulfur sediment environment, which also contained the greatest diversity of different Thermoproteales phylotypes. The widespread presence of introns in the Thermoproteales indicates a high probability of misalignments using different "universal" 16S rRNA primers employed in environmental microbial community analysis.

Downregulation of viral RNA translation by hepatitis C virus non-structural protein NS5A requires the poly(U/UC) sequence in the 3' UTR.

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Hoffman, Brett; Li, Zhubing; Liu, Qiang

2015-08-01

Hepatitis C virus (HCV) non-structural protein 5A (NS5A) is essential for viral replication; however, its effect on HCV RNA translation remains controversial partially due to the use of reporters lacking the 3' UTR, where NS5A binds to the poly(U/UC) sequence. We investigated the role of NS5A in HCV translation using a monocistronic RNA containing a Renilla luciferase gene flanked by the HCV UTRs. We found that NS5A downregulated viral RNA translation in a dose-dependent manner. This downregulation required both the 5' and 3' UTRs of HCV because substitution of either sequence with the 5' and 3' UTRs of enterovirus 71 or a cap structure at the 5' end eliminated the effects of NS5A on translation. Translation of the HCV genomic RNA was also downregulated by NS5A. The inhibition of HCV translation by NS5A required the poly(U/UC) sequence in the 3' UTR as NS5A did not affect translation when it was deleted. In addition, we showed that, whilst the amphipathic α-helix of NS5A has no effect on viral translation, the three domains of NS5A can inhibit translation independently, also dependent on the presence of the poly(U/UC) sequence in the 3' UTR. These results suggested that NS5A downregulated HCV RNA translation through a mechanism involving the poly(U/UC) sequence in the 3' UTR.

Identification of stress responsive genes by studying specific relationships between mRNA and protein abundance.

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Morimoto, Shimpei; Yahara, Koji

2018-03-01

Protein expression is regulated by the production and degradation of mRNAs and proteins but the specifics of their relationship are controversial. Although technological advances have enabled genome-wide and time-series surveys of mRNA and protein abundance, recent studies have shown paradoxical results, with most statistical analyses being limited to linear correlation, or analysis of variance applied separately to mRNA and protein datasets. Here, using recently analyzed genome-wide time-series data, we have developed a statistical analysis framework for identifying which types of genes or biological gene groups have significant correlation between mRNA and protein abundance after accounting for potential time delays. Our framework stratifies all genes in terms of the extent of time delay, conducts gene clustering in each stratum, and performs a non-parametric statistical test of the correlation between mRNA and protein abundance in a gene cluster. Consequently, we revealed stronger correlations than previously reported between mRNA and protein abundance in two metabolic pathways. Moreover, we identified a pair of stress responsive genes ( ADC17 and KIN1 ) that showed a highly similar time series of mRNA and protein abundance. Furthermore, we confirmed robustness of the analysis framework by applying it to another genome-wide time-series data and identifying a cytoskeleton-related gene cluster (keratin 18, keratin 17, and mitotic spindle positioning) that shows similar correlation. The significant correlation and highly similar changes of mRNA and protein abundance suggests a concerted role of these genes in cellular stress response, which we consider provides an answer to the question of the specific relationships between mRNA and protein in a cell. In addition, our framework for studying the relationship between mRNAs and proteins in a cell will provide a basis for studying specific relationships between mRNA and protein abundance after accounting for potential

In depth analysis of the Sox4 gene locus that consists of sense and natural antisense transcripts

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Ling, King-Hwa; Brautigan, Peter J.; Moore, Sarah; Fraser, Rachel; Leong, Melody Pui-Yee; Leong, Jia-Wen; Zainal Abidin, Shahidee; Lee, Han-Chung; Cheah, Pike-See; Raison, Joy M.; Babic, Milena; Lee, Young Kyung; Daish, Tasman; Mattiske, Deidre M.; Mann, Jeffrey R.; Adelson, David L.; Thomas, Paul Q.; Hahn, Christopher N.; Scott, Hamish S.

2016-01-01

SRY (Sex Determining Region Y)-Box 4 or Sox4 is an important regulator of the pan-neuronal gene expression during post-mitotic cell differentiation within the mammalian brain. Sox4 gene locus has been previously characterized with multiple sense and overlapping natural antisense transcripts [1], [2]. Here we provide accompanying data on various analyses performed and described in Ling et al. [2]. The data include a detail description of various features found at Sox4 gene locus, additional experimental data derived from RNA-Fluorescence in situ Hybridization (RNA-FISH), Western blotting, strand-specific reverse-transcription quantitative polymerase chain reaction (RT-qPCR), gain-of-function and in situ hybridization (ISH) experiments. All the additional data provided here support the existence of an endogenous small interfering- or PIWI interacting-like small RNA known as Sox4_sir3, which origin was found within the overlapping region consisting of a sense and a natural antisense transcript known as Sox4ot1. PMID:26958646

In depth analysis of the Sox4 gene locus that consists of sense and natural antisense transcripts

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King-Hwa Ling

2016-06-01

Full Text Available SRY (Sex Determining Region Y-Box 4 or Sox4 is an important regulator of the pan-neuronal gene expression during post-mitotic cell differentiation within the mammalian brain. Sox4 gene locus has been previously characterized with multiple sense and overlapping natural antisense transcripts [1,2]. Here we provide accompanying data on various analyses performed and described in Ling et al. [2]. The data include a detail description of various features found at Sox4 gene locus, additional experimental data derived from RNA-Fluorescence in situ Hybridization (RNA-FISH, Western blotting, strand-specific reverse-transcription quantitative polymerase chain reaction (RT-qPCR, gain-of-function and in situ hybridization (ISH experiments. All the additional data provided here support the existence of an endogenous small interfering- or PIWI interacting-like small RNA known as Sox4_sir3, which origin was found within the overlapping region consisting of a sense and a natural antisense transcript known as Sox4ot1.

A single methyltransferase YefA (RlmCD) catalyses both m5U747 and m5U1939 modifications in Bacillus subtilis 23S rRNA

DEFF Research Database (Denmark)

Desmolaize, Benoit; Fabret, Céline; Brégeon, Damien

2011-01-01

Escherichia coli possesses three paralogues. These comprise the methyltransferases TrmA that targets U54 in tRNAs, RlmC that modifies U747 in 23S rRNA and RlmD that is specific for U1939 in 23S rRNA. The tRNAs and rRNAs of the Gram-positive bacterium Bacillus subtilis have the same three m(5)U modifications....... However, as previously shown, the m(5)U54 modification in B. subtilis tRNAs is catalysed in a fundamentally different manner by the folate-dependent enzyme TrmFO, which is unrelated to the E. coli TrmA. Here, we show that methylation of U747 and U1939 in B. subtilis rRNA is catalysed by a single enzyme...

