Single cut products of Microprocessor on pri-miRNAs
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ORGANISM(S): synthetic construct
PROVIDER: GSE138950 | GEO | 2020/01/24
REPOSITORIES: GEO
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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.
2015-03-02 | GSE58838 | GEO
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
Project description:Anaysis of mRNA changes in HeLa cells following knockdown of Drosha or DGCR8. Drosha is a nuclear RNase III that carries out microRNA (miRNA) processing by cleaving primary microRNA transcript (pri-miRNA). DGCR8 is an essential co-factor of Drosha. Keywords: gene expression array-based (RNA / in situ oligonucleotide)
2009-02-05 | GSE13639 | GEO
Project description:Microprocessor, which consists of a ribonuclease III DROSHA and its cofactor DGCR8, initiates microRNA (miRNA) maturation by cleaving primary miRNA transcripts (pri-miRNAs). We recently demonstrated that the DGCR8 dimer recognizes the apical elements of pri-miRNAs, including the UGU motif, to accurately locate and orient Microprocessor on pri-miRNAs. However, the mechanism underlying the selective RNA binding remains unknown. In this study, we find that hemin, a ferric ion-containing porphyrin, enhances the specific interaction between the apical UGU motif and the DGCR8 dimer, allowing Microprocessor to achieve high efficiency and fidelity of pri-miRNA processing in vitro. Furthermore, by generating a DGCR8 mutant cell line and carrying out rescue experiments, we discover that hemin preferentially stimulates the expression of miRNAs possessing the UGU motif, thereby conferring differential regulation of miRNA maturation. Our findings reveal the molecular action mechanism of hemin in pri-miRNA processing and establish a novel function of hemin in inducing specific RNA-protein interaction.
2018-03-26 | GSE111431 | GEO
Project description:Anaysis of mRNA changes in HeLa cells following knockdown of Drosha or DGCR8. Drosha is a nuclear RNase III that carries out microRNA (miRNA) processing by cleaving primary microRNA transcript (pri-miRNA). DGCR8 is an essential co-factor of Drosha. Experiment Overall Design: siRNA against Drosha or DGCR8 were trasnfected into HeLa cells. siRNA against GFP was used as a control. Biologically duplicated total RNAs were prepared from HeLa cells, 24 hrs and 48 hrs after siRNA transfection.
2009-02-21 | E-GEOD-13639 | biostudies-arrayexpress
Project description:Maturation of canonical microRNA (miRNA) is initiated by DROSHA that cleaves the primary transcript (pri-miRNA). Over 1,800 miRNA loci are annotated in humans, but it remains largely unknown if and at which sites the pri-miRNAs are cleaved by DROSHA. Here we performed in vitro processing on a full set of human pri-miRNAs (miRBase v21) followed by sequencing. This comprehensive profiling enabled us to classify miRNAs based on DROSHA-dependence and map their cleavage sites with respective processing efficiency measures. Only 758 pri-miRNAs are confidently processed by DROSHA, while the majority may be non-canonical or false entries. Analyses of the DROSHA-dependent pri-miRNAs show key cis-elements for processing. We observe widespread alternative processing as well as unproductive cleavage events such as “nick” or “inverse” processing. SRSF3 is a broad-acting auxiliary factor modulating alternative processing and suppressing unproductive processing. The profiling data and methods developed in this study will allow systematic analyses of miRNA regulation.
