A functional connection between the Microprocessor and a variant NEXT complex
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PROVIDER: PRJNA1133631 | ENA |
REPOSITORIES: ENA
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Project description:In mammalian cells, primary miRNAs are cleaved at their hairpin structures by the Microprocessor complex, whose core is composed of DROSHA and DGCR8. Here, we show that 5’flanking regions, resulting from Microprocessor cleavage, are targeted by the RNA exosome in mouse embryonic stem cells (mESCs). This is facilitated by a physical link between DGCR8 and the nuclear exosome targeting (NEXT) component ZCCHC8. Surprisingly, however, both biochemical and mutagenesis studies demonstrate that a variant NEXT complex, containing the RNA helicase MTR4 but devoid of the RNA-binding protein RBM7, is the active entity. This Microprocessor-NEXT variant also targets stem-loop containing RNAs expressed from other genomic regions, such as enhancers. In contrast, Microprocessor does not contribute to the turnover of less structured NEXT substrates. Our results therefore demonstrate that MTR4-ZCCHC8 can link to either RBM7 or DGCR8/DROSHA to target different RNA substrates depending on their structural context.
2025-04-01 | GSE271796 | GEO
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Project description:The human Microprocessor complex cleaves primary microRNA (miRNA) transcripts (pri-miRNAs) to initiate miRNA synthesis. Microprocessor consists of DROSHA (an RNase III enzyme), and DGCR8. DROSHA has two conserved RNase III domains, which make double cuts on each of pri-miRNA strands. In this study, we show that Microprocessor has an unexpected single-cut activity, which creates a single cut on just one of the pri-miRNA strands using one of the two RNase III domains of DROSHA. This cleavage does not lead to the production of miRNA but instead it downregulates miRNA expression. We also demonstrate that certain RNA elements facilitate the single-cut activity of Microprocessor, and by manipulating these elements, we can regulate the ratio of single-cut to double-cut activities, thus controlling miRNA production both in vitro and in vivo.
2020-01-24 | GSE138950 | GEO
Project description:The majority of human microRNAs (miRNAs) are located in the introns of other genes (A. Rodriguez, S. Griffiths-Jones, J. L. Ashurst, and A. Bradley, Genome Res. 14:1902-1910, 2004). Based on the discovery that artificial insertion of pre-miRNAs in introns did not hamper mRNA production and that the miRNA-harboring introns were spliced more slowly than the adjacent introns, a model was previously proposed in which Drosha crops the pre-miRNA and the two cropped fragments from the pre-mRNA are subsequently trans spliced (Y. K. Kim and V. N. Kim, EMBO J. 26:775-783, 2007). However, the molecular basis for this model was not elucidated. To analyze the molecular mechanism of intronic miRNA processing, we developed an in vitro system in which both pre-miRNA processing and mRNA splicing are detected simultaneously. Our analysis using this system showed that pre-miRNA cropping from the pre-mRNA could occur kinetically faster than splicing. Glycerol gradient sedimentation experiments revealed that part of the pre-miRNA was cofractionated with the spliceosome. Furthermore, coimmunoprecipitation experiments with an anti-Drosha antibody demonstrated that Drosha was associated not only with the cropping products but also with a Y-shaped branch intron and a Y-shaped splicing intermediate. These results provide a molecular basis for the postulated existence of a pathway in which the Microprocessor complex becomes associated with the spliceosome, pre-miRNA cropping occurs prior to splicing, and trans splicing takes place between the cropped products.