Mitochondrial 16S ribosomal RNA gene for forensic identification of crocodile species.

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Naga Jogayya, K; Meganathan, P R; Dubey, Bhawna; Haque, I

2013-05-01

All crocodilians are under various threats due to over exploitation and these species have been listed in Appendix I or II of CITES. Lack of molecular techniques for the forensic identification of confiscated samples makes it difficult to enforce the law. Therefore, we herein present a molecular method developed on the basis on 16S rRNA gene of mitochondrial DNA for identification of crocodile species. We have developed a set of 16S rRNA primers for PCR based identification of crocodilian species. These novel primers amplify partial 16S rRNA sequences of six crocodile species which can be later combined to obtain a larger region (1290 bp) of 16S rRNA gene. This 16S rRNA gene could be used as an effective tool for forensic authentication of crocodiles. The described primers hold great promise in forensic identification of crocodile species, which can aid in the effective enforcement of law and conservation of these species. Copyright © 2012 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.

Differential expression of the human thymosin-β4 gene in lymphocytes, macrophages, and granulocytes

International Nuclear Information System (INIS)

Gondo, H.; Kudo, J.; White, J.W.; Barr, C.; Selvanayagam, P.; Saunders, G.F.

1987-01-01

A cDNA clone encoding human thymosin-β 4 was isolated from a cDNA library prepared from peripheral blood leukocytes of a patient with acute lymphocytic leukemia. This clone contained the entire coding sequence of 43 amino acid residues of thymosin-β 4 and had an initiation codon and two termination codons. The amino acid and nucleotide sequences in the coding region were well conserved between rat and human. No signal peptide was found in the deduced protein sequence. Human thymosin-β 4 mRNA, approximately 830 nucleotides in length, was about 30 nucleotides larger than rat thymosin-β 4 mRNA. Expression of the human thymosin-β 4 gene in various primary myeloid and lymphoid malignant cells and in a few human hemopoietic cell lines was studied. Northern blot analyses of different neoplastic B lymphocytes revealed that steady state levels of thymosin-β 4 mRNA varied as a function of differentiation stage. Thymosin-β 4 mRNA levels were decreased in myeloma cells as are class II human leukocyte antigen, Fc receptor, and complement receptor, suggesting a relationship between thymosin-β 4 and the immune response. Treatment of THP-1 cells, a human monocytic cell line, with recombinant human interferon-γ reduced the levels of thymosin-β 4 mRNA. The pattern of thymosin-β 4 gene expression suggests that it may play a fundamental role in the host defense mechanism

Three new shRNA expression vectors targeting the CYP3A4 coding sequence to inhibit its expression

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Siyun Xu

2014-10-01

Full Text Available RNA interference (RNAi is useful for selective gene silencing. Cytochrome P450 3A4 (CYP3A4, which metabolizes approximately 50% of drugs in clinical use, plays an important role in drug metabolism. In this study, we aimed to develop a short hairpin RNA (shRNA to modulate CYP3A4 expression. Three new shRNAs (S1, S2 and S3 were designed to target the coding sequence (CDS of CYP3A4, cloned into a shRNA expression vector, and tested in different cells. The mixture of three shRNAs produced optimal reduction (55% in CYP3A4 CDS-luciferase activity in both CHL and HEK293 cells. Endogenous CYP3A4 expression in HepG2 cells was decreased about 50% at both mRNA and protein level after transfection of the mixture of three shRNAs. In contrast, CYP3A5 gene expression was not altered by the shRNAs, supporting the selectivity of CYP3A4 shRNAs. In addition, HepG2 cells transfected with CYP3A4 shRNAs were less sensitive to Ginkgolic acids, whose toxic metabolites are produced by CYP3A4. These results demonstrate that vector-based shRNAs could modulate CYP3A4 expression in cells through their actions on CYP3A4 CDS, and CYP3A4 shRNAs may be utilized to define the role of CYP3A4 in drug metabolism and toxicity.

Phylogenetic relatedness determined between antibiotic resistance and 16S rRNA genes in actinobacteria.

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Sagova-Mareckova, Marketa; Ulanova, Dana; Sanderova, Petra; Omelka, Marek; Kamenik, Zdenek; Olsovska, Jana; Kopecky, Jan

2015-04-01

Distribution and evolutionary history of resistance genes in environmental actinobacteria provide information on intensity of antibiosis and evolution of specific secondary metabolic pathways at a given site. To this day, actinobacteria producing biologically active compounds were isolated mostly from soil but only a limited range of soil environments were commonly sampled. Consequently, soil remains an unexplored environment in search for novel producers and related evolutionary questions. Ninety actinobacteria strains isolated at contrasting soil sites were characterized phylogenetically by 16S rRNA gene, for presence of erm and ABC transporter resistance genes and antibiotic production. An analogous analysis was performed in silico with 246 and 31 strains from Integrated Microbial Genomes (JGI_IMG) database selected by the presence of ABC transporter genes and erm genes, respectively. In the isolates, distances of erm gene sequences were significantly correlated to phylogenetic distances based on 16S rRNA genes, while ABC transporter gene distances were not. The phylogenetic distance of isolates was significantly correlated to soil pH and organic matter content of isolation sites. In the analysis of JGI_IMG datasets the correlation between phylogeny of resistance genes and the strain phylogeny based on 16S rRNA genes or five housekeeping genes was observed for both the erm genes and ABC transporter genes in both actinobacteria and streptomycetes. However, in the analysis of sequences from genomes where both resistance genes occurred together the correlation was observed for both ABC transporter and erm genes in actinobacteria but in streptomycetes only in the erm gene. The type of erm resistance gene sequences was influenced by linkage to 16S rRNA gene sequences and site characteristics. The phylogeny of ABC transporter gene was correlated to 16S rRNA genes mainly above the genus level. The results support the concept of new specific secondary metabolite

Quantifying Temporal Autocorrelations for the Expression of Geobacter species mRNA Gene Transcripts at Variable Ammonium Levels during in situ U(VI) Bioremediation

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Mouser, P. J.

