Alternative RISC loading complex in Drosophila S2 cells
Ontology highlight
ABSTRACT:
PROVIDER: PRJEB20483 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| ERR1938650.fastq.gz | Fastqsanger.gz | |||
| ERR1938651.fastq.gz | Fastqsanger.gz | |||
| ERR1938652.fastq.gz | Fastqsanger.gz | |||
| ERR1938653.fastq.gz | Fastqsanger.gz | |||
| ERR2116830.fastq.gz | Fastqsanger.gz |
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Project description:In Drosophila, siRNAs are classified as endo- or exo-siRNAs based on their origin. Both are processed from double-stranded RNA precursors by Dcr-2, then loaded into the Argonaute protein Ago2. While exo-siRNAs serve to defend the cell against viruses, endo-siRNAs restrict the spread of selfish DNA in somatic cells, analogous to piRNAs in the germ line. Endo- and exo-siRNAs display a differential requirement for double-stranded RNA binding domain proteins (dsRBPs): R2D2 is needed to load exo-siRNAs into Ago2 while the PD isoform of Loquacious (Loqs-PD) stimulates Dcr-2 during the nucleolytic processing of hairpin-derived endo-siRNAs. In cell culture assays, R2D2 antagonizes Loqs-PD in endo-siRNA silencing and Loqs-PD is an inhibitor of RNA interference. Loqs-PD can interact via the C-terminus unique to this isoform with the DExH/D-helicase domain of Drosophila Dcr-2, where binding of R2D2 has also been localized. Separation of the two pathways is not complete; rather, the dicing and Ago2-loading steps appear uncoupled, analogous to the corresponding steps in miRNA biogenesis. Analysis of deep sequencing data further demonstrates that in r2d2 mutant flies, siRNAs can be loaded into Ago2 but not all siRNA classes are equally proficient for this. Thus, the canonical Ago2-RISC loading complex can be bypassed under certain circumstances.
2011-02-10 | GSE26230 | GEO
Project description:In Drosophila, siRNAs are classified as endo- or exo-siRNAs based on their origin. Both are processed from double-stranded RNA precursors by Dcr-2, then loaded into the Argonaute protein Ago2. While exo-siRNAs serve to defend the cell against viruses, endo-siRNAs restrict the spread of selfish DNA in somatic cells, analogous to piRNAs in the germ line. Endo- and exo-siRNAs display a differential requirement for double-stranded RNA binding domain proteins (dsRBPs): R2D2 is needed to load exo-siRNAs into Ago2 while the PD isoform of Loquacious (Loqs-PD) stimulates Dcr-2 during the nucleolytic processing of hairpin-derived endo-siRNAs. In cell culture assays, R2D2 antagonizes Loqs-PD in endo-siRNA silencing and Loqs-PD is an inhibitor of RNA interference. Loqs-PD can interact via the C-terminus unique to this isoform with the DExH/D-helicase domain of Drosophila Dcr-2, where binding of R2D2 has also been localized. Separation of the two pathways is not complete; rather, the dicing and Ago2-loading steps appear uncoupled, analogous to the corresponding steps in miRNA biogenesis. Analysis of deep sequencing data further demonstrates that in r2d2 mutant flies, siRNAs can be loaded into Ago2 but not all siRNA classes are equally proficient for this. Thus, the canonical Ago2-RISC loading complex can be bypassed under certain circumstances. Examination of small RNAs from two different mutant strains as well as the corresponding heterozygous controls
2011-02-10 | E-GEOD-26230 | biostudies-arrayexpress
Project description:Drosophila melanogaster expresses three classes of small RNAs, which are classified according to their mechanisms of biogenesis. MicroRNAs are ~22-23-nt, ubiquitously expressed small RNAs that are sequentially processed from hairpin-like precursors by Drosha/Pasha and Dcr-1/Loquacious complexes. MicroRNAs usually associate with AGO1 and regulate the expression of protein-coding genes. Piwi-interacting RNAs (piRNAs) of ~24-28-nt associate with Piwi-family proteins and can arise from single-stranded precursors. piRNAs function in transposon silencing and are mainly restricted to gonadal tissues. Endo-siRNAs are found in both germline and somatic tissues. These ~21-nt RNAs are produced by a distinct Dicer, Dcr-2, and do not depend on Drosha/Pasha complexes. They predominantly bind to AGO2 and target both mobile elements and protein-coding genes. Surprisingly, a subset of endo-siRNAs strongly depend for their production on the dsRNA-binding protein Loquacious (Loqs), thought generally to be a partner for Dcr-1 and a co-factor for miRNA biogenesis. Endo-siRNA production depends on a specific Loqs isoform, Loqs-PD, which is distinct from the one, Loqs-PB, required for the production of microRNAs. Paralleling their roles in the biogenesis of distinct small RNA classes, Loqs-PD and Loqs-PB bind to different Dicer proteins, with Dcr-1/Loqs-PB complexes and Dcr-2/Loqs-PD complexes driving microRNA and endo-siRNA biogenesis, respectively. Small RNA profiling by high throughput sequencing
2009-07-31 | GSE17171 | GEO
