SmRNAs generated by single-stranded and double-stranded silencers
Ontology highlight
ABSTRACT:
ORGANISM(S): Arabidopsis thaliana
SUBMITTER: Huan Wang
PROVIDER: E-GEOD-39398 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:To investigate possible smRNAs linked to TMM silencing by single-stranded and double-stranded silencers, we determined sequences of smRNAs by the Illumina high-throughput sequencing platform and compared silencing efficiency of different strategies. We found that single-stranded silencer alone could promote the production of smRNAs.
2013-12-23 | GSE39398 | GEO
Project description:smRNAs generated by single-stranded and double-stranded silencers
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2012-09-19 | E-GEOD-36560 | biostudies-arrayexpress
Project description:ABSTRACT: Exposure to abiotic stresses triggers global changes in the expression of thousands of eukaryotic genes at the transcriptional 70 and post-transcriptional levels. Small RNA (smRNA) pathways and splicing both function as crucial mechanisms regulating stress-responsive gene expression. However, examples of smRNAs regulating gene expression remain largely limited to effects on mRNA stability, translation, and epigenetic regulation. Also, our understanding of the networks controlling plant gene expression in response to environmental changes, and examples of these regulatory pathways intersecting, remains limited. Here, to investigate the role of smRNAs in stress responses we examined smRNA transcriptomes of Brachypodium distachyon plants subjected to various abiotic stresses. We found that exposure to different abiotic stresses specifically induced a group 75 of novel, endogenous small interfering RNAs (stress-induced, UTR-derived siRNAs, or sutr-siRNAs) that originate from the 3M-bM-^@M-2 UTRs of a subset of coding genes. Our bioinformatics analyses predicted that sutr-siRNAs have potential regulatory functions and that over 90% of sutr-siRNAs target intronic regions of many mRNAs in trans. Importantly, a subgroup of these sutr- siRNAs target the important intron regulatory regions, such as branch point sequences, that could affect splicing. Our study indicates that in Brachypodium, sutr-siRNAs may affect splicing by masking or changing accessibility of specific cis-elements 80 through base-pairing interactions to mediate gene expression in response to stresses. We hypothesize that this mode of regulation of gene expression may also serve as a general mechanism for regulation of gene expression in plants and potentially in other eukaryotes. Analysis of smRNA populations in Brachypodium plants challenged by abiotic stresses: To profile the populations of smRNAs in the model monocot Brachypodium distachyon and examine their regulation in response to abiotic stresses, we conducted high-throughput sequencing of small RNAs from plants exposed to four different abiotic stress conditions, cold, heat (air), heat (water immersion), and salt, in the wild type Brachypodium cultivar Bd21. For our experiments we used information from the literature to select two time-points for stress durations, short and long, which differed for each stress: cold (6 and 24 hours), heat-air (1 and 3 hours), heat-water (1 and 3 hours), and salt (48 hours). We generated small RNA libraries for Illumina sequencing (GAII) from the leaves of Brachypodium plants subjected to stresses and selected smRNAs between 15 and 40 nt in length, which we mapped to the Brachypodium genome.
2015-01-20 | E-GEOD-55217 | biostudies-arrayexpress
Project description:The functional structure of all biologically active molecules is dependent on intra- and inter-molecular interactions. This is especially evident for RNA molecules whose functionality, maturation, and regulation requires formation of correct secondary structure through encoded base-pairing interactions. Unfortunately, intra- and inter-molecular base-pairing information is lacking for most RNAs. Here, we use high-throughput sequencing to interrogate all base-paired RNA in Arabidopsis thaliana, and identify ~200 new small (sm)RNA-producing substrates of RNA-DEPENDENT RNA POLYMERASE 6. Our comprehensive analysis of paired RNAs reveals conserved functionality within introns and both 5’ and 3’ untranslated regions (UTRs) of mRNAs, as well as a novel population of functional RNAs, many of which are the precursors of smRNAs. Finally, we identify intra-molecular base-pairing interactions to produce a genome-wide collection of RNA secondary structure models. These findings highlight the importance of base-paired RNAs in eukaryotes, and present an approach that should be widely applicable for the analysis of this key structural feature of RNA. Double-stranded (dsRNA) specific RNA sequencing (dsRNA-seq) in the unopened flowerbuds of wild-type col-0 plants and rdr6 mutant plants, including 2 samples of col-0 dsRNA (1X- or 2X- Ribominus-treated samples) and 1 sample of rdr6 dsRNA. Corresponding two smRNA libraries (smRNA-seq) of both wild-type col-0 plants and rdr6 mutant plants of the same tissue are also presented.
