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| Status |
Public on Apr 23, 2017 |
| Title |
Genome-wide mapping of DROSHA cleavage sites on primary microRNAs and novel substrates [RNA-seq] |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
|
| Summary |
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.
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| Overall design |
RNA-seq of total RNA in siRNA-transfected HEK293T cells (3 replicates)
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| Contributor(s) |
Kim B, Jeong K, Kim VN |
| Citation(s) |
28431232 |
| Submission date |
Jan 16, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Kyowon Jeong |
| Organization name |
Seoul National University
|
| Street address |
1 Gwanak-ro
|
| City |
Seoul |
| ZIP/Postal code |
08826 |
| Country |
South Korea |
| |
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| Platforms (1) |
| GPL16791 |
Illumina HiSeq 2500 (Homo sapiens) |
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| Samples (9)
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| This SubSeries is part of SuperSeries: |
| GSE93653 |
Genome-wide mapping of DROSHA cleavage sites on primary microRNAs and novel substrates |
|
| Relations |
| BioProject |
PRJNA361474 |
| SRA |
SRP096819 |
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