Project description:Drosophila melanogaster expresses three classes of small RNAs, which are classified according to their mechanisms of biogenesis. MicroRNAs are ~22-23-nt, ubiquitously expressed small RNAs that are sequentially processed from hairpin-like precursors by Drosha/Pasha and Dcr-1/Loquacious complexes. MicroRNAs usually associate with AGO1 and regulate the expression of protein-coding genes. Piwi-interacting RNAs (piRNAs) of ~24-28-nt associate with Piwi-family proteins and can arise from single-stranded precursors. piRNAs function in transposon silencing and are mainly restricted to gonadal tissues. Endo-siRNAs are found in both germline and somatic tissues. These ~21-nt RNAs are produced by a distinct Dicer, Dcr-2, and do not depend on Drosha/Pasha complexes. They predominantly bind to AGO2 and target both mobile elements and protein-coding genes. Surprisingly, a subset of endo-siRNAs strongly depend for their production on the dsRNA-binding protein Loquacious (Loqs), thought generally to be a partner for Dcr-1 and a co-factor for miRNA biogenesis. Endo-siRNA production depends on a specific Loqs isoform, Loqs-PD, which is distinct from the one, Loqs-PB, required for the production of microRNAs. Paralleling their roles in the biogenesis of distinct small RNA classes, Loqs-PD and Loqs-PB bind to different Dicer proteins, with Dcr-1/Loqs-PB complexes and Dcr-2/Loqs-PD complexes driving microRNA and endo-siRNA biogenesis, respectively. Small RNA profiling by high throughput sequencing Total RNA was isolated using Trizol reagent (Invitrogen) and size-fractionated by PAGE into 19-24nt. These were independently processed and sequenced using the Illumina GAII platform. In total, six libraries were analyzed.
2009-07-30 | E-GEOD-17171 | biostudies-arrayexpress
Project description:In Drosophila, the siRNA pathway is initiated when exogenous or endogenous double stranded RNA (dsRNA) is processed into siRNAs by Dicer-2 (Dcr-2) and a dsRNA-binding protein (dsRBP) cofactor called Loquacious (Loqs). The siRNAs are then loaded onto Argonaute-2 (Ago2) protein by the action of Dcr-2 with another dsRBP cofactor called R2D2. Loaded Ago2 executes the destruction of target RNAs that have sequence complementarity to the siRNA. Dcr-2, R2D2, and Ago2 have also been shown to be required for innate antiviral defense in Drosophila. However, the biogenesis of virus-derived siRNAs (vsiRNAs) and their targets in virus-infected cells remain unclear. Here, we analyzed the antiviral response in Drosophila by monitoring the replication of different RNA viruses and deep sequencing of small RNAs in infected animals. We show that vsiRNAs are generated by Dcr-2 processing of dsRNA formed during viral genome replication and transcription. These vsiRNAs then directly target viral transcripts but not genomes, to inhibit viral replication. The biogenesis of vsiRNAs was virtually independent of Loqs and R2D2. R2D2, however, was essential for sorting and loading of vsiRNAs onto Ago2 and effective silencing of viral RNA expression. Loqs was completely dispensable for silencing of viruses in contrast to its role in silencing of endogenous targets. Our results suggest the existence of a specific siRNA pathway triggered by viral infection independent of conserved dsRBP cofactors and separate from the endogenous pathway. Inhibition of virus replication resulting from direct injection of viral RNA into Drosophila embryos was also not dependent on Loqs, suggesting the distinction of the two pathways is not related to the mode of entry but recognition of intrinsic features of viral RNA or its mode of replication. We speculate that this unique framework might be necessary for a prompt and efficient antiviral response
2012-03-16 | GSE36449 | GEO
Project description:While mammalian somatic cells are incapable of mounting an effective RNA interference (RNAi) response to viral infections, plants and invertebrates are able to generate high levels of functional short interfering RNAs (siRNAs) of viral origin that can effectively control many infections. In Drosophila, the RNAi response is mediated by the Dicer 2 enzyme (dDcr2) acting in concert with two co-factors called Loqs-PD and R2D2. To examine whether a functional RNAi response could be reconstituted in human somatic cells by expression of these insect proteins, we expressed dDcr2, in the presence or absence of Loqs-PD and/or R2D2, in a previously described human cell line, NoDice/ΔPKR, that lacks functional forms of both the human Dicer (dcr) and EIF2AK2 (pkr) gene. Upon expression of dDcr2, Loqs-PD and R2D2 in these human cells, we observed the production of ~21-nt long siRNAs, derived from a co-transfected double stranded RNA (dsRNA) expression vector, that were loaded into the human RNA-induced silencing complex (RISC) and were able to significantly reduce the expression of a cognate indicator gene. We conclude that it is possible to at least partly rescue the ability of mammalian somatic cells to express functional siRNAs by using gene products of invertebrate origin.