2021-07-28 | PXD025767 | Pride
Project description:Human Microprocessor is a trimeric complex composed of the RNase III enzyme, DROSHA, and a dimer of DGCR8 (its cofactor). Microprocessor initiates the biogenesis of microRNA by processing primary microRNAs (pri-miRNAs), during which, its cleavage efficiency and accuracy are enhanced because DGCR8 interacts with the apical UGU motif of pri-miRNAs. However, the mechanism of DGCR8-UGU interaction and influence of this interaction on the expression of cellular miRNA are still elusive. In this study, we demonstrated that the Rhed domain (i.e., the RNA-binding heme domain, amino acids 285-478) of DGCR8 is sufficient to recognize and interact with UGU. In addition, we identified three amino acid residues in Rhed (amino acids 361-363), which are critical for the UGU interaction, and we showed that these residues are essential for the Microprocessor complex to accurately and efficiently process pri-miRNAs in vitro. Furthermore, we found that within the DGCR8 dimer, the UGU-binding site from just one monomer is capable of discriminating between UGU- and non-UGU-containing pri-miRNAs. Finally, we showed that these amino acids are more important for the expression of UGU miRNAs than non-UGU miRNAs in human cells. This study improves our understanding of the substrate-recognizing mechanism of DGCR8, and implicates the roles of this recognition in differentiating miRNA expression in human cells.
2020-07-08 | GSE140209 | GEO
Project description:MicroRNA (miRNA) maturation is initiated by DROSHA, a double-stranded RNA (dsRNA)-specific RNase III enzyme. By cleaving primary miRNAs (pri-miRNAs) at specific positions, DROSHA serves as a main determinant of miRNA sequences and a highly selective gate-keeper for the canonical miRNA pathway. However, the sites of DROSHA-mediated processing have not been annotated on a genomic scale, and it remains unclear to what extent DROSHA functions outside the miRNA pathway. Here we establish a protocol termed ‘formaldehyde crosslinking and immunoprecipitation followed by sequencing (fCLIP-seq)’ that allows identification of DROSHA cleavage sites at single nucleotide resolution. fCLIP identifies numerous cleavage sites that do not match the ends of annotated mature miRNAs, particularly at the 3′ termini, suggesting widespread end modifications during miRNA maturation such as trimming and tailing. fCLIP also finds many pri-miRNAs undergoing alternative processing, yielding multiple miRNA isoforms. Moreover, we discover dozens of novel substrates that are bound and cleaved by DROSHA. These substrates are processed less efficiently than canonical pri-miRNAs, and produce only minute amounts of small RNAs. Depletion of DROSHA results in an increase of the hairpin-containing transcripts. Thus, the hairpins may serve mainly as cis-elements for DROSHA-mediated gene regulation rather than as miRNA genes. fCLIP-seq not only accurately maps the cleavage sites of DROSHA and suggests noncanonical functions of DROSHA, but also could be a general tool for investigating interactions between dsRNA binding proteins and structured RNAs.
2017-04-23 | GSE93651 | GEO
Project description:MicroRNA (miRNA) maturation is initiated by DROSHA, a double-stranded RNA (dsRNA)-specific RNase III enzyme. By cleaving primary miRNAs (pri-miRNAs) at specific positions, DROSHA serves as a main determinant of miRNA sequences and a highly selective gate-keeper for the canonical miRNA pathway. However, the sites of DROSHA-mediated processing have not been annotated on a genomic scale, and it remains unclear to what extent DROSHA functions outside the miRNA pathway. Here we establish a protocol termed ‘formaldehyde crosslinking and immunoprecipitation followed by sequencing (fCLIP-seq)’ that allows identification of DROSHA cleavage sites at single nucleotide resolution. fCLIP identifies numerous cleavage sites that do not match the ends of annotated mature miRNAs, particularly at the 3′ termini, suggesting widespread end modifications during miRNA maturation such as trimming and tailing. fCLIP also finds many pri-miRNAs undergoing alternative processing, yielding multiple miRNA isoforms. Moreover, we discover dozens of novel substrates that are bound and cleaved by DROSHA. These substrates are processed less efficiently than canonical pri-miRNAs, and produce only minute amounts of small RNAs. Depletion of DROSHA results in an increase of the hairpin-containing transcripts. Thus, the hairpins may serve mainly as cis-elements for DROSHA-mediated gene regulation rather than as miRNA genes. fCLIP-seq not only accurately maps the cleavage sites of DROSHA and suggests noncanonical functions of DROSHA, but also could be a general tool for investigating interactions between dsRNA binding proteins and structured RNAs.
2017-04-23 | GSE93650 | GEO
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
2008-12-25 | GSE14215 | GEO