Sindbis virus induces the production of a novel class of endogenous siRNAs in Aedes aegypti mosquitoes
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ABSTRACT:
ORGANISM(S): Aedes aegypti
PROVIDER: GSE35161 | GEO | 2012/04/01
SECONDARY ACCESSION(S): PRJNA150789
REPOSITORIES: GEO
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Project description:We examined the biogenesis of mRNA-derived endogenous short-interfering RNAs (endo-siRNAs) in the disease vector mosquito, Aedes aegypti. Under standard conditions, mRNA-derived endo-siRNAs were produced from the bidirectional transcription of tail-tail overlapping gene pairs. Upon infection with the alphavirus, Sindbis virus (SINV), another class of mRNA-derived endo-siRNAs was observed. Genes producing SINV-induced endo-siRNAs were not enriched for overlapping partners or nearby genes, but were enriched for transcripts with long 3'UTRs. Endo-siRNAs from this class derived uniformly from the entire length of the target transcript, and were found to regulate the transcript levels of the genes from which they were derived. Strand-specific qPCR experiments demonstrated that antisense strands of targeted mRNA genes were produced to exonic, but not intronic regions. Finally, small RNAs mapped to both sense and antisense strands of exon-exon junctions, suggesting double-stranded RNA precursors to SINV-induced endo-siRNAs may be synthesized from mature mRNA templates. These results suggest additional complexity in small RNA pathways and gene regulation in the presence of an infecting virus in disease vector mosquitoes. Examination of endo-siRNA production in Aedes aegypti mosquitoes
2012-04-01 | E-GEOD-35161 | biostudies-arrayexpress
Project description:Background: Eukaryotic cells express a complex layer of noncoding RNAs. An intriguing family of regulatory RNAs includes transcripts from the opposite strand of protein coding genes, so called natural antisense transcripts (NATs). Here, we test the hypothesis that antisense transcription triggers RNA interference and gives rise to endogenous short RNAs (endo-siRNAs). Methods/Results: We used cloned human embryonic kidney cells (HEK293) followed by short RNAseq to investigate the small genic RNA transcriptome. 378 genes gave rise to short RNA reads that mapped to exons of RefSeq genes. The length profile of short RNAs showed a broad peak of 20-24 nucleotides, indicative of endo-siRNAs. Collapsed reads mapped predominantly to the first and the last exon of genes (74%). RNAs reads were intersected with sequences occupied by RNAPolII or bound to Argonaute (AGO1 by crosslinking, ligation, and sequencing of hybrids, CLASH). In the first exon, 94% of the reads correlated with PolII occupancy with an average density of 130 (relative units); this decreased to 65%/20 in middle exons and 54%/12 in the last exon. CLASH reads mapping to multi-exon genes showed little distribution bias with an average of about 5 CLASH reads overlapping with 60% of the endo-siRNA reads. However, endo-siRNAs (21-25 nt) intersecting with CLASH reads were enriched at the 5'end and decreased towards the 3'end. We then investigated the 378 genes with particular focus on features indicative for short RNA production; however, found that endo-siRNA numbers did not correlate with gene structures that favor convergent transcription. In contrast, our gene set was found notably over-represented in the NATsDB sense/antisense group as compared to non-overlapping and non-bidirectional groups. Moreover, read counts showed no correlation with the steady-state levels of the related mRNAs and the pattern of endo-siRNAs proved reproducible after an induced mutagenic insult. Conclusions: Our results suggest that antisense transcripts contribute to low levels of endo-siRNAs in fully differentiated human cells. A characteristic endo-siRNA footprint is being produced at sites of RNAPolII transcription which is also related to AGO1. This endo-siRNA signature represents an intriguing finding and its reproducibility suggests that the production of endo-siRNAs is a regulated process with potential homoeostatic impact.