2016-12-23 | GSE83433 | GEO
Project description:In Drosophila, the siRNA pathway is initiated when exogenous or endogenous double stranded RNA (dsRNA) is processed into siRNAs by Dicer-2 (Dcr-2) and a dsRNA-binding protein (dsRBP) cofactor called Loquacious (Loqs). The siRNAs are then loaded onto Argonaute-2 (Ago2) protein by the action of Dcr-2 with another dsRBP cofactor called R2D2. Loaded Ago2 executes the destruction of target RNAs that have sequence complementarity to the siRNA. Dcr-2, R2D2, and Ago2 have also been shown to be required for innate antiviral defense in Drosophila. However, the biogenesis of virus-derived siRNAs (vsiRNAs) and their targets in virus-infected cells remain unclear. Here, we analyzed the antiviral response in Drosophila by monitoring the replication of different RNA viruses and deep sequencing of small RNAs in infected animals. We show that vsiRNAs are generated by Dcr-2 processing of dsRNA formed during viral genome replication and transcription. These vsiRNAs then directly target viral transcripts but not genomes, to inhibit viral replication. The biogenesis of vsiRNAs was virtually independent of Loqs and R2D2. R2D2, however, was essential for sorting and loading of vsiRNAs onto Ago2 and effective silencing of viral RNA expression. Loqs was completely dispensable for silencing of viruses in contrast to its role in silencing of endogenous targets. Our results suggest the existence of a specific siRNA pathway triggered by viral infection independent of conserved dsRBP cofactors and separate from the endogenous pathway. Inhibition of virus replication resulting from direct injection of viral RNA into Drosophila embryos was also not dependent on Loqs, suggesting the distinction of the two pathways is not related to the mode of entry but recognition of intrinsic features of viral RNA or its mode of replication. We speculate that this unique framework might be necessary for a prompt and efficient antiviral response We analyzed the small RNA reponse to viral infection by deep sequencing of small RNA libraries from wild type and RNAi mutant adult flies infected with Sindbis birus and Vesicular Stomatatis virus.
2012-03-16 | E-GEOD-36449 | biostudies-arrayexpress
Project description:Small interfering RNAs (siRNAs) are the key components for RNA interference (RNAi), a conserved RNA silencing mechanism in many eukaryotes. In Drosophila, an RNase III enzyme Dicer-2 (Dcr-2), aided by its cofactor Loquacious-PD (Loqs-PD), plays major role in generating 21 base-pair (bp) siRNA duplexes from long double-stranded RNAs (dsRNAs). The ATP hydrolysis by the helicase domain of Dcr-2 is critical to the successful processing of a long dsRNA into consecutive siRNA duplexes. Here we report the cryo-EM structures of Dcr-2/Loqs-PD in the apo state and in multiple processing states of a 50-bp dsRNA substrate. The structures elucidated interactions between Dcr-2 and Loqs-PD, and significant conformational changes of Dcr-2 during a dsRNA processing cycle. The N-terminal helicase and DUF283 domains undergo conformational change upon initial dsRNA binding, forming an ATP binding pocket and a 5’-phosphate binding pocket. The overall conformation of Dcr-2/Loqs-PD is relatively rigid during translocating along the dsRNA in the presence of ATP, while the interaction between DUF283 and RIIIDb domains prevents non-specific cleavage during translocation by blocking the access of dsRNA to the RNase active center. Additional ATP-dependent conformational changes are required to form an active-dicing state and precisely cleave the dsRNA into 21-bp siRNA duplex confirmed by the structure in the post-dicing state. Collectively, this study revealed the molecular mechanism for the full-cycle of ATP-dependent dsRNA processing by Dcr-2/Loqs-PD.
2022-04-21 | PXD033365 |
Project description:Transposable elements are a serious threat for genome integrity and their control via small RNA mediated silencing pathways is an ancient strategy. The fruit fly Drosophila melanogaster has two silencing mechanisms that repress TEs expression: endogenous siRNAs (esiRNAs or endo-siRNAs) and Piwi-interacting small RNAs (piRNAs). The biogenesis of endo-siRNAs involves Loqs-PD, which acts predominantly during processing of dsRNA by Dcr-2, and R2D2 that primarily helps to direct siRNAs for loading into Ago2. We provide deep sequencing evidence consistent with the idea that R2D2 and Loqs-PD can function in part redundantly. Certain transposons display a preference for either dsRBD-protein for production or loading; this appeared to correlate neither with overall abundance, classification of the transposon or a specific site of genomic origin. The endo-siRNA biogenesis pathway in the germline operates according to the same principles as the existing model for the soma, and its impairment does not significantly affect piRNAs. Expanding the analysis, we confirmed the occurrence of somatic piRNA-like RNAs (pilRNAs) that show a ping-pong signature. We detected expression of the Piwi-family protein mRNAs only barely above background, indicating that the somatic pilRNAs may arise from a small sub-population of somatic cells that express a functional piRNA pathway.
2015-01-19 | GSE45290 | GEO
Project description:We observed several hundreds of thousand reads matching the IIV6 genome in IIV6 infected S2 and wild-type flies. The large majority of these reads have a size of 21 nt. This peak was absent from the library prepared from S2 non-infected and infected Dcr-2-/- mutant flies. The virus-derived siRNAs were not uniformally distributed along the viral genome, but rather several hotspots were observed, revealing that specific regions of the viral genome generate the siRNAs.
2013-02-06 | GSE41007 | GEO