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Microprocessor mediates transcription termination in long noncoding microRNA genes

ABSTRACT: MicroRNA (miRNA) play a major role in the post-transcriptional regulation of gene expression. In mammals most miRNA derive from the introns of protein coding genes where they exist as hairpin structures in the primary gene transcript, synthesized by RNA polymerase II (Pol II). These are cleaved co-transcriptionally by the Microprocessor complex, comprising DGCR8 and the RNase III endonuclease Drosha, to release the precursor (pre-)miRNA hairpin, so generating both miRNA and spliced messenger RNA1-4. However, a substantial minority of miRNA originate from Pol II-synthesized long non coding (lnc) RNA where transcript processing is largely uncharacterized5. Here, we show that most lnc-pri-miRNA do not use the canonical cleavage and polyadenylation (CPA) transcription termination pathway6, but instead use Microprocessor cleavage both to release pre-miRNA and terminate transcription. We present a detailed characterization of one such lnc-pri-miRNA that generates the highly expressed liver-specific miR-1227. Genome-wide analysis then reveals that Microprocessor-mediated transcription termination is commonly used by lnc-pri-miRNA but not by protein coding miRNA genes. This identifies a fundamental difference between lncRNA and pre-mRNA processing. Remarkably, inactivation of the Microprocessor can lead to extensive transcriptional readthrough of lnc-pri-miRNA, resulting in inhibition of downstream genes by transcriptional interference. Consequently we define a novel RNase III-mediated, polyadenylation-independent mechanism of Pol II transcription termination in mammalian cells.

ORGANISM(S): Homo sapiens

PROVIDER: GSE58838 | GEO | 2015/03/02

SECONDARY ACCESSION(S): PRJNA253670

REPOSITORIES: GEO

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Microprocessor mediates transcription termination in long noncoding microRNA genes

Project description:MicroRNA (miRNA) play a major role in the post-transcriptional regulation of gene expression. In mammals most miRNA derive from the introns of protein coding genes where they exist as hairpin structures in the primary gene transcript, synthesized by RNA polymerase II (Pol II). These are cleaved co-transcriptionally by the Microprocessor complex, comprising DGCR8 and the RNase III endonuclease Drosha, to release the precursor (pre-)miRNA hairpin, so generating both miRNA and spliced messenger RNA1-4. However, a substantial minority of miRNA originate from Pol II-synthesized long non coding (lnc) RNA where transcript processing is largely uncharacterized5. Here, we show that most lnc-pri-miRNA do not use the canonical cleavage and polyadenylation (CPA) transcription termination pathway6, but instead use Microprocessor cleavage both to release pre-miRNA and terminate transcription. We present a detailed characterization of one such lnc-pri-miRNA that generates the highly expressed liver-specific miR-1227. Genome-wide analysis then reveals that Microprocessor-mediated transcription termination is commonly used by lnc-pri-miRNA but not by protein coding miRNA genes. This identifies a fundamental difference between lncRNA and pre-mRNA processing. Remarkably, inactivation of the Microprocessor can lead to extensive transcriptional readthrough of lnc-pri-miRNA, resulting in inhibition of downstream genes by transcriptional interference. Consequently we define a novel RNase III-mediated, polyadenylation-independent mechanism of Pol II transcription termination in mammalian cells. Chromatin associated RNA-seq from sicntrl,siDrosha,siDGCR8 treated Hela cells. Same for sicntrl and siDGCR8 from Huh7 cells. Nuclear polyA + and polyA- RNA-seq from sicntrl and siDGCR8 in HeLa cells. Chromatin associated RNA-seq from siDicer treated Hela cells.

2015-03-02 | E-GEOD-58838 | biostudies-arrayexpress

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Project description:Microprocessor is responsible for conversion of pri-miRNA transcripts into pre-miRNA hairpins in miRNA biogenesis. The in vivo properties of this process remain enigmatic. Here, we present the first study of in vivo transcriptome-wide pri-miRNA processing using next-generation sequencing of chromatin-associated pri-miRNAs. We identify a distinctive Microprocessor signature in the transcriptome profile, from which efficiency of the endogenous processing event can be accurately quantified. This analysis reveals differential susceptibility to Microprocessor cleavage as a key regulatory step in miRNA biogenesis. Processing is highly variable among pri-miRNAs and a better predictor of miRNA abundance than primary transcription itself. Processing is also largely stable across three cell lines, suggesting a major contribution of sequence determinants. Based on differential processing efficiencies we define functionality for short sequence features adjacent to the pre-miRNA hairpin. In conclusion, we identify Microprocessor as the main hub for diversified miRNA output and suggest a role for uncoupling miRNA biogenesis from host gene expression.

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TGF-beta-upregulated Lnc-Nr6a1 acts as a reservoir of miR-181 and mediates assembly of a glycolytic complex [ChIP-seq]

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TGF-beta-upregulated Lnc-Nr6a1 acts as a reservoir of miR-181 and mediates assembly of a glycolytic complex [RNA-seq]

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Project description:MicroRNAs (miRNAs) are small RNAs that regulate gene expression. miRNAs are produced from primary miRNAs (pri-miRNAs), the cleavage of which is catalyzed by the Microprocessor complex. Microprocessor therefore plays a key role in determining the efficiency and precision of miRNA production, and thus the function of the final miRNA product. In this study, we utilized high-throughput sequencing-integrated enzymology with purified Microprocessor proteins and randomized pri-miRNA sequences to investigate the catalytic mechanism of Microprocessor. We identified multiple mismatches and wobble base pairs in the upper stem of pri-miRNAs, which determine the efficiency and accuracy of pri-miRNA processing. The existence of these RNA elements helps to explain the alternative cleavage mechanism of Microprocessor, which occurs for some human pri-miRNAs. We also showed that these RNA elements are targets of RNA-editing or single nucleotide polymorphisms (SNPs) for regulating miRNA biogenesis. These findings considerably improve our understanding of pri-miRNA processing mechanisms, and provide a foundation for interpreting differential miRNA expression by several mechanisms, such as RNA modifications and SNPs.

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