Diverse endonucleolytic cleavage sites in the mammalian transcriptome depend upon microRNAs, Drosha, and additional nucleases
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ABSTRACT:
PROVIDER: PRJNA127179 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR052657.fastq.gz | Fastqsanger.gz | |||
| SRR052658.fastq.gz | Fastqsanger.gz | |||
| SRR052659.fastq.gz | Fastqsanger.gz | |||
| SRR052660.fastq.gz | Fastqsanger.gz | |||
| SRR052661.fastq.gz | Fastqsanger.gz |
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Project description:Messenger RNAs are regulated by a variety of degradation mechanisms in mammalian cells. In the canonical animal microRNA pathway, microRNAs in complex with Argonaute proteins bind to many mRNA targets with imperfect complementarity, leading to degradation of the mRNA through the regular decay machinery. The ancestral “slicer” endonuclease activity of Argonaute2 itself, which requires more extensive complementarity with the target RNA, is not used in this pathway, and has only been observed in two microRNA-guided cases. Nevertheless, the cleavage capacity of mammalian Ago2 is conserved and essential for viability. Here, we assess the endonucleolytic function of Ago2 and other nucleases by identifying cleavage products retaining 5`-phosphate groups in mouse ES cells on a transcriptome-wide scale. We detect a significant signature of Ago2-dependent cleavage events and validate several targets. Unexpectedly, a broader class of Ago2-independent cleavage sites is also observed, indicating participation of additional nucleases in this mode of mRNA regulation. Within this class, we identify a cohort of Drosha-dependent mRNA cleavage events, including one in the Dgcr8 mRNA, that functionally regulate mRNA levels in mES cells. Together, these results highlight the underappreciated role of endonucleolytic cleavage in controlling mRNA fates in mammals. Global 5`-phosphate-dependent RACE in WT, Ago2-KO and Drosha-excised mouse ES cells and human 293S cells
2010-06-25 | E-GEOD-21975 | biostudies-arrayexpress
Project description:Messenger RNAs are regulated by a variety of degradation mechanisms in mammalian cells. In the canonical animal microRNA pathway, microRNAs in complex with Argonaute proteins bind to many mRNA targets with imperfect complementarity, leading to degradation of the mRNA through the regular decay machinery. The ancestral “slicer” endonuclease activity of Argonaute2 itself, which requires more extensive complementarity with the target RNA, is not used in this pathway, and has only been observed in two microRNA-guided cases. Nevertheless, the cleavage capacity of mammalian Ago2 is conserved and essential for viability. Here, we assess the endonucleolytic function of Ago2 and other nucleases by identifying cleavage products retaining 5`-phosphate groups in mouse ES cells on a transcriptome-wide scale. We detect a significant signature of Ago2-dependent cleavage events and validate several targets. Unexpectedly, a broader class of Ago2-independent cleavage sites is also observed, indicating participation of additional nucleases in this mode of mRNA regulation. Within this class, we identify a cohort of Drosha-dependent mRNA cleavage events, including one in the Dgcr8 mRNA, that functionally regulate mRNA levels in mES cells. Together, these results highlight the underappreciated role of endonucleolytic cleavage in controlling mRNA fates in mammals.
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Project description:The life span of a mammalian mRNA is determined, in part, by the binding of regulatory proteins and small RNA-guided complexes. The conserved endonuclease activity of Argonaute2 requires extensive complementarity between a small RNA and its target and is not used by animal microRNAs, which pair with their targets imperfectly. Here we investigate the endonucleolytic function of Ago2 and other nucleases by transcriptome-wide profiling of mRNA cleavage products retaining 5' phosphate groups in mouse embryonic stem cells (mESCs). We detect a prominent signature of Ago2-dependent cleavage events and validate several such targets. Unexpectedly, a broader class of Ago2-independent cleavage sites is also observed, indicating participation of additional nucleases in site-specific mRNA cleavage. Within this class, we identify a cohort of Drosha-dependent mRNA cleavage events that functionally regulate mRNA levels in mESCs, including one in the Dgcr8 mRNA. Together, these results highlight the underappreciated role of endonucleolytic cleavage in controlling mRNA fates in mammals.
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Project description:Neural stem cell regulation is essential for the formation of the central nervous system and homeostatic neurogenesis in the adult mammalian brain. The RNAseIII Drosha, a key component of the miRNA microprocessor, plays a central role in regulating NSC maintenance partially through a miRNA-independent mechanism. Drosha controls mRNA expression levels by targeting and cleaving evolutionary conserved stem-loop hairpins located in the mRNAs of stem cell-related transcription factors. However, it is unknown how the Drosha-mediated endonucleolytic cleavage of mRNA is regulated. Here, we identify novel Drosha and NFIB interactors in hippocampal NSCs by in vitro pull-down assays followed by Mass Spectrometry. We unravel the RNA binding proteins implicated in Drosha-mediated regulation of neurogenesis and we find Scaffold Attachment Factor B1 to play a novel and essential role in NFIB mRNA regulation during neural stem cell differentiation.
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Project description:Drosha is a type III RNAse, which plays a critical role in miRNA biogenesis. Drosha and its double-stranded RNA-binding partner protein Pasha/DGCR8 likely recognize and cleave miRNA precursor RNAs or pri-miRNA hairpins co-transcriptionally. To identify RNAs processed by Drosha, we used tiling microarrays to examine transcripts after depletion of drosha mRNA with dsRNA in Drosophila Schneider S2 cells. This strategy identified 137 Drosha-regulated RNAs, including 11 putative pri-miRNAs comprising 15 annotated miRNAs. Most of the identified pri-miRNAs seem extremely large, >10 kilobases as revealed by both the Drosha knock down strategy and by RNA PolII chromatin IP followed by Drosophila tiling microarrays. Surprisingly, more than a hundred additional RNAs not annotated as miRNAs are under Drosha control and are likely to be direct targets of Drosha action. This is because many of them encode annotated genes, and unlike bona fide pri-miRNAs, they are not affected by depletion of the miRNA processing factor, dicer-1. Moreover, application of the evofold analysis software indicates that at least 25 of the Drosha-regulated RNAs contain evolutionarily conserved hairpins similar to those recognized by the Drosha-Pasha/DGCR8 complex in pri-miRNAs. One of these hairpins is located in the 5′ UTR of both pasha and mammalian DGCR8. These observations suggest that a negative feedback loop acting on pasha mRNA may regulate the miRNA-biogenesis pathway: i.e., excess Drosha cleaves pasha/DGCR8 primary transcripts and leads to a reduction in pasha/DGCR8 mRNA levels and Pasha/DGCR8 synthesis. Keywords: time course, ChIP-chip
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