Genome-wide mapping of DROSHA cleavage sites on primary microRNAs and novel substrates
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA361461 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR5179100_1.fastq.gz | Fastqsanger.gz | |||
| SRR5179100_2.fastq.gz | Fastqsanger.gz | |||
| SRR5179101_1.fastq.gz | Fastqsanger.gz | |||
| SRR5179101_2.fastq.gz | Fastqsanger.gz | |||
| SRR5179102_1.fastq.gz | Fastqsanger.gz |
Items per page: 5 1 - 5 of 23 |
Similar Datasets
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:Messenger RNAs are regulated by a variety of degradation mechanisms in mammalian cells. In the canonical animal microRNA pathway, microRNAs in complex with Argonaute proteins bind to many mRNA targets with imperfect complementarity, leading to degradation of the mRNA through the regular decay machinery. The ancestral “slicer” endonuclease activity of Argonaute2 itself, which requires more extensive complementarity with the target RNA, is not used in this pathway, and has only been observed in two microRNA-guided cases. Nevertheless, the cleavage capacity of mammalian Ago2 is conserved and essential for viability. Here, we assess the endonucleolytic function of Ago2 and other nucleases by identifying cleavage products retaining 5`-phosphate groups in mouse ES cells on a transcriptome-wide scale. We detect a significant signature of Ago2-dependent cleavage events and validate several targets. Unexpectedly, a broader class of Ago2-independent cleavage sites is also observed, indicating participation of additional nucleases in this mode of mRNA regulation. Within this class, we identify a cohort of Drosha-dependent mRNA cleavage events, including one in the Dgcr8 mRNA, that functionally regulate mRNA levels in mES cells. Together, these results highlight the underappreciated role of endonucleolytic cleavage in controlling mRNA fates in mammals. Global 5`-phosphate-dependent RACE in WT, Ago2-KO and Drosha-excised mouse ES cells and human 293S cells
2010-06-25 | E-GEOD-21975 | biostudies-arrayexpress
Project description:Messenger RNAs are regulated by a variety of degradation mechanisms in mammalian cells. In the canonical animal microRNA pathway, microRNAs in complex with Argonaute proteins bind to many mRNA targets with imperfect complementarity, leading to degradation of the mRNA through the regular decay machinery. The ancestral “slicer” endonuclease activity of Argonaute2 itself, which requires more extensive complementarity with the target RNA, is not used in this pathway, and has only been observed in two microRNA-guided cases. Nevertheless, the cleavage capacity of mammalian Ago2 is conserved and essential for viability. Here, we assess the endonucleolytic function of Ago2 and other nucleases by identifying cleavage products retaining 5`-phosphate groups in mouse ES cells on a transcriptome-wide scale. We detect a significant signature of Ago2-dependent cleavage events and validate several targets. Unexpectedly, a broader class of Ago2-independent cleavage sites is also observed, indicating participation of additional nucleases in this mode of mRNA regulation. Within this class, we identify a cohort of Drosha-dependent mRNA cleavage events, including one in the Dgcr8 mRNA, that functionally regulate mRNA levels in mES cells. Together, these results highlight the underappreciated role of endonucleolytic cleavage in controlling mRNA fates in mammals.
2010-06-25 | GSE21975 | GEO
Project description:Diverse endonucleolytic cleavage sites in the mammalian transcriptome depend upon microRNAs, Drosha, and additional nucleases
| PRJNA127179 | ENA
Project description:This SuperSeries is composed of the SubSeries listed below.
2017-04-23 | GSE93653 | GEO
Project description:The RNase III enzyme Drosha has a central role in microRNA (miRNA) biogenesis, where it required to release the stem-loop intermediate from primary (pri)-miRNA transcripts. However, it can also cleave stem-loops embedded within messenger (m)RNAs. This destabilizes the mRNA causing target gene repression and has been found to occur primarily in stem cells. While pri-miRNA stem-loops have been extensively studied, such non-canonical substrates of Drosha has yet to characterized in detail. In this study, we employed high-throughput sequencing to capture all polyA-tailed RNAs that are cleaved by Drosha in mouse embryonic stem cells (ESCs). We then compared the features of non-canonical versus miRNA stem-loop substrates. First, these mRNA substrates are less efficiently processed than miRNA stem-loops. Sequence and structural analyses revealed that these substrates are also less stable and more likely to fold into alternative structures than miRNA stem-loops. Moreover, they lack the sequence and structural motifs found in miRNAs stem-loops that are required for precise cleavage. Notably, we discovered a non-canonical Drosha substrate that is cleaved in an inverse manner, which is a process that is normally inhibited by features in miRNA stem-loops. Our study thus provides valuable insights into the recognition of non-canonical targets by Drosha.