2014-02-28 | GSE52996 | GEO
Project description:Background: Eukaryotic cells express a complex layer of noncoding RNAs. An intriguing family of regulatory RNAs includes transcripts from the opposite strand of protein coding genes, so called natural antisense transcripts (NATs). Here, we test the hypothesis that antisense transcription triggers RNA interference and gives rise to endogenous short RNAs (endo-siRNAs). Methods/Results: We used cloned human embryonic kidney cells (HEK293) followed by short RNAseq to investigate the small genic RNA transcriptome. 378 genes gave rise to short RNA reads that mapped to exons of RefSeq genes. The length profile of short RNAs showed a broad peak of 20-24 nucleotides, indicative of endo-siRNAs. Collapsed reads mapped predominantly to the first and the last exon of genes (74%). RNAs reads were intersected with sequences occupied by RNAPolII or bound to Argonaute (AGO1 by crosslinking, ligation, and sequencing of hybrids, CLASH). In the first exon, 94% of the reads correlated with PolII occupancy with an average density of 130 (relative units); this decreased to 65%/20 in middle exons and 54%/12 in the last exon. CLASH reads mapping to multi-exon genes showed little distribution bias with an average of about 5 CLASH reads overlapping with 60% of the endo-siRNA reads. However, endo-siRNAs (21-25 nt) intersecting with CLASH reads were enriched at the 5'end and decreased towards the 3'end. We then investigated the 378 genes with particular focus on features indicative for short RNA production; however, found that endo-siRNA numbers did not correlate with gene structures that favor convergent transcription. In contrast, our gene set was found notably over-represented in the NATsDB sense/antisense group as compared to non-overlapping and non-bidirectional groups. Moreover, read counts showed no correlation with the steady-state levels of the related mRNAs and the pattern of endo-siRNAs proved reproducible after an induced mutagenic insult. Conclusions: Our results suggest that antisense transcripts contribute to low levels of endo-siRNAs in fully differentiated human cells. A characteristic endo-siRNA footprint is being produced at sites of RNAPolII transcription which is also related to AGO1. This endo-siRNA signature represents an intriguing finding and its reproducibility suggests that the production of endo-siRNAs is a regulated process with potential homoeostatic impact. Size selected RNASeq of 3 human embryonic kidney cell (HEK293) samples. 1 control and 2 samples exposed to 100 μg/ml ethyl methanesulfonate for 24 hrs.
2014-02-28 | E-GEOD-52996 | biostudies-arrayexpress
Project description:Unrestricted movement of mobile genetic elements could cause pre-mature lethality in Drosophila melanogaster. Specifically, retro transposons can disrupt genomic integrity through insertions, deletions and chromosomal rearrangements. Therefore, eukaryotes have developed defense mechanisms to silence these elements. In Drosophila, endogenous small interfering (endo-siRNAs) repress retro transposon mobility in somatic cells. The generation of endo-siRNAs requires Dicer-2 processing of double-stranded RNA precursors, yet the origins of this precursor are unknown. Here we show that retro transposons in Dmel-2 cells produce sense and antisense transcripts and identify bonafide transcription start sites for these RNAs. We determine that retro transposon antisense transcripts are less polyadenylated than sense transcripts. RNA-seq and small RNA-seq upon Dicer-2 depletion showed global decrease in endo-siRNAs mapping to retro transposons and increased expression of both S and AS retro transposon transcripts. These data support a model in which double-stranded RNA precursors are derived from convergent transcription and retained in the nucleus. Dicer-2 processes these precursors into endo-siRNAs that silence both sense and antisense retro transposon transcripts. Reduction of sense retro transposon transcripts potentially lowers element specific protein levels required for movement. This mechanism preserves genomic integrity and is especially important for Drosophila fitness because mobile genetic elements are highly active.
2016-01-26 | GSE67725 | GEO
Project description:Small interfering RNAs (siRNAs) direct RNA interference (RNAi) in eukaryotes. In flies, somatic cells produce siRNAs from exogenous double-stranded RNA as a defense against viral infection. Here, we identify 21-nt long, endogenous siRNAs (endo-siRNAs) corresponding to transposons and heterochromatic sequences in the somatic cells of Drosophila melanogaster. We also detected endo-siRNAs complementary to mRNAs: these siRNAs disproportionately mapped to the complementary regions of overlapping mRNAs predicted to form dsRNA in vivo. Normal accumulation of somatic endo-siRNAs requires the siRNA-generating ribonuclease, Dicer-2, and the RNAi effector protein, Ago2. We propose that endo-siRNAs generated by the fly RNAi pathway silence selfish genetic elements in the soma much as piRNAs do in the germ line. Keywords: Small RNA detection and quantification.
2008-04-11 | GSE11019 | GEO
Project description:Proteomic analysis of Ago2-/- oocytes revealed that Ago2 interacted with endogenous small interfering RNAs (endo-siRNAs) to repress mRNA translation globally.
2021-05-07 | PXD025846 | iProX
Project description:Small noncoding RNA (sncRNA), including microRNAs (miRNAs) and endogenous small-interfering RNAs (endo-siRNAs) are key gene regulators in eukaryotes, playing critical roles in plant development and stress tolerance. Trans-acting siRNAs (ta-siRNAs), which are secondary siRNAs triggered by miRNAs, and siRNAs from natural antisense transcripts (nat-siRNAs) are two well-studied classes of endo-siRNAs. In order to understand sncRNAs’ roles in plant cold response and stress acclimation, we studied miRNAs and endo-siRNAs in Cassava (Manihot esculenta), a major source of food for the world populations in tropical regions. Combining Next-Generation sequencing and computational and experimental analyses, we profiled and characterized sncRNA species and mRNA genes from the plants that experienced severe and moderate cold stresses, that underwent further severe cold stress after cold acclimation at moderate stress, and that grew under the normal condition. We also included Castor bean (Ricinus communis) to understand conservation of sncRNAs. In addition to known miRNAs, we identified dozens of novel miRNAs as well as ta-siRNA-yielding and nat-siRNA-yielding loci in Cassava and Castor bean, respectively. Among the expressed sncRNAs, many sncRNAs were differentially expressed under cold stresses. Our study provided the results on gene regulation by sncRNAs in cold acclimation of Euphorbiaceous plants and the role of sncRNA-mediated pathways affected by cold stress and stress acclimation in Cassava.