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2011-04-19 | E-GEOD-28591 | biostudies-arrayexpress
Project description:In plants, heterochromatin is maintained by a small RNA-based gene silencing mechanism known as RNA-directed DNA methylation (RdDM). RdDM requires the non-redundant functions of two plant-specific DNA-dependent RNA polymerases (Pol) Pol IV and Pol V. Pol IV plays a major role in siRNA biogenesis, while Pol V may recruit DNA methylation machinery to target endogenous loci for silencing. Although small RNA-generating regions which are dependent on both Pol IV and Pol V have been identified previously, the genomic loci targeted Pol V for siRNA accumulation and silencing have not been described extensively. To characterize the Pol V-dependent, heterochromatic siRNA-generating regions in the Arabidopsis genome, we deeply sequenced the small RNA populations of wild-type and Pol V mutant plants. Furthermore, we characterized the siRNA-generating regions which were dependent on RdDM effectors and examined their dependency on Pol V. Small RNA libraries were generated and deeply sequenced from mutant alleles dms4-1, drd1-1, dms3-1, and rdm1-4, along with their control library (“Wt(T+S)”) which has been described previously (Kanno et al. 2004 Current Biology). More than 2,000 small RNA-generating loci were identified which were greatly suppressed in Pol V mutants. The Pol V-dependent, heterochromatic siRNA-generating regions were characterized in the Arabidopsis genome by deep sequencing the small RNA populations of wild-type and Pol V mutant plants. Deep SBS sequencing was used for small RNA profiling of immature inflorescence tissues from RNA polymerase V and RdDM mutants.
2012-06-11 | E-GEOD-36424 | biostudies-arrayexpress
Project description:Background: RNA silencing pathways play critical roles in gene regulation, virus infection, and transposon control. RNA interference (RNAi) is mediated by small interfering RNAs (siRNAs), which are liberated from double stranded (ds) RNA precursors by Dicer and direct the RNA-induced silencing complex (RISC) to target transcripts. Recent efforts have uncovered important principles governing small RNA (smRNA) sorting into RISC, yet mechanisms defining substrate selection by Dicer proteins remain uncharacterized. Methodology: To better characterize Dicer-2 substrates in Drosophila, we examined the antiviral RNAi response, which generates virus-derived siRNAs from viral RNA. Using high-throughput sequencing, we found that diverse viruses were uniquely targeted; substrates included dsRNA replication intermediates and intramolecular RNA stem loops. smRNA distribution patterns from viral and synthetic dsRNA precursors were highly reproducible, and machine learning techniques identified characteristics of precursor molecules and smRNA duplexes important in determining relative smRNA abundance. Significance: To our knowledge, this study provides the first description of the rules governing Dicer-2 substrate selection, which has important implications for exogenous RNA silencing technologies and the development of smRNA-based antiviral therapeutics. virus-derived siRNA (vsiRNA) expression comparison between control and 4 different virus-infected cells in control as well as 5 different RNAi pathway protein knock-downs in Drosophila dl1 cells
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Project description:Whole genome small RNA sequencing (wild-type Col-0, abh1-1, ein5-6, ein5-6 abh1-1) and 5'-RACE (GMUCT) (wild-type Col-0 and ein5-6) sequencing of parenthetically indicated genotypes of Arabidopsis using the Illumina Genetic Analyzer. Whole-genome oligonucleotide tiling microarrays were used for gene expression studies of the entire transcriptome for wildtype Col-0, abh1-1, ein5-6, and ein5-6 abh1-1 plants. Micro (mi)RNAs and small-interfering (si)RNAs are abundant endogenous small (sm)RNAs that control transcript expression by post-transcriptional gene silencing. Here, we show that concomitant loss of XRN4/EIN5, a 5’-3’ exoribonuclease, and CBP80/ABH1, the largest subunit of the mRNA cap binding complex, results in Arabidopsis plants manifesting myriad developmental defects. Through the analysis of ein5 abh1 double mutant plants, we find that CBP80/ABH1 regulates the levels of mature miRNAs, which suggests this protein is a novel component of the miRNA-mediated RNA silencing pathway. Additionally, we show that a novel class of smRNAs are processed from both sense and anti-sense strands of ~130 endogenous transcripts that apparently are converted to double-stranded RNA and subsequently processed into smRNAs, and accumulate in the absence of XRN4/EIN5. Moreover, we demonstrate that accumulation of these smRNAs is often synergistically increased in ein5 abh1 double mutant plants, which suggests that these proteins act coordinately to regulate the substrates from which they are processed. Finally, we find that the parent transcripts of these novel smRNAs accumulate in an uncapped form upon loss of XRN4/EIN5. These results suggest that uncapped endogenous transcripts can shuttle into an RNA silencing pathway where they become smRNA biogenesis substrates. Overall, our results reveal unexpected connections between RNA metabolism and silencing. Keywords: transcriptome analysis using tiling array; whole genome small RNA sequencing; 5'-RACE sequencing
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