2010-12-01

In order to develop decision-making tools for the prediction and optimization of subsurface bioremediation strategies, we must be able to link the molecular-scale activity of microorganisms involved in remediation processes with biogeochemical processes observed at the field-scale. This requires the ability to quantify changes in the in situ metabolic condition of dominant microbes and associate these changes to fluctuations in nutrient levels throughout the bioremediation process. It also necessitates a need to understand the spatiotemporal variability of the molecular-scale information to develop meaningful parameters and constraint ranges in complex bio-physio-chemical models. The expression of three Geobacter species genes (ammonium transporter (amtB), nitrogen fixation (nifD), and a housekeeping gene (recA)) were tracked at two monitoring locations that differed significantly in ammonium (NH4+) concentrations during a field-scale experiment where acetate was injected into the subsurface to simulate Geobacteraceae in a uranium-contaminated aquifer. Analysis of amtB and nifD mRNA transcript levels indicated that NH4+ was the primary form of fixed nitrogen during bioremediation. Overall expression levels of amtB were on average 8-fold higher at NH4+ concentrations of 300 μM or more than at lower NH4+ levels (average 60 μM). The degree of temporal correlation in Geobacter species mRNA expression levels was calculated at both locations using autocorrelation methods that describe the relationship between sample semi-variance and time lag. At the monitoring location with lower NH4+, a temporal correlation lag of 8 days was observed for both amtB and nifD transcript patterns. At the location where higher NH4+ levels were observed, no discernable temporal correlation lag above the sampling frequency (approximately every 2 days) was observed for amtB or nifD transcript fluctuations. Autocorrelation trends in recA expression levels at both locations indicated that

Seeing the forest for the trees: annotating small RNA producing genes in plants.

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Coruh, Ceyda; Shahid, Saima; Axtell, Michael J

2014-04-01

A key goal in genomics is the complete annotation of the expressed regions of the genome. In plants, substantial portions of the genome make regulatory small RNAs produced by Dicer-Like (DCL) proteins and utilized by Argonaute (AGO) proteins. These include miRNAs and various types of endogenous siRNAs. Small RNA-seq, enabled by cheap and fast DNA sequencing, has produced an enormous volume of data on plant miRNA and siRNA expression in recent years. In this review, we discuss recent progress in using small RNA-seq data to produce stable and reliable annotations of miRNA and siRNA genes in plants. In addition, we highlight key goals for the future of small RNA gene annotation in plants. Copyright © 2014 Elsevier Ltd. All rights reserved.

Macrophage activation and differentiation signals regulate schlafen-4 gene expression: evidence for Schlafen-4 as a modulator of myelopoiesis.

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Wendy J van Zuylen

Full Text Available BACKGROUND: The ten mouse and six human members of the Schlafen (Slfn gene family all contain an AAA domain. Little is known of their function, but previous studies suggest roles in immune cell development. In this report, we assessed Slfn regulation and function in macrophages, which are key cellular regulators of innate immunity. METHODOLOGY/PRINCIPAL FINDINGS: Multiple members of the Slfn family were up-regulated in mouse bone marrow-derived macrophages (BMM by the Toll-like Receptor (TLR4 agonist lipopolysaccharide (LPS, the TLR3 agonist Poly(I∶C, and in disease-affected joints in the collagen-induced model of rheumatoid arthritis. Of these, the most inducible was Slfn4. TLR agonists that signal exclusively through the MyD88 adaptor protein had more modest effects on Slfn4 mRNA levels, thus implicating MyD88-independent signalling and autocrine interferon (IFN-β in inducible expression. This was supported by the substantial reduction in basal and LPS-induced Slfn4 mRNA expression in IFNAR-1⁻/⁻ BMM. LPS causes growth arrest in macrophages, and other Slfn family genes have been implicated in growth control. Slfn4 mRNA levels were repressed during macrophage colony-stimulating factor (CSF-1-mediated differentiation of bone marrow progenitors into BMM. To determine the role of Slfn4 in vivo, we over-expressed the gene specifically in macrophages in mice using a csf1r promoter-driven binary expression system. Transgenic over-expression of Slfn4 in myeloid cells did not alter macrophage colony formation or proliferation in vitro. Monocyte numbers, as well as inflammatory macrophages recruited to the peritoneal cavity, were reduced in transgenic mice that specifically over-expressed Slfn4, while macrophage numbers and hematopoietic activity were increased in the livers and spleens. CONCLUSIONS: Slfn4 mRNA levels were up-regulated during macrophage activation but down-regulated during differentiation. Constitutive Slfn4 expression in the

RDE-4 preferentially binds long dsRNA and its dimerization is necessary for cleavage of dsRNA to siRNA.

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Parker, Greg S; Eckert, Debra M; Bass, Brenda L

2006-05-01

In organisms ranging from Arabidopsis to humans, Dicer requires dsRNA-binding proteins (dsRBPs) to carry out its roles in RNA interference (RNAi) and micro-RNA (miRNA) processing. In Caenorhabditis elegans, the dsRBP RDE-4 acts with Dicer during the initiation of RNAi, when long dsRNA is cleaved to small interfering RNAs (siRNAs). RDE-4 is not required in subsequent steps, and how RDE-4 distinguishes between long dsRNA and short siRNA is unclear. We report the first detailed analysis of RDE-4 binding, using purified recombinant RDE-4 and various truncated proteins. We find that, similar to other dsRBPs, RDE-4 is not sequence-specific. However, consistent with its in vivo roles, RDE-4 binds with higher affinity to long dsRNA. We also observe that RDE-4 is a homodimer in solution, and that the C-terminal domain of the protein is required for dimerization. Using extracts from wild-type and rde-4 mutant C. elegans, we show that the C-terminal dimerization domain is required for the production of siRNA. Our findings suggest a model for RDE-4 function during the initiation of RNAi.

Extensive 16S rRNA gene sequence diversity in Campylobacter hyointestinalis strains: taxonomic and applied implications

DEFF Research Database (Denmark)

Harrington, C.S.; On, Stephen L.W.

1999-01-01

Phylogenetic relationships of Campylobacter hyointestinalis subspecies were examined by means of 16S rRNA gene sequencing. Sequence similarities among C. hyointestinalis subsp. lawsonii strains exceeded 99.0 %, but values among C. hyointestinalis subsp. hyointestinalis strains ranged from 96...... of the genus Campylobacter, emphasizing the need for multiple strain analysis when using 16S rRNA gene sequence comparisons for taxonomic investigations........4 to 100 %. Sequence similarites between strains representing the two different subspecies ranged from 95.7 to 99.0 %. An intervening sequence was identified in certain of the C. hyointestinalis subsp. lawsonii strains. C. hyointestinalis strains occupied two distinct branches in a phylogenetic analysis...

Re-inspection of small RNA sequence datasets reveals several novel human miRNA genes.

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Thomas Birkballe Hansen

Full Text Available BACKGROUND: miRNAs are key players in gene expression regulation. To fully understand the complex nature of cellular differentiation or initiation and progression of disease, it is important to assess the expression patterns of as many miRNAs as possible. Thereby, identifying novel miRNAs is an essential prerequisite to make possible a comprehensive and coherent understanding of cellular biology. METHODOLOGY/PRINCIPAL FINDINGS: Based on two extensive, but previously published, small RNA sequence datasets from human embryonic stem cells and human embroid bodies, respectively [1], we identified 112 novel miRNA-like structures and were able to validate miRNA processing in 12 out of 17 investigated cases. Several miRNA candidates were furthermore substantiated by including additional available small RNA datasets, thereby demonstrating the power of combining datasets to identify miRNAs that otherwise may be assigned as experimental noise. CONCLUSIONS/SIGNIFICANCE: Our analysis highlights that existing datasets are not yet exhaustedly studied and continuous re-analysis of the available data is important to uncover all features of small RNA sequencing.