2014-07-05 | GSE52177 | GEO
Project description:MicroRNA (miRNA) and endogenous siRNA (endo-siRNA) are two essential classes of small noncoding RNAs (sncRNAs) in eukaryotic organisms. The class of miRNA is diverse and there exist noncanonical miRNAs that bypass the canonical miRNA biogenesis pathway. In order to identify noncanonical miRNAs and endo-siRNAs responding to virus infection and study their potential function, we sequenced small-RNA species from cells lytically infected with murine gammaherpesvirus 68. In addition to 3 novel canonical miRNAs in mouse, two antisense miRNAs in virus and 25 novel noncanonical miRNAs, including miRNAs derived from tRNAs, snoRNAs and introns, in the host were identified. These noncanonical miRNAs exhibited features distinct from canonical miRNAs in the lengths and structures of miRNA hairpins as well as base pairings and first nucleotide preference. Many of the novel miRNAs are conserved in mammals. In addition to several known murine endo-siRNAs detected by the sequencing profiling, a novel locus in the mouse genome was identified to give rise to endo-siRNAs. This novel endo-siRNA locus is comprised of two tandem inverted B4 short interspersed nuclear elements (SINEs). Unexpectedly, the SINE-derived endo-siRNAs were found in a variety of sequencing data as well as virus-infected cells. Moreover, a murine miRNA was up-regulated more than 35 fold in infected than in mock-treated cells. The putative target genes of the viral and the up-regulated murine miRNAs were potentially involved in processes of gene transcription and protein phosphorylation and localized to membranes, suggesting their role in manipulating the host basal immune system during lytic infection. Our results extended the number of noncanonical miRNAs in mammals and shed new lights on their potential functions of lytic infection of MHV68. Mouse NIH 3T12 cells infectd with MHV68 (3 samples) and mock-treated (2 samples) were examined. Noncanonical microRNAs and endogenous siRNAs discovery in lytic infection of murine gammaherpesvirus MHV68 (NC_001826.2).
2012-12-31 | E-GEOD-36639 | biostudies-arrayexpress
Project description:Small interfering RNAs (siRNAs) direct RNA interference (RNAi) in eukaryotes. In flies, somatic cells produce siRNAs from exogenous double-stranded RNA as a defense against viral infection. Here, we identify 21-nt long, endogenous siRNAs (endo-siRNAs) corresponding to transposons and heterochromatic sequences in the somatic cells of Drosophila melanogaster. We also detected endo-siRNAs complementary to mRNAs: these siRNAs disproportionately mapped to the complementary regions of overlapping mRNAs predicted to form dsRNA in vivo. Normal accumulation of somatic endo-siRNAs requires the siRNA-generating ribonuclease, Dicer-2, and the RNAi effector protein, Ago2. We propose that endo-siRNAs generated by the fly RNAi pathway silence selfish genetic elements in the soma much as piRNAs do in the germ line. Keywords: Small RNA detection and quantification. Small RNAs (18-30 nt) from fly heads (WT, ago2 mutants, dcr-2 homozygous and heterozygous mutants, and WT expressing an inverted repeat directed against exon 3 of the gene "white") and S2 cells (transgenic for a construct expressing siRNAs against white and GFP) were sequenced using a Solexa Genome Analyzer instrument. Raw sequence data files for this study are available for download from the SRA FTP site at ftp://ftp.ncbi.nlm.nih.gov/sra/Studies/SRP000/SRP000181
2008-04-10 | E-GEOD-11019 | biostudies-arrayexpress
Project description:Small non-coding RNA biogenesis typically involves cleavage of structured precursors by RNase III-like endonucleases. However, guide RNAs that direct U-insertion/deletion mRNA editing in mitochondria of trypanosomes maintain 5′ triphosphate characteristic of transcription start site and possess U-tail indicative of 3′ processing and uridylation. Here, we identified a protein complex composed of RET1 TUTase and 3′-5′ DSS1 exonuclease, and three additional subunits. This complex, termed mitochondrial 3′ processome (MPsome), is responsible for primary uridylation of ~800-nt gRNA precursors, their processive degradation to a mature length of 50-60 nt, and secondary U-tail addition. Both strands of gRNA gene are transcribed giving rise to sense and antisense precursors of similar size. Head-to-head hybridization of these transcripts blocks symmetrical 3′-5′ degradation at the fixed distance from the double-stranded region. Together, our findings suggest a model in which gRNA is derived from the 5′ extremity of a primary molecule by uridylation-induced, antisense transcription-controlled exonucleolytic degradation.
2016-02-08 | GSE71292 | GEO