DROSHA complementation experiments using small RNA sequencing
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE117603 | GEO | 2018/12/13
REPOSITORIES: GEO
Similar Datasets
Project description:DROSHA complementation experiments using small RNA sequencing
| PRJNA482632 | ENA
Project description:The experiments were conducted to check whether mismatches and wobble base pairs in the upper stem of pri-miRNAs affects the DROSHA cleavage and thereby miRNA biogenesis.
2020-02-28 | GSE142136 | 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:Regulation of microRNA (miR) biogenesis is complex and stringently controlled. Here we identify the kinase GSK3B as an important modulator of global miR biogenesis at microprocessor level. Repression of GSK3B activity reduces Drosha activity towards pri-miRs, leading to accumulation of unprocessed pri-miRs and reduction of pre-miRs and mature miRs without altering levels or cellular localisationof miR biogenesisproteins. Conversly, GSK3B activation increases Drosha activityand mature miR accumulation. GSK3B achieves this through promoting Drosha:cofactor and Drosha:pri-miR interactions: it binds to DGCR8 and p72 in the Microprocessor, an effect dependent of RNA. Indeed GSK3B itself can immunoprecipitate pri-miRs, suggesting possible RNA binding capacity. Kinase assays identify the mechanism for GSK3B-enhansed Drosha activity, which requires GSK3B nuclear localisation, as phosphorylation of Drosha at s300 and/or s302 confirmed by enhanced Drosha activity and association with cofactors, and increased abundance of mature microRNAs in the presence of phospho-mimetic Drosha. Functional implicatons of GSK3B-enhanced miR biogenesis are illustrated by increased levels of GSK3B-upregulatd miR targets following GSK3B inhibition. These data, the first link GSK3B with the miR cascade in humans, hilight a novel pro-biogenesis role for GSK3B in increasing miR biogenesis as a component of the Microprocessor complex with wide ranging consiquences.
2016-05-20 | GSE81141 | 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
Project description:Biogenesis of canonical microRNAs (miRNAs) involves multiple steps: nuclear processing of primary miRNA (pri-miRNA) by DROSHA, nuclear export of precursor miRNA (pre-miRNA) by Exportin 5 (XPO5), and cytoplasmic processing of pre-miRNA by DICER. To gain a deeper understanding of the contribution of each of these maturation steps, we deleted DROSHA, XPO5, and DICER in the same human cell line, and analyzed their effects on miRNA biogenesis. Canonical miRNA production was completely abolished in DROSHA-deleted cells while we detected a few DROSHA-independent miRNAs including three previously unidentified noncanonical miRNAs (miR-7706, miR-3615, and miR-1254). In contrast to DROSHA knockout, many canonical miRNAs were still detected without DICER albeit at markedly reduced levels. In the absence of DICER, pre-miRNAs are loaded directly onto AGO and trimmed at the 3â² end, yielding miRNAs from the 5â² strand (5p miRNAs). Interestingly, in XPO5 knockout cells, most miRNAs are affected only modestly, suggesting that XPO5 is necessary but not critical for miRNA maturation. Our study demonstrates an essential role of DROSHA and an important contribution of DICER in the canonical miRNA pathway, and reveals that the function of XPO5 can be complemented by alternative mechanisms. Thus, this study allows us to understand differential contributions of key biogenesis factors, and provides with valuable resources for miRNA research. Two independent sequencing experiments (set 1 and set 2, respectively) were performed using 9 samples.
2016-03-17 | E-GEOD-77989 | 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:Biogenesis of canonical microRNAs (miRNAs) involves multiple steps: nuclear processing of primary miRNA (pri-miRNA) by DROSHA, nuclear export of precursor miRNA (pre-miRNA) by Exportin 5 (XPO5), and cytoplasmic processing of pre-miRNA by DICER. To gain a deeper understanding of the contribution of each of these maturation steps, we deleted DROSHA, XPO5, and DICER in the same human cell line, and analyzed their effects on miRNA biogenesis. Canonical miRNA production was completely abolished in DROSHA-deleted cells while we detected a few DROSHA-independent miRNAs including three previously unidentified noncanonical miRNAs (miR-7706, miR-3615, and miR-1254). In contrast to DROSHA knockout, many canonical miRNAs were still detected without DICER albeit at markedly reduced levels. In the absence of DICER, pre-miRNAs are loaded directly onto AGO and trimmed at the 3′ end, yielding miRNAs from the 5′ strand (5p miRNAs). Interestingly, in XPO5 knockout cells, most miRNAs are affected only modestly, suggesting that XPO5 is necessary but not critical for miRNA maturation. Our study demonstrates an essential role of DROSHA and an important contribution of DICER in the canonical miRNA pathway, and reveals that the function of XPO5 can be complemented by alternative mechanisms. Thus, this study allows us to understand differential contributions of key biogenesis factors, and provides with valuable resources for miRNA research.
2016-03-17 | GSE77989 | GEO
Project description:MicroRNA (miRNA) biogenesis is a tightly controlled multi-step process operated in the nucleus by the activity of the Large Drosha Complex (LDC). Through high resolution mass spectrometry (MS) analysis we discovered that the LDC is extensively methylated, with 82 distinct methylated sites associated to 16 out of 23 subunits of the LDC. The majority of these modifications occurs on arginine (R)- residues (61), leading to 86 methylation events, while 29 lysine (K)-methylation events occurs on 21 sites of the complex. Interestingly, the depletion and pharmacological inhibition of PRMT1 lead to a widespread alteration of the methylation state of the complex and induce global decrease of miRNA expression, as a consequence of the specific impairment of the pri-to-pre-miRNA processing step. In particular, we show that the reduced methylation of the ILF3 subunit of the complex is linked to its diminished binding to the target pri-miRNAs. Overall, our study uncovers a previously uncharacterized role of R-methylation in the regulation of the LDC activity in mammalian cells, thus effecting global miRNA levels.
2019-10-23 | PXD011617 | Pride
Project description:We report the ability of the Drosha null/conditional-null mouse model to enable the identification of pri-miRNA transcripts. The conditional-null allele of Drosha phenocopies the null allele both in mESC and in mice, upon conversion to the null state with Cre. Examination of the effects of Drosha deficiency in mouse embryonic stem cells.
2015-02-09 | E-GEOD-55735 | biostudies-arrayexpress