Novel, abundant Drosha isoforms are deficient in miRNA processing in cancer cells
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ABSTRACT:
PROVIDER: PRJNA599131 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR10832427_subreads.fastq.gz | Fastqsanger.gz | |||
| SRR10832428_subreads.fastq.gz | Fastqsanger.gz | |||
| SRR10832429_subreads.fastq.gz | Fastqsanger.gz | |||
| SRR10832430_subreads.fastq.gz | Fastqsanger.gz | |||
| SRR10832431_subreads.fastq.gz | Fastqsanger.gz |
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Project description:MicroRNAs (miRNAs) are a class of small noncoding RNAs about 22-nucleotide (nt) in length that collectively regulate more than 60% of coding genes. Aberrant miRNA expression is associated with numerous diseases, including cancer. miRNA biogenesis is licensed by the ribonuclease (RNase) III enzyme Drosha, the regulation of which is critical in determining miRNA levels. We and others have previously revealed that alternative splicing regulates the subcellular localization of Drosha. To further investigate the alternative splicing landscape of Drosha transcripts, we performed PacBio sequencing in different human cell lines. We identified two novel isoforms resulting from partial intron-retention in the region encoding the Drosha catalytic domain. One isoform (AS27a) generates a truncated protein that is unstable in cells. The other (AS32a) produces a full-length Drosha with a 14 amino acid insertion in the RIIID domain. By taking advantage of Drosha knockout cells in combination with a previously established reporter assay, we demonstrated that Drosha-AS32a lacks cleavage activity. Furthermore, neither Drosha-27a nor Drosha-32a were able to rescue miRNA expression in the Drosha knockout cells. Interestingly, both isoforms were abundantly detected in a wide range of cancer cell lines (up to 15% of all Drosha isoforms). Analysis of the RNA-seq data from over 1000 breast cancer patient samples revealed that the AS32a is relatively more abundant in tumors than in normal tissue, suggesting that AS32a may play a role in cancer development.
2022-01-01 | GSE143169 | GEO
Project description:MicroRNAs (miRNAs) are a class of small noncoding RNAs about 22-nucleotide (nt) in length that collectively regulate more than 60% of coding genes. Aberrant miRNA expression is associated with numerous diseases, including cancer. miRNA biogenesis is licenced by the ribonuclease (RNase) III enzyme Drosha, the regulation of which is critical in determining miRNA levels. We and others have previously revealed that alternative splicing regulates the subcellular localization of Drosha. To further investigate the alternative splicing landscape of Drosha transcripts, we performed PacBio sequencing in different human cell lines. We identified two novel isoforms resulting from partial intron-retention in the region encoding the Drosha catalytic domain. One isoform (AS27a) generates a truncated protein that is unstable in cells. The other (AS32a) produces a full-length Drosha with a 14 amino acid insertion in the RIIID domain. By taking advantage of Drosha knockout cells in combination with a previously established reporter assay, we demonstrated that Drosha-AS32a lacks cleavage activity. Furthermore, neither Drosha-27a nor Drosha-32a were able to rescue miRNA expression in the Drosha knockout cells. Interestingly, both isoforms were abundantly detected in a wide range of cancer cell lines (up to 15% of all Drosha isoforms). Analysis of the RNA-seq data from over 1000 breast cancer patient samples revealed that the AS32a is relatively more abundant in tumours than in normal tissue, suggesting that AS32a may play a role in cancer development.
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Project description:The in vitro high-throughput human pri-miRNA processing assays were conducted to check whether mismatches and wobble base pairs in the upper stem of pri-miRNAs affects the DROSHA cleavage.
