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A specific dsRNA-binding protein complex selectively sequesters endogenous inverted-repeat siRNA precursors and inhibits their processing.

ABSTRACT: In plants, several dsRNA-binding proteins (DRBs) have been shown to play important roles in various RNA silencing pathways, mostly by promoting the efficiency and/or accuracy of Dicer-like proteins (DCL)-mediated small RNA production. Among the DRBs encoded by the Arabidopsis genome, we recently identified DRB7.2 whose function in RNA silencing was unknown. Here, we show that DRB7.2 is specifically involved in siRNA production from endogenous inverted-repeat (endoIR) loci. This function requires its interacting partner DRB4, the main cofactor of DCL4 and is achieved through specific sequestration of endoIR dsRNA precursors, thereby repressing their access and processing by the siRNA-generating DCLs. The present study also provides multiple lines of evidence showing that DRB4 is partitioned into, at least, two distinct cellular pools fulfilling different functions, through mutually exclusive binding with either DCL4 or DRB7.2. Collectively, these findings revealed that plants have evolved a specific DRB complex that modulates selectively the production of endoIR-siRNAs. The existence of such a complex and its implication regarding the still elusive biological function of plant endoIR-siRNA will be discussed.

SUBMITTER: Montavon T

PROVIDER: S-EPMC5388410 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

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A specific dsRNA-binding protein complex selectively sequesters endogenous inverted-repeat siRNA precursors and inhibits their processing.

Montavon Thomas T Kwon Yerim Y Zimmermann Aude A Hammann Philippe P Vincent Timothée T Cognat Valérie V Michel Fabrice F Dunoyer Patrice P

Nucleic acids research 20170201 3

In plants, several dsRNA-binding proteins (DRBs) have been shown to play important roles in various RNA silencing pathways, mostly by promoting the efficiency and/or accuracy of Dicer-like proteins (DCL)-mediated small RNA production. Among the DRBs encoded by the Arabidopsis genome, we recently identified DRB7.2 whose function in RNA silencing was unknown. Here, we show that DRB7.2 is specifically involved in siRNA production from endogenous inverted-repeat (endoIR) loci. This function requires ...[more]

PMID: 28180322

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Project description:BackgroundEndogenous small interfering (esi)RNAs repress mRNA levels and retrotransposon mobility in Drosophila somatic cells by poorly understood mechanisms. 21 nucleotide esiRNAs are primarily generated from retrotransposons and two inverted repeat (hairpin) loci in Drosophila culture cells in a Dicer2 dependent manner. Additionally, proteins involved in 3' end processing, such as Symplekin, CPSF73 and CPSR100, have been recently implicated in the esiRNA pathway.ResultsHere we present evidence of overlap between two essential RNA metabolic pathways: esiRNA biogenesis and mRNA 3' end processing. We have identified a nucleus-specific interaction between the essential esiRNA cleavage enzyme Dicer2 (Dcr2) and Symplekin, a component of the core cleavage complex (CCC) required for 3' end processing of all eukaryotic mRNAs. This interaction is mediated by the N-terminal 271 amino acids of Symplekin; CCC factors CPSF73 and CPSF100 do not contact Dcr2. While Dcr2 binds the CCC, Dcr2 knockdown does not affect mRNA 3' end formation. RNAi-depletion of CCC components Symplekin and CPSF73 causes perturbations in esiRNA abundance that correlate with fluctuations in retrotransposon and hairpin esiRNA precursor levels. We also discovered that esiRNAs generated from retrotransposons and hairpins have distinct physical characteristics including a higher predominance of 22 nucleotide hairpin-derived esiRNAs and differences in 3' and 5' base preference. Additionally, retrotransposon precursors and derived esiRNAs are highly enriched in the nucleus while hairpins and hairpin derived esiRNAs are predominantly cytoplasmic similar to canonical mRNAs. RNAi-depletion of either CPSF73 or Symplekin results in nuclear retention of both hairpin and retrotransposon precursors suggesting that polyadenylation indirectly affects cellular localization of Dcr2 substrates.ConclusionsTogether, these observations support a novel mechanism in which differences in localization of esiRNA precursors impacts esiRNA biogenesis. Hairpin-derived esiRNAs are generated in the cytoplasm independent of Dcr2-Symplekin interactions, while retrotransposons are processed in the nucleus.

