MicroRNA-seq of MDA-MB-231 cells transduced with anti-ELOVL5 and control shRNAs
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE173709 | GEO | 2021/05/03
REPOSITORIES: GEO
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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.
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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.
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