2020-02-28 | GSE142140 | GEO
Project description:High-throughput in vitro DROSHA processing on artificial variants
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Project description:DROSHA serves as a gatekeeper of the microRNA (miRNA) pathway by processing primary transcripts (pri-miRNAs). While the functions of structured domains of DROSHA have been well-documented, the contribution of N-terminal proline-rich disordered domain (PRD) remains elusive. Here we show that the PRD promotes the processing of miRNA hairpins located within introns. We identified a DROSHA isoform (p140) lacking the PRD, which is produced by proteolytic cleavage. Small RNA sequencing revealed that p140 is significantly impaired in the maturation of intronic miRNAs. Consistently, our minigene constructs demonstrated that PRD enhances the processing of intronic hairpins, but not those in exons. Splice site mutations did not affect the PRD’s enhancing effect on intronic constructs, suggesting that the PRD acts independently of splicing reaction by interacting with sequences residing within introns. The N-terminal regions from zebrafish and Xenopus DROSHA can replace the human counterpart, indicating functional conservation despite poor sequence alignment. Moreover, we found that rapidly evolving intronic miRNAs are generally more dependent on PRD than conserved ones, suggesting a role of PRD in miRNA evolution. Our study reveals a new layer of miRNA regulation mediated by a low-complexity disordered domain that senses the genomic contexts of miRNA loci.
2023-05-28 | GSE230544 | GEO
Project description:We asked whether the human drosha protein, an RNase III homolog known to process microRNAs (miRNAs), might also be a small nuclear RNA (snRNA) 3' processing factor. Using retroviral siRNA silencing constructs, we stably knocked down drosha protein to nearly undetectable levels. Knockdown cells exhibited reduced growth rates and viability compared to controls, but no accumulation of unprocessed U2 snRNA precursors. In fungi, RNase III homologs process rRNA precursors and certain mRNAs. Although rRNA processing appears to be normal in the drosha knockdown cells, expression microarray analysis revealed misregulation of several mRNAs involved in cell growth and proliferation. Curiously, drosha knockdown appeared to downregulate the predicted mRNA targets of several miRNAs Keywords: siRNA knockdown
2008-01-01 | GSE6767 | GEO
Project description:Using a protein interaction screen, we identify the Microprocessor component Drosha as a novel Dnmt1-interactor. Drosha-deficient ES cells display genomic hypomethylation which is not accounted for by changes in the levels of Dnmt proteins. Both genetic and transfection studies show that Drosha stimulates Dnmt1 methyltransferase activity. We identify two transcripts that are specifically upregulated in Drosha but not Dicer-deficient ES cells. Regions within these transcripts predicted to form stem-loop structures are processed by Microprocessor and can inhibit DNMT1-mediated methylation in vitro. Our results highlight Drosha as a novel regulator of mammalian DNA methylation and we propose that Drosha-mediated processing of RNA is necessary to ensure full Dnmt1 activity. This adds to the Drosha repertoire of non-miRNA dependent functions as well as implicating RNA in regulating Dnmt1 activity and correct levels of genomic methylation.
2017-07-27 | GSE86907 | GEO
Project description:We asked whether the human drosha protein, an RNase III homolog known to process microRNAs (miRNAs), might also be a small nuclear RNA (snRNA) 3' processing factor. Using retroviral siRNA silencing constructs, we stably knocked down drosha protein to nearly undetectable levels. Knockdown cells exhibited reduced growth rates and viability compared to controls, but no accumulation of unprocessed U2 snRNA precursors. In fungi, RNase III homologs process rRNA precursors and certain mRNAs. Although rRNA processing appears to be normal in the drosha knockdown cells, expression microarray analysis revealed misregulation of several mRNAs involved in cell growth and proliferation. Curiously, drosha knockdown appeared to downregulate the predicted mRNA targets of several miRNAs Experiment Overall Design: The experimental goal was to evaluate gene expression changes induced by siRNA knockdown of drosha. Four samples of HeLa cells were transfected with retroviral siRNA expression vectors: two replicates of an anti-GFP siRNA vector (siGFP) and two different anti-drosha siRNA vectors (sidroshaB and sidroshaC). Cells were selected with puromycin 24 hours after transfection and harvested 72 hours after transfection. Trizol-harvested RNA was processed with standard Affymetrix protocols and hybridized to U133Plus2.0 GeneChips. Signals were scaled to an arbitrary global mean value of 800.
2008-06-15 | E-GEOD-6767 | biostudies-arrayexpress
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