Project description:DNA replication errors are a major driver of evolutionâ??from single nucleotide polymorphisms to large-scale copy number variations (CNVs). Here we test a specific replication-based model to explain the generation of interstitial, inverted triplications. While no genetic information is lost, the novel inversion junctions and increased copy number of the included sequences create the potential for adaptive phenotypes. The modelâ??Origin-Dependent Inverted-Repeat Amplification (ODIRA)â??proposes that a replication error at pre-existing short, interrupted, inverted repeats in genomic sequences generates an extrachromosomal, inverted dimeric, autonomously replicating intermediate; subsequent genomic integration of the dimer yields this class of CNV without loss of distal chromosomal sequences. We used a combination of in vitro and in vivo approaches to test the feasibility of the proposed replication error and its downstream consequences on chromosome structure in the yeast Saccharomyces cerevisiae. We show that the proposed replication errorâ??the ligation of leading and lagging nascent strands to create a "closed" forkâ??can occur in vitro at short, interrupted inverted repeats. The removal of molecules with closed forks results in a hairpin-capped linear duplex that we show replicates in vivo to create an inverted, dimeric plasmid that subsequently integrates into the genome by homologous recombination, creating an inverted triplication. While other models have been proposed to explain inverted triplications and their derivatives, our model can also explain the generation of human, de novo, inverted amplicons that have a 2:1 mixture of sequences from both homologues of a single parentâ??a feature readily explained by a plasmid intermediate that arises from one homologue and integrates into the other homolog prior to meiosis. Our tests of key features of ODIRA lend support to this mechanism and suggest further avenues of enquiry to unravel the origins of interstitial, inverted CNVs pivotal in human health and evolution These are all CGH arrays comparing DNA copy number between evolved yeast strains and a euploid wt strain.

Project description:DNA replication errors are a major driver of evolution—from single nucleotide polymorphisms to large-scale copy number variations (CNVs). Here we test a specific replication-based model to explain the generation of interstitial, inverted triplications. While no genetic information is lost, the novel inversion junctions and increased copy number of the included sequences create the potential for adaptive phenotypes. The model—Origin-Dependent Inverted-Repeat Amplification (ODIRA)—proposes that a replication error at pre-existing short, interrupted, inverted repeats in genomic sequences generates an extrachromosomal, inverted dimeric, autonomously replicating intermediate; subsequent genomic integration of the dimer yields this class of CNV without loss of distal chromosomal sequences. We used a combination of in vitro and in vivo approaches to test the feasibility of the proposed replication error and its downstream consequences on chromosome structure in the yeast Saccharomyces cerevisiae. We show that the proposed replication error—the ligation of leading and lagging nascent strands to create “closed” forks—can occur in vitro at short, interrupted inverted repeats. The removal of molecules with two closed forks results in a hairpin-capped linear duplex that we show replicates in vivo to create an inverted, dimeric plasmid that subsequently integrates into the genome by homologous recombination, creating an inverted triplication. While other models have been proposed to explain inverted triplications and their derivatives, our model can also explain the generation of human, de novo, inverted amplicons that have a 2:1 mixture of sequences from both homologues of a single parent—a feature readily explained by a plasmid intermediate that arises from one homologue and integrates into the other homologue prior to meiosis. Our tests of key features of ODIRA lend support to this mechanism and suggest further avenues of enquiry to unravel the origins of interstitial, inverted CNVs pivotal in human health and evolution.

Project description:Next-generation Illumina sequencing technology was used to analyze small RNA associated with post-transcriptional gene silencing induced by intron-spliced hairpin RNA (ihpRNA) in Arabidopsis. The experimental induction of RNA silencing in plants often involves expression of transgenes encoding inverted repeat (IR) sequences to produce abundant dsRNAs that are processed into small RNAs (sRNAs). These sRNAs are key mediators of post-transcriptional gene silencing (PTGS) and determine the specificity of the inhibition of gene expression. Despite its broad utility as a research tool, IR-PTGS is only a partially understood mechanism of RNA silencing in plants. We generated four sets of 60 Arabidopsis plants, each containing IR transgenes expressing different configurations of uidA and CHALCONE SYNTHASE (CHS) gene fragments. The levels of PTGS were dependent on the orientation and position of the fragment in the IR construct. To investigate these differences, we characterized the sRNA profiles by Illumina sequencing of seven libraries generated from transgenic families showing different levels of IR-PTGS. Mapping of sRNA sequences to their corresponding transgene-derived and endogenous transcripts identified distinctive patterns of differential sRNA accumulation. Analyses of these patterns and peaks revealed similarities among sRNAs associated with IR-PTGS and endogenous sRNAs linked to uncapped mRNA decay. We also found unexpected associations between sRNA accumulation and the presence of predicted open reading frames in the trigger sequence. Our observations provide new guidelines for designing constructs to increase the efficiency of IR-PTGS. In addition, strong IR-PTGS affected the prevalence of endogenous sRNAs, which has implications for the use of PTGS for experimental or applied purposes. Sequencing of small RNA from Arabidopsis plants transformed with ihpRNA constructs. Seven small RNA libraries were sequenced: Lib 0, made from non-transgenic Arabidopsis plants, and Libs 1-6, made from plants transformed with different configurations of ihpRNA.

Dataset Information

A specific dsRNA-binding protein complex selectively sequesters endogenous inverted-repeat siRNA precursors and inhibits their processing.

Publications

A specific dsRNA-binding protein complex selectively sequesters endogenous inverted-repeat siRNA precursors and inhibits their processing.

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