ReliefSeq: a gene-wise adaptive-K nearest-neighbor feature selection tool for finding gene-gene interactions and main effects in mRNA-Seq gene expression data.

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Brett A McKinney

Full Text Available Relief-F is a nonparametric, nearest-neighbor machine learning method that has been successfully used to identify relevant variables that may interact in complex multivariate models to explain phenotypic variation. While several tools have been developed for assessing differential expression in sequence-based transcriptomics, the detection of statistical interactions between transcripts has received less attention in the area of RNA-seq analysis. We describe a new extension and assessment of Relief-F for feature selection in RNA-seq data. The ReliefSeq implementation adapts the number of nearest neighbors (k for each gene to optimize the Relief-F test statistics (importance scores for finding both main effects and interactions. We compare this gene-wise adaptive-k (gwak Relief-F method with standard RNA-seq feature selection tools, such as DESeq and edgeR, and with the popular machine learning method Random Forests. We demonstrate performance on a panel of simulated data that have a range of distributional properties reflected in real mRNA-seq data including multiple transcripts with varying sizes of main effects and interaction effects. For simulated main effects, gwak-Relief-F feature selection performs comparably to standard tools DESeq and edgeR for ranking relevant transcripts. For gene-gene interactions, gwak-Relief-F outperforms all comparison methods at ranking relevant genes in all but the highest fold change/highest signal situations where it performs similarly. The gwak-Relief-F algorithm outperforms Random Forests for detecting relevant genes in all simulation experiments. In addition, Relief-F is comparable to the other methods based on computational time. We also apply ReliefSeq to an RNA-Seq study of smallpox vaccine to identify gene expression changes between vaccinia virus-stimulated and unstimulated samples. ReliefSeq is an attractive tool for inclusion in the suite of tools used for analysis of mRNA-Seq data; it has power to

Nucleolin is required for DNA methylation state and the expression of rRNA gene variants in Arabidopsis thaliana.

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Frédéric Pontvianne

2010-11-01

Full Text Available In eukaryotes, 45S rRNA genes are arranged in tandem arrays in copy numbers ranging from several hundred to several thousand in plants. Although it is clear that not all copies are transcribed under normal growth conditions, the molecular basis controlling the expression of specific sets of rRNA genes remains unclear. Here, we report four major rRNA gene variants in Arabidopsis thaliana. Interestingly, while transcription of one of these rRNA variants is induced, the others are either repressed or remain unaltered in A. thaliana plants with a disrupted nucleolin-like protein gene (Atnuc-L1. Remarkably, the most highly represented rRNA gene variant, which is inactive in WT plants, is reactivated in Atnuc-L1 mutants. We show that accumulated pre-rRNAs originate from RNA Pol I transcription and are processed accurately. Moreover, we show that disruption of the AtNUC-L1 gene induces loss of symmetrical DNA methylation without affecting histone epigenetic marks at rRNA genes. Collectively, these data reveal a novel mechanism for rRNA gene transcriptional regulation in which the nucleolin protein plays a major role in controlling active and repressed rRNA gene variants in Arabidopsis.

Gene expression correlates with process rates quantified for sulfate- and Fe(III-reducing bacteria in U(VI-contaminated sediments

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Denise M Akob

2012-08-01

Full Text Available Though iron- and sulfate-reducing bacteria are well known for mediating uranium(VI reduction in contaminated subsurface environments, quantifying the in situ activity of the microbial groups responsible remains a challenge. The objective of this study was to demonstrate the use of quantitative molecular tools that target mRNA transcripts of key genes related to Fe(III and sulfate reduction pathways in order to monitor these processes during in situ U(VI remediation in the subsurface. Expression of the Geobacteraceae-specific citrate synthase gene (gltA and the dissimilatory (bisulfite reductase gene (dsrA, were correlated with the activity of iron- or sulfate-reducing microorganisms, respectively, under stimulated bioremediation conditions in microcosms of sediments sampled from the U.S. Department of Energy’s Oak Ridge Integrated Field Research Challenge (OR-IFRC site at Oak Ridge, Tennessee. In addition, Geobacteraceae-specific gltA and dsrA transcript levels were determined in parallel with the predominant electron acceptors present in moderately and highly contaminated subsurface sediments from the OR-IFRC. Phylogenetic analysis of the cDNA generated from dsrA mRNA, sulfate-reducing bacteria-specific 16S rRNA, and gltA mRNA identified activity of specific microbial groups. Active sulfate reducers were members of the Desulfovibrio, Desulfobacterium, and Desulfotomaculum genera. Members of the subsurface Geobacter clade, closely related to uranium-reducing Geobacter uraniireducens and Geobacter daltonii, were the metabolically-active iron-reducers in biostimulated microcosms and in situ core samples. Direct correlation of transcripts and process rates demonstrated evidence of competition between the functional guilds in subsurface sediments. We further showed that active populations of Fe(III-reducing bacteria and sulfate-reducing bacteria are present in OR-IFRC sediments and are good potential targets for in situ bioremediation.

Reanalysis of RNA-sequencing data reveals several additional fusion genes with multiple isoforms.

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Kangaspeska, Sara; Hultsch, Susanne; Edgren, Henrik; Nicorici, Daniel; Murumägi, Astrid; Kallioniemi, Olli

2012-01-01

RNA-sequencing and tailored bioinformatic methodologies have paved the way for identification of expressed fusion genes from the chaotic genomes of solid tumors. We have recently successfully exploited RNA-sequencing for the discovery of 24 novel fusion genes in breast cancer. Here, we demonstrate the importance of continuous optimization of the bioinformatic methodology for this purpose, and report the discovery and experimental validation of 13 additional fusion genes from the same samples. Integration of copy number profiling with the RNA-sequencing results revealed that the majority of the gene fusions were promoter-donating events that occurred at copy number transition points or involved high-level DNA-amplifications. Sequencing of genomic fusion break points confirmed that DNA-level rearrangements underlie selected fusion transcripts. Furthermore, a significant portion (>60%) of the fusion genes were alternatively spliced. This illustrates the importance of reanalyzing sequencing data as gene definitions change and bioinformatic methods improve, and highlights the previously unforeseen isoform diversity among fusion transcripts.

