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In depth sequencing of the siRNAs associated with peach latent mosaic viroid infection

ABSTRACT: Using a high-throughput sequencing approach we quantitatively analyzed the content of viroid-derived siRNAs of an infected tree. Our results show that the entire PLMVd genome is found in the siRNA population. Also both polarities are susceptible to be targeted by the RNAi machinery but specific regions for each polarity are over represented. Those regions, that are not the same for each polarity, do not necessarily correlate with double stranded regions that could be substrate for Dicer-like enzymes. Finally the analysis of the first 5’ nucleotide revealed a bias toward a C or a U in viroid-derived siRNAs, indicating that at least AGO5 and AGO1 can recruit these small RNAs. Analysis of siRNAs population from RNA sample isolated from a viroid-infected tree

ORGANISM(S): Prunus persica

SUBMITTER: Jean-Pierre Perreault

PROVIDER: E-GEOD-18764 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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In-depth sequencing of the siRNAs associated with peach latent mosaic viroid infection.

Bolduc François F Hoareau Christopher C St-Pierre Patrick P Perreault Jean-Pierre JP

BMC molecular biology 20100216

<h4>Background</h4>It has been observed that following viroid infection, there is an accumulation of viroid-derived siRNAs in infected plants. Some experimental results suggest that these small RNAs may be produced by the plant defense system to protect it from infection, indicating that viroids can elicit the RNA-silencing pathways. The objective of this study is to identify in the peach latent mosaic viroid (PLMVd), a model RNA genome, the regions that are most susceptible to RNA interference ...[more]

PMID: 20158907

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In depth sequencing of the siRNAs associated with peach latent mosaic viroid infection

Project description:Using a high-throughput sequencing approach we quantitatively analyzed the content of viroid-derived siRNAs of an infected tree. Our results show that the entire PLMVd genome is found in the siRNA population. Also both polarities are susceptible to be targeted by the RNAi machinery but specific regions for each polarity are over represented. Those regions, that are not the same for each polarity, do not necessarily correlate with double stranded regions that could be substrate for Dicer-like enzymes. Finally the analysis of the first 5’ nucleotide revealed a bias toward a C or a U in viroid-derived siRNAs, indicating that at least AGO5 and AGO1 can recruit these small RNAs.

2010-02-20 | GSE18764 | GEO

Dicer Partner Proteins Tune the Length of Mature miRNAs in Flies and Mammals

Project description:Drosophila Dicer-1 produces microRNAs (miRNAs) from pre-miRNA, whereas Dicer-2 generates small interfering RNAs (siRNAs) from long dsRNA. loquacious (loqs) encodes three Dicer partner proteins, Loqs-PA, Loqs-PB, and, Loqs-PD, generated by alternative splicing. To understand the function of each Loqs isoform, we constructed loqs isoform-specific rescue flies. Loqs-PD promotes siRNA production in vivo by Dicer-2. Loqs-PA or Loqs-PB is required for viability, but the proteins are not fully redundant: Loqs-PB is required to produce a specific subset of miRNAs. Surprisingly, Loqs-PB tunes the product size cleaved by Dicer-1 from pre-miR-307a, generating a longer miRNA isoform with a distinct seed sequence and target specificity. The mouse and human Dicer-binding partner TRBP, a homolog of Loqs-PB, similarly tunes the site of pre-miR-132 cleavage by mammalian Dicer. Thus, Dicer-binding partner proteins can change the choice of cleavage site by Dicer, producing miRNAs with different target specificities than those that would be made by Dicer alone. Examination of Dicer-binding proteins on small RNA profiles of female fly heads, fly ovaries, mouse embryonic fibroblasts, and mouse tail fibroblasts.

