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Two distinct mechanisms generate endogenous siRNAs from bidirectional transcription in Drosophila melanogaster.

ABSTRACT: Cis-natural antisense transcripts (cis-NATs) have been speculated to be substrates for endogenous RNA interference (RNAi), but little experimental evidence for such a pathway in animals has been reported. Analysis of massive Drosophila melanogaster small RNA data sets now reveals two mechanisms that yield endogenous small interfering RNAs (siRNAs) via bidirectional transcription. First, >100 cis-NATs with overlapping 3' exons generate 21-nt, and, based on previously published small RNA data [corrected] Dicer-2 (Dcr-2)-dependent, 3'-end modified siRNAs. The processing of cis-NATs by RNA interference (RNAi) seems to be actively restricted, and the selected loci are enriched for nucleic acid-based functions and include Argonaute-2 (AGO2) itself. Second, we report that extended intervals of the thickveins and klarsicht genes generate exceptionally abundant siRNAs from both strands. These siRNA clusters derive from atypical cis-NAT arrangements involving introns and 5' or internal exons, but their biogenesis is similarly Dcr-2- and AGO2-dependent. These newly recognized siRNA pathways broaden the scope of regulatory networks mediated by small RNAs.

SUBMITTER: Okamura K

PROVIDER: S-EPMC2713754 | biostudies-literature | 2008 Jun

REPOSITORIES: biostudies-literature

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Two distinct mechanisms generate endogenous siRNAs from bidirectional transcription in Drosophila melanogaster.

Okamura Katsutomo K Balla Sudha S Martin Raquel R Liu Na N Lai Eric C EC

Nature structural & molecular biology 20080525 6

Cis-natural antisense transcripts (cis-NATs) have been speculated to be substrates for endogenous RNA interference (RNAi), but little experimental evidence for such a pathway in animals has been reported. Analysis of massive Drosophila melanogaster small RNA data sets now reveals two mechanisms that yield endogenous small interfering RNAs (siRNAs) via bidirectional transcription. First, >100 cis-NATs with overlapping 3' exons generate 21-nt, and, based on previously published small RNA data [cor ...[more]

PMID: 18500351

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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:Genome-wide pervasive transcription has been reported in many eukaryotic organisms, revealing a highly interleaved transcriptome organization that involves hundreds of previously unknown non-coding RNAs. These recently identified transcripts either exist stably in cells (stable unannotated transcripts, SUTs) or are rapidly degraded by the RNA surveillance pathway (cryptic unstable transcripts, CUTs). One characteristic of pervasive transcription is the extensive overlap of SUTs and CUTs with previously annotated features, which prompts questions regarding how these transcripts are generated, and whether they exert function. Single-gene studies have shown that transcription of SUTs and CUTs can be functional, through mechanisms involving the generated RNAs or their generation itself. So far, a complete transcriptome architecture including SUTs and CUTs has not been described in any organism. Knowledge about the position and genome-wide arrangement of these transcripts will be instrumental in understanding their function. Here we provide a comprehensive analysis of these transcripts in the context of multiple conditions, a mutant of the exosome machinery and different strain backgrounds of Saccharomyces cerevisiae. We show that both SUTs and CUTs display distinct patterns of distribution at specific locations. Most of the newly identified transcripts initiate from nucleosome-free regions (NFRs) associated with the promoters of other transcripts (mostly protein-coding genes), or from NFRs at the 3' ends of protein-coding genes. Likewise, about half of all coding transcripts initiate from NFRs associated with promoters of other transcripts. These data change our view of how a genome is transcribed, indicating that bidirectionality is an inherent feature of promoters. Such an arrangement of divergent and overlapping transcripts may provide a mechanism for local spreading of regulatory signals-that is, coupling the transcriptional regulation of neighbouring genes by means of transcriptional interference or histone modification.

Project description:BackgroundConstant hypoxia (CH) and intermittent hypoxia (IH) occur during several pathological conditions such as asthma and obstructive sleep apnea. Our research is focused on understanding the molecular mechanisms that lead to injury or adaptation to hypoxic stress using Drosophila as a model system. Our current genome-wide study is designed to investigate gene expression changes and identify protective mechanism(s) in D. melanogaster after exposure to severe (1% O(2)) intermittent or constant hypoxia.Methodology/principal findingsOur microarray analysis has identified multiple gene families that are up- or down-regulated in response to acute CH or IH. We observed distinct responses to IH and CH in gene expression that varied in the number of genes and type of gene families. We then studied the role of candidate genes (up-or down-regulated) in hypoxia tolerance (adult survival) for longer periods (CH-7 days, IH-10 days) under severe CH or IH. Heat shock proteins up-regulation (specifically Hsp23 and Hsp70) led to a significant increase in adult survival (as compared to controls) of P-element lines during CH. In contrast, during IH treatment the up-regulation of Mdr49 and l(2)08717 genes (P-element lines) provided survival advantage over controls. This suggests that the increased transcript levels following treatment with either paradigm play an important role in tolerance to severe hypoxia. Furthermore, by over-expressing Hsp70 in specific tissues, we found that up-regulation of Hsp70 in heart and brain play critical role in tolerance to CH in flies.Conclusions/significanceWe observed that the gene expression response to IH or CH is specific and paradigm-dependent. We have identified several genes Hsp23, Hsp70, CG1600, l(2)08717 and Mdr49 that play an important role in hypoxia tolerance whether it is in CH or IH. These data provide further clues about the mechanisms by which IH or CH lead to cell injury and morbidity or adaptation and survival.

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Two distinct mechanisms generate endogenous siRNAs from bidirectional transcription in Drosophila melanogaster.

Publications

Two distinct mechanisms generate endogenous siRNAs from bidirectional transcription in Drosophila melanogaster.

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