Reanalysis of RNA-sequencing data reveals several additional fusion genes with multiple isoforms.

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Sara Kangaspeska

Full Text Available RNA-sequencing and tailored bioinformatic methodologies have paved the way for identification of expressed fusion genes from the chaotic genomes of solid tumors. We have recently successfully exploited RNA-sequencing for the discovery of 24 novel fusion genes in breast cancer. Here, we demonstrate the importance of continuous optimization of the bioinformatic methodology for this purpose, and report the discovery and experimental validation of 13 additional fusion genes from the same samples. Integration of copy number profiling with the RNA-sequencing results revealed that the majority of the gene fusions were promoter-donating events that occurred at copy number transition points or involved high-level DNA-amplifications. Sequencing of genomic fusion break points confirmed that DNA-level rearrangements underlie selected fusion transcripts. Furthermore, a significant portion (>60% of the fusion genes were alternatively spliced. This illustrates the importance of reanalyzing sequencing data as gene definitions change and bioinformatic methods improve, and highlights the previously unforeseen isoform diversity among fusion transcripts.

Transcriptomic-Wide Discovery of Direct and Indirect HuR RNA Targets in Activated CD4+ T Cells.

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Patsharaporn Techasintana

Full Text Available Due to poor correlation between steady state mRNA levels and protein product, purely transcriptomic profiling methods may miss genes posttranscriptionally regulated by RNA binding proteins (RBPs and microRNAs (miRNAs. RNA immunoprecipitation (RIP methods developed to identify in vivo targets of RBPs have greatly elucidated those mRNAs which may be regulated via transcript stability and translation. The RBP HuR (ELAVL1 and family members are major stabilizers of mRNA. Many labs have identified HuR mRNA targets; however, many of these analyses have been performed in cell lines and oftentimes are not independent biological replicates. Little is known about how HuR target mRNAs behave in conditional knock-out models. In the present work, we performed HuR RIP-Seq and RNA-Seq to investigate HuR direct and indirect targets using a novel conditional knock-out model of HuR genetic ablation during CD4+ T activation and Th2 differentiation. Using independent biological replicates, we generated a high coverage RIP-Seq data set (>160 million reads that was analyzed using bioinformatics methods specifically designed to find direct mRNA targets in RIP-Seq data. Simultaneously, another set of independent biological replicates were sequenced by RNA-Seq (>425 million reads to identify indirect HuR targets. These direct and indirect targets were combined to determine canonical pathways in CD4+ T cell activation and differentiation for which HuR plays an important role. We show that HuR may regulate genes in multiple canonical pathways involved in T cell activation especially the CD28 family signaling pathway. These data provide insights into potential HuR-regulated genes during T cell activation and immune mechanisms.

quatre-quart1 is an indispensable U12 intron-containing gene that plays a crucial role in Arabidopsis development.

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Kwak, Kyung Jin; Kim, Bo Mi; Lee, Kwanuk; Kang, Hunseung

2017-05-17

Despite increasing understanding of the importance of the splicing of U12-type introns in plant development, the key question of which U12 intron-containing genes are essential for plant development has not yet been explored. Here, we assessed the functional role of the quatre-quart1 (QQT1) gene, one of the ~230 U12 intron-containing genes in Arabidopsis thaliana. Expression of QQT1 in the U11/U12-31K small nuclear ribonucleoprotein mutant (31k) rescued the developmental-defect phenotypes of the 31k mutant, whereas the miRNA-mediated qqt1 knockdown mutants displayed severe defects in growth and development, including severely arrested stem growth, small size, and the formation of serrated leaves. The structures of the shoot apical meristems in the qqt1 mutants were abnormal and disordered. Identification of QQT1-interacting proteins via a yeast two-hybrid screening and a firefly luciferase complementation-imaging assay revealed that a variety of proteins, including many chloroplast-targeted proteins, interacted with QQT1. Importantly, the levels of chloroplast-targeted proteins in the chloroplast were reduced, and the chloroplast structure was abnormal in the qqt1 mutant. Collectively, these results provide clear evidence that QQT1 is an indispensable U12 intron-containing gene whose correct splicing is crucial for the normal development of Arabidopsis. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Genome-wide annotation of porcine microRNA genes and transcriptome profiling during Actinobacillus infection

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Nielsen, Mathilde

MicroRNAs are small single stranded non-coding RNA molecules which contributes to the regulation of gene expression by primarily binding to the 3´end of protein coding mRNA, hereby inhibiting the translation process or promting degradation of the mRNA. The main focus of this PhD project was to ex......MicroRNAs are small single stranded non-coding RNA molecules which contributes to the regulation of gene expression by primarily binding to the 3´end of protein coding mRNA, hereby inhibiting the translation process or promting degradation of the mRNA. The main focus of this PhD project...

Systematic and quantitative mRNA expression analysis of TRP channel genes at the single trigeminal and dorsal root ganglion level in mouse

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Vandewauw Ine

2013-02-01

Full Text Available Abstract Background Somatosensory nerve fibres arising from cell bodies within the trigeminal ganglia (TG in the head and from a string of dorsal root ganglia (DRG located lateral to the spinal cord convey endogenous and environmental stimuli to the central nervous system. Although several members of the transient receptor potential (TRP superfamily of cation channels have been implicated in somatosensation, the expression levels of TRP channel genes in the individual sensory ganglia have never been systematically studied. Results Here, we used quantitative real-time PCR to analyse and compare mRNA expression of all TRP channels in TG and individual DRGs from 27 anatomically defined segments of the spinal cord of the mouse. At the mRNA level, 17 of the 28 TRP channel genes, TRPA1, TRPC1, TRPC3, TRPC4, TRPC5, TRPM2, TRPM3, TRPM4, TRPM5, TRPM6, TRPM7, TRPM8, TRPV1, TRPV2, TRPV4, TRPML1 and TRPP2, were detectable in every tested ganglion. Notably, four TRP channels, TRPC4, TRPM4, TRPM8 and TRPV1, showed statistically significant variation in mRNA levels between DRGs from different segments, suggesting ganglion-specific regulation of TRP channel gene expression. These ganglion-to-ganglion differences in TRP channel transcript levels may contribute to the variability in sensory responses in functional studies. Conclusions We developed, compared and refined techniques to quantitatively analyse the relative mRNA expression of all TRP channel genes at the single ganglion level. This study also provides for the first time a comparative mRNA distribution profile in TG and DRG along the entire vertebral column for the mammalian TRP channel family.