2012-12-07 | E-GEOD-37443 | biostudies-arrayexpress

Role of RccR in the transcriptional control of primary carbon metabolism in Pseudomonas

Project description:To characterise the RccR regulon, we performed a ChIP-seq assay using a polyclonal RccR antiserum on SBW25 WT/rccR strains grown in minimal pyruvate and glycerol media. We were able to identify 8 RccR binding sites from this experiment including one in the rccR promoter region itself. The peaks identified in our RccR ChIP-seq assays corresponded to strongly enriched regions relative to the respective rccR controls. All 8 peaks were localized in intergenic regions, upstream of one or more genes.

2017-08-24 | E-MTAB-5745 | biostudies-arrayexpress

RNA-seq of total royal jelly and MRJP-3-bound RNA

Project description:MRJP-3 is an RNA binding protein that binds both double- and single-stranded RNA in vitro. To test whether i) MRJP-3 binds RNA in its natural environment, the royal jelly (RJ) and ii) to characterize its RJ RNA partners; a pull down followed by RNA-seq system was developed. MRJP-3 bound RNA was compared to total RJ RNA extracted from the same hive. To establish the system, we first incubated biotinylated MRJP-3, or biotinylated BSA in RJ and pulled the proteins out with strepavidin coated magnetic beads followed by RNA extraction and bioanalyzer profiling. MRJP-3 pull out was enriched for RNA compare to the BSA and just beads controls. The RNA population pulled down with MRJP-3 has similar profile to the total RJ RNA, further demonstrating the MRJP-3 has no affinity to a certain RNA species.

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Three groups of transposable elements with contrasting copy number dynamics and host responses in the maize (Zea mays ssp. mays) genome

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2014-03-11 | E-GEOD-55730 | biostudies-arrayexpress

Global effects of the CSR-1 RNA interference pathway on transcriptional landscape

Project description:Argonaute proteins and their small RNA co-factors short interfering RNAs (siRNAs) are known to inhibit gene expression at the transcriptional and post-transcriptional levels. In Caenorhabditis elegans, the Argonaute CSR-1 binds thousands of endogenous siRNAs (endo-siRNAs) antisense to germline transcripts and associates with chromatin in a siRNA-dependent manner. However, its role in gene expression regulation remains controversial. Here, we used a genome-wide profiling of nascent RNA transcripts to demonstrate that the CSR-1 RNAi pathway promotes sense-oriented Pol II transcription. Moreover, a loss of CSR-1 function resulted in global increase in antisense transcription and ectopic transcription of silent chromatin domains, which led to reduced chromatin incorporation of centromere-specific histone H3. Based on these findings, we propose that the CSR-1 pathway has a role in maintaining the directionality of active transcription thereby propagating the distinction between transcriptionally active and silent genomic regions. GRO-seq (Global Run-On sequencing) for detection of nascent transcripts. Two biological replicates were generated for csr-1 hypomorphic mutant and the corresponding WT samples using ~300,000 worms for each experiment, and two biological replicates were prepared for drh-3 (ne4253) mutant and the corresponding WT samples using ~100,000 worms for each experiment. The GRO-seq were performed in late L3/early L4 stage worms. (8 samples total)

2014-02-28 | E-GEOD-49946 | biostudies-arrayexpress

Small RNA and gene expression profile in the adult testes of the common marmoset

Project description:Small RNAs mediate gene silencing by binding Argonaute/Piwi proteins to regulate target RNAs. Here we describe small RNA profiling of the adult testes of Callithrix jacchus, the common marmoset. The most abundant class of small RNAs in the adult testis was piRNAs, while 353 novel miRNAs but few endo-siRNAs were also identified. MARWI, a marmoset homolog of mouse MIWI and a very abundant PIWI in adult testes, associates with piRNAs that show characteristics of mouse pachytene piRNAs. As in other mammals, most marmoset piRNAs are derived from conserved clustered regions in the genome, which are annotated as intergenic regions. However, some of these piRNA cluster regions contain antisense-orientated pseudogenes, suggesting regulation of parental functional protein-coding genes. More piRNAs map to transposable element (TE) subfamilies when they have copies in piRNA clusters. In addition, the strand-bias observed for piRNAs mapped to each TE subfamily correlates with the polarity of copies inserted in clusters. These findings suggest that pachytene piRNA clusters determine the abundance and strand-bias of TE-derived piRNAs, and also regulate protein-coding genes via pseudogene-derived piRNAs. small RNA levels in the adult marmoset testis, and MARWI-IP small RNA levels and RNA levels from the adult marmoset testis with two replicates.

