Characterisation of the AGO2 binding sites in HeLa cell line using eiCLIP
Ontology highlight
ABSTRACT:
INSTRUMENT(S): NextSeq 500, NextSeq500
ORGANISM(S): Homo sapiens
SUBMITTER: Nejc Haberman
PROVIDER: E-MTAB-12945 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Here we investigate the RNA-binding protein AGO2 in HeLa by using a combination of AGO2 enhanced individual nucleotide resolution UV crosslinking and immunoprecipitation (eiCLIP). We find that 5’ capped RNAs derived from 3’UTRs likely originate from AGO2-mediated cleavage, and most often occur at locations with potential to form G-quadruplexes.
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Project description:Argonaute (Ago) proteins mediate post-transcriptional gene repression by binding guide microRNAs (miRNAs) to regulate targeted RNAs. To confidently assess Agobound small RNAs, we adapted a mouse embryonic stem cell system to express a single inducible epitope-tagged Ago protein. Here, we report the small RNA profile of Agodeficient cells and determine Ago-dependent stability is a common feature of mammalian miRNAs. Considering both in vivo Ago-dependence for stability and Ago2 binding as defined by immunopurification, we have identified a novel class of non-canonical miRNAs derived from protein-coding gene promoters, which we name transcriptional start site miRNAs (TSS-miRNAs). A subset of promoter-proximal RNA polymerase II complexes produce hairpin RNAs that are processed in a DGCR8/Drosha-independent, but Dicer-dependent manner. TSS-miRNA activity is detectable endogenously, upon transfection of a mimic or by mRNA overexpression. Finally, we present evidence of differential expression and conservation in humans, suggesting important roles in gene regulation. Examination of Ago immunoprecipitations and mESC without Ago proteins
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