2024-03-27 | GSE228299 | GEO
Project description:Background: The expression of microRNAs (miRNAs) is primarily regulated during their transcription. However, the transcriptional regulation of miRNA genes has not been studied extensively owing to the lack of sufficient information about the promoters and transcription start sites of most miRNAs. Results: In this study, we identified the transcription start sites of human primary miRNAs (pri-miRNAs) using DROSHA knockout cells. DROSHA knockout resulted in increased accumulation of pri-miRNAs and facilitated the precise mapping of their 5′ end nucleotides using the rapid amplification of cDNA ends (RACE) technique. By analyzing the promoter region encompassing the transcription start sites of miRNAs, we found that the unrelated miRNAs in their sequences have many common elements in their promoters for binding the same transcription factors. Moreover, by analyzing intronic miRNAs, we also obtained comprehensive evidence that miRNA-harboring introns are spliced more slowly than other introns. Conclusions: The precisely mapped transcription start sites of pri-miRNAs, and the list of transcription factors for pri-miRNAs regulation, will be valuable resources for future studies to understand the regulatory network of miRNAs.
2016-10-05 | GSE80258 | GEO
Project description:Mutations in Fused in Sarcoma (FUS) gene cause the familial and progressive form of amyotrophic lateral sclerosis (ALS). FUS is a nuclear RNA-binding protein involved in RNA processing and the biogenesis of a specific set of microRNAs. Here we report that Drosha and two previously uncharacterized Drosha-dependent miRNAs are strong modulators of FUS expression and prevent the cytoplasmic segregation of insoluble mutant FUS in vivo. We demonstrate that depletion of Drosha mitigates FUS-mediated degeneration, survival, and motor defects in Drosophila. Mutant FUS strongly interacts with Drosha and causes its cytoplasmic mis-localization into the insoluble FUS inclusions. Reduction in Drosha levels increases the solubility of mutant FUS. Interestingly, we found two Drosha dependent microRNAs, miR-378i and miR-6832-5p, which differentially regulate the expression, solubility, and cytoplasmic aggregation of mutant FUS in iPSC neurons and mammalian cells. More importantly, we report different modes of action of these miRNAs against mutant FUS. Whereas miR-378i may regulate mutant FUS inclusions by preventing G3BP-mediated stress granule formation, miR-6832-5p may affect FUS expression via other proteins or pathways. Overall, our research reveals a possible association between ALS-linked FUS mutations and the Drosha-dependent miRNA regulatory circuit, as well as a useful perspective on potential ALS treatment via microRNAs.
2023-09-09 | GSE242360 | GEO
Project description:Microprocessor (MP), DROSHA-DGCR8, processes primary miRNA transcripts (pri-miRNAs) to initiate miRNA biogenesis. The canonical cleavage mechanism of MP has been extensively investigated and comprehensively validated for two decades. However, this canonical mechanism cannot account for the processing of certain pri-miRNAs in animals. In this study, by conducting high-throughput pri-miRNA cleavage assays for approximately 260,000 pri-miRNA sequences, we discovered and comprehensively characterized a noncanonical cleavage mechanism of MP. This noncanonical mechanism does not need several RNA and protein elements essential for the canonical mechanism; instead, it utilizes previously unrecognized DROSHA dsRNA recognition sites (DRES). Interestingly, the noncanonical mechanism is conserved across animals and plays a particularly significant role in C. elegans. Our established noncanonical mechanism elucidates MP cleavage in numerous RNA substrates unaccounted for by the canonical mechanism in animals. This study suggests a broader substrate repertoire of animal MPs and an expanded regulatory landscape for miRNA biogenesis.
2023-08-01 | GSE216012 | GEO