Identification of stress responsive genes by studying specific relationships between mRNA and protein abundance

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Shimpei Morimoto

2018-03-01

Full Text Available Protein expression is regulated by the production and degradation of mRNAs and proteins but the specifics of their relationship are controversial. Although technological advances have enabled genome-wide and time-series surveys of mRNA and protein abundance, recent studies have shown paradoxical results, with most statistical analyses being limited to linear correlation, or analysis of variance applied separately to mRNA and protein datasets. Here, using recently analyzed genome-wide time-series data, we have developed a statistical analysis framework for identifying which types of genes or biological gene groups have significant correlation between mRNA and protein abundance after accounting for potential time delays. Our framework stratifies all genes in terms of the extent of time delay, conducts gene clustering in each stratum, and performs a non-parametric statistical test of the correlation between mRNA and protein abundance in a gene cluster. Consequently, we revealed stronger correlations than previously reported between mRNA and protein abundance in two metabolic pathways. Moreover, we identified a pair of stress responsive genes (ADC17 and KIN1 that showed a highly similar time series of mRNA and protein abundance. Furthermore, we confirmed robustness of the analysis framework by applying it to another genome-wide time-series data and identifying a cytoskeleton-related gene cluster (keratin 18, keratin 17, and mitotic spindle positioning that shows similar correlation. The significant correlation and highly similar changes of mRNA and protein abundance suggests a concerted role of these genes in cellular stress response, which we consider provides an answer to the question of the specific relationships between mRNA and protein in a cell. In addition, our framework for studying the relationship between mRNAs and proteins in a cell will provide a basis for studying specific relationships between mRNA and protein abundance after

In Vivo Testing of MicroRNA-Mediated Gene Knockdown in Zebrafish

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Ivone Un San Leong

2012-01-01

Full Text Available The zebrafish (Danio rerio has become an attractive model for human disease modeling as there are a large number of orthologous genes that encode similar proteins to those found in humans. The number of tools available to manipulate the zebrafish genome is limited and many currently used techniques are only effective during early development (such as morpholino-based antisense technology or it is phenotypically driven and does not offer targeted gene knockdown (such as chemical mutagenesis. The use of RNA interference has been met with controversy as off-target effects can make interpreting phenotypic outcomes difficult; however, this has been resolved by creating zebrafish lines that contain stably integrated miRNA constructs that target the desired gene of interest. In this study, we show that a commercially available miRNA vector system with a mouse-derived miRNA backbone is functional in zebrafish and is effective in causing eGFP knockdown in a transient in vivo eGFP sensor assay system. We chose to apply this system to the knockdown of transcripts that are implicated in the human cardiac disorder, Long QT syndrome.

Dynamic transcription of long non-coding RNA genes during CD4+ T cell development and activation.

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Fei Xia

Full Text Available BACKGROUND: Recent evidence shows that long non-coding RNA (LncRNA play important regulatory roles in many biology process, including cell development, activation and oncogenesis. However, the roles of these LncRNAs in the development and activation of CD4+ T cells, which is an important component of immune response, remain unknown. RESULTS: To predict the function of LncRNA in the development and activation of CD4+ T cells, first, we examined the expression profiles of LncRNAs and mRNAs in CD4-CD8- (DN, CD4+CD8+ (DP, CD4+CD8-, and activated CD4+CD8- T cells in a microarray analysis and verified these results by real time PCRs (qPCR. We found that the expression of hundreds of LncRNAs significantly changed in each process of developmental transition, including DN into DP, DP into CD4+CD8-, and CD4+CD8- into activated CD4+ T cells. A Kendall distance analysis suggested that the expression of LncRNAs in DN, DP, CD4+CD8- T cells and activated CD4+ T cells were correlated with the expression of mRNAs in these T cells. The Blat algorithm and GO analysis suggested that LncRNAs may exert important roles in the development and activation of CD4+ T cells. These roles included proliferation, homeostasis, maturation, activation, migration, apoptosis and calcium ion transportation. CONCLUSION: The present study found that the expression profiles of LncRNAs in different stages of CD4+ T cells are distinguishable. LncRNAs are involved in the key biological process in CD4+ T cell development and activation.

The dsRNA binding protein RDE-4 interacts with RDE-1, DCR-1, and a DExH-box helicase to direct RNAi in C. elegans.

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Tabara, Hiroaki; Yigit, Erbay; Siomi, Haruhiko; Mello, Craig C

2002-06-28

Double-stranded (ds) RNA induces potent gene silencing, termed RNA interference (RNAi). At an early step in RNAi, an RNaseIII-related enzyme, Dicer (DCR-1), processes long-trigger dsRNA into small interfering RNAs (siRNAs). DCR-1 is also required for processing endogenous regulatory RNAs called miRNAs, but how DCR-1 recognizes its endogenous and foreign substrates is not yet understood. Here we show that the C. elegans RNAi pathway gene, rde-4, encodes a dsRNA binding protein that interacts during RNAi with RNA identical to the trigger dsRNA. RDE-4 protein also interacts in vivo with DCR-1, RDE-1, and a conserved DExH-box helicase. Our findings suggest a model in which RDE-4 and RDE-1 function together to detect and retain foreign dsRNA and to present this dsRNA to DCR-1 for processing.

Identification of novel microRNA genes in freshwater and marine ecotypes of the three-spined stickleback (Gasterosteus aculeatus).

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Rastorguev, S M; Nedoluzhko, A V; Sharko, F S; Boulygina, E S; Sokolov, A S; Gruzdeva, N M; Skryabin, K G; Prokhortchouk, E B

2016-11-01

The three-spined stickleback (Gasterosteus aculeatus L.) is an important model organism for studying the molecular mechanisms of speciation and adaptation to salinity. Despite increased interest to microRNA discovery and recent publication on microRNA prediction in the three-spined stickleback using bioinformatics approaches, there is still a lack of experimental support for these data. In this paper, high-throughput sequencing technology was applied to identify microRNA genes in gills of the three-spined stickleback. In total, 595 miRNA genes were discovered; half of them were predicted in previous computational studies and were confirmed here as microRNAs expressed in gill tissue. Moreover, 298 novel microRNA genes were identified. The presence of miRNA genes in selected 'divergence islands' was analysed and 10 miRNA genes were identified as not randomly located in 'divergence islands'. Regulatory regions of miRNA genes were found enriched with selective SNPs that may play a role in freshwater adaptation. © 2016 John Wiley & Sons Ltd.

Simultaneous DNA-RNA Extraction from Coastal Sediments and Quantification of 16S rRNA Genes and Transcripts by Real-time PCR.