2014-06-01 | E-GEOD-52927 | biostudies-arrayexpress

Function, targets and evolution of Caenorhabditis elegans piRNAs (small RNA-Seq)

Project description:piRNAs are required to maintain germline integrity and fertility but their mechanism of action is poorly understood. Here we demonstrate that C. elegans piRNAs silence transcripts in trans through imperfectly complementary sites. We find that target silencing is independent of Piwi endonuclease activity or “slicing”. Instead, we show that piRNAs initiate a localized secondary endogenous small interfering RNA (endo-siRNA) response. Endogenous protein-coding gene, pseudogene and transposon transcripts exhibit Piwi-dependent endo-siRNAs at sites complementary to piRNAs and are derepressed in Piwi mutants. Genomic loci of piRNA biogenesis are depleted of protein-coding genes but not pseudogenes or transposons. Our data suggest that nematode piRNA clusters are evolving to generate piRNAs against active mobile elements. Thus, piRNAs provide heritable, sequence-specific triggers for RNAi in C. elegans. 7 small RNA libraries were sequenced as part of 25 flow cell lanes on the Illumina GA II platform. Samples were treated with tobacco acid pyrophosphatase to allow cloning of small RNAs with a 5'-triphosphate. Samples were labelled for multiplexing using 4-bp 5'-barcodes or barcodes included in Illumina TruSeq adapters. In most cases a single flow cell lane included several multiplexed libraries.

2012-06-18 | E-GEOD-28617 | biostudies-arrayexpress

Endogenous, tissue-specific short-interfering RNAs silence the chalcone synthase gene family in Glycine max seed coats

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2010-05-13 | E-GEOD-21825 | biostudies-arrayexpress

Analysis of 22G siRNA triggered siRNA amplification and 3' 2'-O-methylated small RNAs in Caenorhabditis elegans

Project description:Small RNAs, including piRNAs, miRNAs and endogenous siRNAs, bind Argonaute proteins to form RNA-silencing complexes that target coding genes, transposons and aberrant RNAs. To assess the requirements for endogenous siRNA formation and activity in C. elegans, we developed a GFP-based sensor for the endogenous siRNA 22G siR-1, one of a set of abundant siRNAs processed from a precursor RNA mapping to the X chromosome, the X-cluster. Silencing of the sensor is also dependent on the partially complementary, unlinked 26G siR-O7 siRNA. We show that 26G siR-O7 acts in trans to initiate 22G siRNA formation from the X-cluster. The presence of several mispairs between 26G siR-O7 and the X-cluster mRNA, as well as mutagenesis of the siRNA sensor, indicates that siRNA target recognition is permissive to a degree of mispairing. From a candidate reverse genetic screen, we identified several factors required for 22G siR-1 activity, including the Argonaute ergo-1 and the 3' methyltransferase henn-1. Quantitative RT-PCR of small RNAs in a henn-1 mutant and deep sequencing of methylated small RNAs indicate that siRNAs and piRNAs that associate with PIWI clade Argonautes are methylated by HENN-1, while siRNAs and miRNAs that associate with non-PIWI clade Argonautes are not. Thus, PIWI-class Argonaute proteins are specifically adapted to associate with methylated small RNAs in C. elegans. This SuperSeries is composed of the following subset Series: GSE34320: Analysis of 22G siRNA triggered siRNA amplification in Caenorhabditis elegans GSE34321: Analysis of 3' 2'-O-methylated small RNAs in Caenorhabditis elegans Refer to individual Series

2012-02-03 | E-GEOD-35550 | biostudies-arrayexpress

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