Abstract
Many microRNAs regulate gene expression via atypical mechanisms, which are difficult to discern using native cross-linking methods. To ascertain the scope of non-canonical miRNA targeting, methods are needed that identify all targets of a given miRNA. We designed a new class of miR-CLIP probe, whereby psoralen is conjugated to the 3p arm of a pre-microRNA to capture targetomes of miR-124 and miR-132 in HEK293T cells. Processing of pre-miR-124 yields miR-124 and a 5'-extended isoform, iso-miR-124. Using miR-CLIP, we identified overlapping targetomes from both isoforms. From a set of 16 targets, 13 were differently inhibited at mRNA/protein levels by the isoforms. Moreover, delivery of pre-miR-124 into cells repressed these targets more strongly than individual treatments with miR-124 and iso-miR-124, suggesting that isomirs from one pre-miRNA may function synergistically. By mining the miR-CLIP targetome, we identified nine G-bulged target-sites that are regulated at the protein level by miR-124 but not isomiR-124. Using structural data, we propose a model involving AGO2 helix-7 that suggests why only miR-124 can engage these sites. In summary, access to the miR-124 targetome via miR-CLIP revealed for the first time how heterogeneous processing of miRNAs combined with non-canonical targeting mechanisms expand the regulatory range of a miRNA. © The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research. Show more
Permanent link
https://doi.org/10.3929/ethz-b-000466843Publication status
publishedExternal links
Journal / series
Nucleic Acids ResearchVolume
Pages / Article No.
Publisher
Oxford University PressOrganisational unit
03760 - Hall, Jonathan / Hall, Jonathan
Funding
169612 - Chemical Approaches to Functionalize Human microRNAs (SNF)
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