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Project description:Molecular connection between the TUTase URT1 and decapping activators
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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. 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:The Microprocessor plays an essential role in canonical miRNA biogenesis by facilitating cleavage of stem-loop structures in primary transcripts to yield pre-miRNAs. Although miRNA biogenesis has been extensively studied through biochemical and molecular genetic approaches, it has yet to be addressed to what extent the current miRNA biogenesis models hold true in intact cells. To address the issues of in vivo recognition and cleavage by the Microprocessor, we investigate RNAs that are associated with DGCR8 and Drosha by using immunoprecipitation coupled with next-generation sequencing. Here, we present global protein-RNA interactions with unprecedented sensitivity and specificity. Our data indicate that precursors of canonical miRNAs and miRNA-like hairpins are the major substrates of the Microprocessor. As a result of specific enrichment of nascent cleavage products, we are able to pinpoint the Microprocessor-mediated cleavage sites per se at single-nucleotide resolution. Unexpectedly, a 2-nt 3M-bM-^@M-^Y overhang invariably exists at the ends of cleaved bases instead of nascent pre-miRNAs. Besides canonical miRNA precursors, we find that two novel miRNA-like structures embedded in mRNAs are cleaved to yield pre-miRNA-like hairpins, uncoupled from miRNA maturation. Our data provide a framework for in vivo Microprocessor-mediated cleavage and a foundation for experimental and computational studies on miRNA biogenesis in living cells. CLIP-seq for DGCR8 and Drosha, RIP-seq for DGCR8, sequencing of AGO2-assocated miRNA
2014-10-02 | E-GEOD-61979 | biostudies-arrayexpress
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:The Microprocessor complex, consisting of DROSHA and DGCR8, is essential for miRNA maturation and plays a critical role in gene regulation. Mutations in this complex's components are frequently associated with Wilms tumor (WiT), a common pediatric kidney cancer. Understanding the functional impacts of these mutations is key to elucidating WiT pathogenesis. To this end, we developed an innovative Microsensor system to dynamically evaluate Microprocessor function in human cellular environments. Using this tool, we introduced and analyzed the DGCR8-E518K mutation, previously identified in WiT patients. This mutation was shown to significantly disrupt cellular homeostasis, affecting proliferation, apoptosis, and migration. On a molecular level, we demonstrated that the E518K mutation impairs the Microprocessor's efficiency in processing a specific subset of pri-miRNAs that lack the canonical 16-20 nucleotide mismatch feature, leading to abnormal miRNA expression profiles. Additionally, cells expressing the E518K mutant exhibited increased susceptibility to ferroptosis, as indicated by heightened sensitivity to the pro-ferroptotic agent RSL3. Our findings provide new insights into the Microprocessor's role in WiT, with the Microsensor system offering a robust platform for exploring the molecular mechanisms of Microprocessor-associated mutations. This study lays the groundwork for future in vivo research and the potential development of therapeutic strategies targeting the Microprocessor pathway in WiT.
2025-03-10 | GSE276799 | GEO
Project description:The Microprocessor complex, consisting of DROSHA and DGCR8, is essential for miRNA maturation and plays a critical role in gene regulation. Mutations in this complex's components are frequently associated with Wilms tumor (WiT), a common pediatric kidney cancer. Understanding the functional impacts of these mutations is key to elucidating WiT pathogenesis. To this end, we developed an innovative Microsensor system to dynamically evaluate Microprocessor function in human cellular environments. Using this tool, we introduced and analyzed the DGCR8-E518K mutation, previously identified in WiT patients. This mutation was shown to significantly disrupt cellular homeostasis, affecting proliferation, apoptosis, and migration. On a molecular level, we demonstrated that the E518K mutation impairs the Microprocessor's efficiency in processing a specific subset of pri-miRNAs that lack the canonical 16-20 nucleotide mismatch feature, leading to abnormal miRNA expression profiles. Additionally, cells expressing the E518K mutant exhibited increased susceptibility to ferroptosis, as indicated by heightened sensitivity to the pro-ferroptotic agent RSL3. Our findings provide new insights into the Microprocessor's role in WiT, with the Microsensor system offering a robust platform for exploring the molecular mechanisms of Microprocessor-associated mutations. This study lays the groundwork for future in vivo research and the potential development of therapeutic strategies targeting the Microprocessor pathway in WiT.
2025-03-10 | GSE276798 | GEO