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Tatti, Enrico; McKew, Boyd A; Whitby, Corrine; Smith, Cindy J

2016-06-11

Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of taxonomic and functional groups in environmental samples. Used in combination with a reverse transcriptase reaction (RT-q-PCR), it can also be employed to quantify gene transcripts. q-PCR makes use of highly sensitive fluorescent detection chemistries that allow quantification of PCR amplicons during the exponential phase of the reaction. Therefore, the biases associated with 'end-point' PCR detected in the plateau phase of the PCR reaction are avoided. A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. First, a method for the co-extraction of DNA and RNA from coastal sediments, including the additional steps required for the preparation of DNA-free RNA, is outlined. Second, a step-by-step guide for the quantification of 16S rRNA genes and transcripts from the extracted nucleic acids via q-PCR and RT-q-PCR is outlined. This includes details for the construction of DNA and RNA standard curves. Key considerations for the use of RT-q-PCR assays in microbial ecology are included.

Determination of the number of copies of genes coding for 5s-rRNA and tRNA in the genomes of 43 species of wheat and Aegilops

International Nuclear Information System (INIS)

Vakhitov, V.A.; Gimalov, F.R.; Nikonorov, Yu.M.

1986-01-01

The number of 5s-rRNA and tRNA genes has been studied in 43 species of wheat and Aegilops differing in ploidy level, genomic composition and origin. It has been demonstrated that the repeatability of the 5s-rRNA and tRNA genes increases in wheat with increasing ploidy level, but not in proportion to the genome size. In Aegilops, in distinction from wheat, the relative as well as absolute number of 5s-RNA genes increases with increasing ploidy level. The proportion of the sequences coding for tRNA in the dipoloid and polyploid Aegilops species is practically similar, while the number of tRNA genes increases almost 2-3 times with increasing ploidy level. Large variability has been recorded between the species with similar genomic composition and ploidy level in respect of the number of the 5s-rRNA and tRNA genes. It has been demonstrated that integration of the initial genomes of the amphidiploids is accompanied by elimination of a particular part of these genomes. It has been concluded that the mechanisms of establishment and evolution of genomes in the intra- and intergeneric allopolyploids are not identical

Valyl-tRNA synthetase gene of Escherichia coli K12: Molecular genetic characterization and homology within a family of aminoacyl-tRNA synthetases

International Nuclear Information System (INIS)

Heck, J.D. III.

1988-01-01

This work reports the subcloning and characterization of the molecular elements necessary for the expression of the Escherichia coli valS gene encoding valyl-tRNA synthetase. The valS gene was subcloned from plasmid pLC26-22 by genetic complementation of a valS ts strain. The DNA region encoding the valS structural gene was determined by in vitro coupled transcription-translation assays. Cells transformed with a plasmid containing a full length copy of the valS gene enhanced in vivo valyl-tRNA synthetase specific activity twelve-fold. DNA sequences flanking the valS structural gene are presented. The transcription initiation sites of the valS gene were determined, in vivo and in vitro, by S1 nuclease protection studies, primer-extension analysis and both [α- 32 P]labeled and [γ- 32 P]end-labeled in vitro transcription assays. The DNA sequence of the valS gene of Escherichia coli has been determined. Significant similarity at the primary sequence level was detected between valyl-tRNA synthetase of E. coli and other known branched-chain aminoacyl-tRNA synthetases. An extended open reading frame (ORF) encoded on the DNA strand opposite the valS structural gene is described

A renaissance for the pioneering 16S rRNA gene

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Tringe, Susannah; Hugenholtz, Philip

2008-09-07

Culture-independent molecular surveys using the 16S rRNA gene have become a mainstay for characterizing microbial community structure over the last quarter century. More recently this approach has been overshadowed by metagenomics, which provides a global overview of a community's functional potential rather than just an inventory of its inhabitants. However, the pioneering 16S rRNA gene is making a comeback in its own right thanks to a number of methodological advancements including higher resolution (more sequences), analysis of multiple related samples (e.g. spatial and temporal series) and improved metadata and use of metadata. The standard conclusion that microbial ecosystems are remarkably complex and diverse is now being replaced by detailed insights into microbial ecology and evolution based only on this one historically important marker gene.

A renaissance for the pioneering 16S rRNA gene.

Science.gov (United States)

Tringe, Susannah G; Hugenholtz, Philip

2008-10-01

Culture-independent molecular surveys using the 16S rRNA gene have become a mainstay for characterizing microbial community structure over the past quarter century. More recently this approach has been overshadowed by metagenomics, which provides a global overview of a community's functional potential rather than just an inventory of its inhabitants. However, the pioneering 16S rRNA gene is making a comeback in its own right thanks to a number of methodological advancements including higher resolution (more sequences), analysis of multiple related samples (e.g. spatial and temporal series) and improved metadata, and use of metadata. The standard conclusion that microbial ecosystems are remarkably complex and diverse is now being replaced by detailed insights into microbial ecology and evolution based only on this one historically important marker gene.

Genomic organization of the rat alpha 2u-globulin gene cluster.

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McFadyen, D A; Addison, W; Locke, J

1999-05-01

The alpha 2u-globulin are a group of similar proteins, belonging to the lipocalin superfamily of proteins, that are synthesized in a subset of secretory tissues in rats. The many alpha 2u-globulin isoforms are encoded by a multigene family that exhibits extensive homology. Despite a high degree of sequence identity, individual family members show diverse expression patterns involving complex hormonal, tissue-specific, and developmental regulation. Analysis suggests that there are approximately 20 alpha 2u-globulin genes in the rat genome. We have used fluorescence in situ hybridization (FISH) to show that the alpha 2u-globulin genes are clustered at a single site on rat Chromosome (Chr) 5 (5q22-24). Southern blots of rat genomic DNA separated by pulsed field gel electrophoresis indicated that the alpha 2u-globulin genes are contained on two NruI fragments with a total size of 880 kbp. Analysis of three P1 clones containing alpha 2u-globulin genes indicated that the alpha 2u-globulin genes are tandemly arranged in a head-to-tail fashion. The organization of the alpha 2u-globulin genes in the rat as a tandem array of single genes differs from the homologous major urinary protein genes in the mouse, which are organized as tandem arrays of divergently oriented gene pairs. The structure of these gene clusters may have consequences for the proposed function, as a pheromone transporter, for the protein products encoded by these genes.

Identification of pathogenic Nocardia species by reverse line blot hybridization targeting the 16S rRNA and 16S-23S rRNA gene spacer regions.

