Role of the proline-rich disordered domain of DROSHA in intronic microRNA processing
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA961731 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR24302008.fastq.gz | Fastqsanger.gz | |||
| SRR24302009.fastq.gz | Fastqsanger.gz | |||
| SRR24302010.fastq.gz | Fastqsanger.gz | |||
| SRR24302011.fastq.gz | Fastqsanger.gz | |||
| SRR24302012.fastq.gz | Fastqsanger.gz |
Items per page: 5 1 - 5 of 20 |
Similar Datasets
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: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.
| S-EPMC10270192 | biostudies-literature
Project description:MicroRNAs (miRNAs) are approximately 22-nucleotide endogenous RNAs that often repress the expression of complementary messenger RNAs. In animals, miRNAs derive from characteristic hairpins in primary transcripts through two sequential RNase III-mediated cleavages; Drosha cleaves near the base of the stem to liberate a approximately 60-nucleotide pre-miRNA hairpin, then Dicer cleaves near the loop to generate a miRNA:miRNA* duplex. From that duplex, the mature miRNA is incorporated into the silencing complex. Here we identify an alternative pathway for miRNA biogenesis, in which certain debranched introns mimic the structural features of pre-miRNAs to enter the miRNA-processing pathway without Drosha-mediated cleavage. We call these pre-miRNAs/introns 'mirtrons', and have identified 14 mirtrons in Drosophila melanogaster and another four in Caenorhabditis elegans (including the reclassification of mir-62). Some of these have been selectively maintained during evolution with patterns of sequence conservation suggesting important regulatory functions in the animal. The abundance of introns comparable in size to pre-miRNAs appears to have created a context favourable for the emergence of mirtrons in flies and nematodes. This suggests that other lineages with many similarly sized introns probably also have mirtrons, and that the mirtron pathway could have provided an early avenue for the emergence of miRNAs before the advent of Drosha.
| S-EPMC2475599 | biostudies-literature
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: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: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: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:Human basic proline-rich proteins and basic glycosylated proline-rich proteins are the most complex family of human adult salivary proteins, secreted only by the parotid gland, expressed by the polymorphic PRB1-4 genes. This study reports the results obtained for their characterization in our laboratory utilizing integrated top-down and bottom-up HPLC-ESI-MS and MS/MS platform. The family include 11 parent peptides/proteins and more than 6 parent glycosylated proteins. Despite the complexity, on the basis of their primary structure they result the product of the duplication of three repeats and we propose that they can be classified in two main groups, the first including the peptides called P-E, P-Ko, IB-6, Ps-1, Ps-2, P-H, P-F, P-J, and P-D, the second including the peptides called IB-1, II-2 and the glycosylated basic proline-rich proteins codified by PRB3 and PRB4 genes. A third minor hybrid group includes the proteins called IB-8a Con1- and Con1+. Some of the proteins of the family (IB-1, II-2 and P-J) are almost always detectable in adult human saliva, while the others are expressed with different frequencies according to the phenotype. Various isoforms are detectable in the adult saliva and three of them, until now not reported (P-H S1→A, P-Ko P36→S, P-Ko A41→S), were characterized by MS/MS fragmentation.
2018-08-08 | PXD009813 | Pride
Project description:High-throughput in vitro DROSHA processing on artificial variants
| PRJNA544246 | ENA
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.
2024-06-03 | PXD017677 | Pride