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Xiao, Meng; Kong, Fanrong; Sorrell, Tania C; Cao, Yongyan; Lee, Ok Cha; Liu, Ying; Sintchenko, Vitali; Chen, Sharon C A

2010-02-01

Although 16S rRNA gene sequence analysis is employed most often for the definitive identification of Nocardia species, alternate molecular methods and polymorphisms in other gene targets have also enabled species determinations. We evaluated a combined Nocardia PCR-based reverse line blot (RLB) hybridization assay based on 16S and 16S-23S rRNA gene spacer region polymorphisms to identify 12 American Type Culture Collection and 123 clinical Nocardia isolates representing 14 species; results were compared with results from 16S rRNA gene sequencing. Thirteen 16S rRNA gene-based (two group-specific and 11 species-specific) and five 16S-23S spacer-targeted (two taxon-specific and three species-specific) probes were utilized. 16S rRNA gene-based probes correctly identified 124 of 135 isolates (sensitivity, 92%) but were unable to identify Nocardia paucivorans strains (n = 10 strains) and a Nocardia asteroides isolate with a novel 16S rRNA gene sequence. Nocardia farcinica and Nocardia cyriacigeorgica strains were identified by the sequential use of an N. farcinica-"negative" probe and a combined N. farcinica/N. cyriacigeorgica probe. The assay specificity was high (99%) except for weak cross-reactivity between the Nocardia brasiliensis probe with the Nocardia thailandica DNA product; however, cross-hybridization with closely related nontarget species may occur. The incorporation of 16S-23S rRNA gene spacer-based probes enabled the identification of all N. paucivorans strains. The overall sensitivity using both probe sets was >99%. Both N. farcinica-specific 16S-23S rRNA gene spacer-directed probes were required to identify all N. farcinica stains by using this probe set. The study demonstrates the utility of a combined PCR/RLB assay for the identification of clinically relevant Nocardia species and its potential for studying subtypes of N. farcinica. Where species assignment is ambiguous or not possible, 16S rRNA gene sequencing is recommended.

Rift Valley fever virus NSS gene expression correlates with a defect in nuclear mRNA export.

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Copeland, Anna Maria; Van Deusen, Nicole M; Schmaljohn, Connie S

2015-12-01

We investigated the localization of host mRNA during Rift Valley fever virus (RVFV) infection. Fluorescence in situ hybridization revealed that infection with RVFV altered the localization of host mRNA. mRNA accumulated in the nuclei of RVFV-infected but not mock-infected cells. Further, overexpression of the NSS gene, but not the N, GN or NSM genes correlated with mRNA nuclear accumulation. Nuclear accumulation of host mRNA was not observed in cells infected with a strain of RVFV lacking the gene encoding NSS, confirming that expression of NSS is likely responsible for this phenomenon. Published by Elsevier Inc.

Mitochondrial and cytoplasmic isoleucyl-, glutamyl- and arginyl-tRNA synthetases of yeast are encoded by separate genes.

Science.gov (United States)

Tzagoloff, A; Shtanko, A

1995-06-01

Three complementation groups of a pet mutant collection have been found to be composed of respiratory-deficient deficient mutants with lesions in mitochondrial protein synthesis. Recombinant plasmids capable of restoring respiration were cloned by transformation of representatives of each complementation group with a yeast genomic library. The plasmids were used to characterize the complementing genes and to institute disruption of the chromosomal copies of each gene in respiratory-proficient yeast. The sequences of the cloned genes indicate that they code for isoleucyl-, arginyl- and glutamyl-tRNA synthetases. The properties of the mutants used to obtain the genes and of strains with the disrupted genes indicate that all three aminoacyl-tRNA synthetases function exclusively in mitochondrial proteins synthesis. The ISM1 gene for mitochondrial isoleucyl-tRNA synthetase has been localized to chromosome XVI next to UME5. The MSR1 gene for the arginyl-tRNA synthetase was previously located on yeast chromosome VIII. The third gene MSE1 for the mitochondrial glutamyl-tRNA synthetase has not been localized. The identification of three new genes coding for mitochondrial-specific aminoacyl-tRNA synthetases indicates that in Saccharomyces cerevisiae at least 11 members of this protein family are encoded by genes distinct from those coding for the homologous cytoplasmic enzymes.

Thermal and x-ray studies on Tl2U(MoO4)3 and Tl4U(MoO4)4

International Nuclear Information System (INIS)

Dahale, N.D.; Keskar, Meera; Kulkarni, N.K.; Singh Mudher, K.D.

2006-01-01

In the quaternary Tl-U(IV)-Mo-O system, two new compounds namely Tl 2 U(MoO 4 ) 3 and Tl 4 U(MoO 4 ) 4 were prepared and characterized by powder X-ray diffraction and thermal methods. These compounds were prepared by solid state reactions of Tl 2 MoO 4 , UMoO 5 and MoO 3 in the required stoichiometric ratio at 500 deg C in evacuated sealed quartz ampoule. The XRD data of Tl 2 U(MoO 4 ) 3 and Tl 4 U(MoO 4 ) 4 were indexed on orthorhombic cell. TG curves of Tl 2 U(MoO 4 ) 3 and Tl 4 U(MoO 4 ) 4 did not show any weight change up to 700 deg C in an inert atmosphere. During heating in an inert atmosphere, Tl 2 U(MoO 4 ) 3 and Tl 4 U(MoO 4 ) 4 showed endothermic Dta peaks due to melting of the compounds at 519 and 565 deg C, respectively. (author)

Loss of wobble uridine modification in tRNA anticodons interferes with TOR pathway signaling

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Viktor Scheidt

2014-11-01

Full Text Available Previous work in yeast has suggested that modification of tRNAs, in particular uridine bases in the anticodon wobble position (U34, is linked to TOR (target of rapamycin signaling. Hence, U34 modification mutants were found to be hypersensitive to TOR inhibition by rapamycin. To study whether this involves inappropriate TOR signaling, we examined interaction between mutations in TOR pathway genes (tip41∆, sap190∆, ppm1∆, rrd1∆ and U34 modification defects (elp3∆, kti12∆, urm1∆, ncs2∆ and found the rapamycin hypersensitivity in the latter is epistatic to drug resistance of the former. Epistasis, however, is abolished in tandem with a gln3∆ deletion, which inactivates transcription factor Gln3 required for TOR-sensitive activation of NCR (nitrogen catabolite repression genes. In line with nuclear import of Gln3 being under control of TOR and dephosphorylation by the Sit4 phosphatase, we identify novel TOR-sensitive sit4 mutations that confer rapamycin resistance and importantly, mislocalise Gln3 when TOR is inhibited. This is similar to gln3∆ cells, which abolish the rapamycin hypersensitivity of U34 modification mutants, and suggests TOR deregulation due to tRNA undermodification operates through Gln3. In line with this, loss of U34 modifications (elp3∆, urm1∆ enhances nuclear import of and NCR gene activation (MEP2, GAP1 by Gln3 when TOR activity is low. Strikingly, this stimulatory effect onto Gln3 is suppressed by overexpression of tRNAs that usually carry the U34 modifications. Collectively, our data suggest that proper TOR signaling requires intact tRNA modifications and that loss of U34 modifications impinges on the TOR-sensitive NCR branch via Gln3 misregulation.

Loss of wobble uridine modification in tRNA anticodons interferes with TOR pathway signaling.