MiR-CLIP reveals iso-miR selective regulation in the miR-124 targetome

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 MicroRNA Family Gets Wider: The IsomiRs Classification and Role

“…Therefore, the improvement of new strategies to discriminate selective targets for these novel microRNA variants is primary. In this context, the miR-CLIP technique ( Imig et al, 2015 ), based on the transfection of pre-miRNAs conjugated with biotin and psoralen to trap mRNA targets in cells, aided the identification of targets differentially regulated by miR-124 and its 5′ isoform ( Wang et al, 2020 ). After the transfection of a modified pre-miRNA-124 miR-CLIP probe in HEK293T cells, supported by the subsequent individual transfection of canonical and isomiR-124, the authors identified 16 potential targets ( Wang et al, 2020 ).…”

“…In this context, the miR-CLIP technique ( Imig et al, 2015 ), based on the transfection of pre-miRNAs conjugated with biotin and psoralen to trap mRNA targets in cells, aided the identification of targets differentially regulated by miR-124 and its 5′ isoform ( Wang et al, 2020 ). After the transfection of a modified pre-miRNA-124 miR-CLIP probe in HEK293T cells, supported by the subsequent individual transfection of canonical and isomiR-124, the authors identified 16 potential targets ( Wang et al, 2020 ). Out of these 16 selected candidates, 12 were mostly regulated by canonical miR-124, three were regulated by both isoforms, and just one was strongly inhibited exclusively by isomiR-124 ( Wang et al, 2020 ).…”

“…After the transfection of a modified pre-miRNA-124 miR-CLIP probe in HEK293T cells, supported by the subsequent individual transfection of canonical and isomiR-124, the authors identified 16 potential targets ( Wang et al, 2020 ). Out of these 16 selected candidates, 12 were mostly regulated by canonical miR-124, three were regulated by both isoforms, and just one was strongly inhibited exclusively by isomiR-124 ( Wang et al, 2020 ).…”

MicroRNAs and Stem-like Properties: The Complex Regulation Underlying Stemness Maintenance and Cancer Development

“…An example of how isomiRs can promote noncanonical gene targeting is provided by miR-124. The processing of pri-miR-124 generates two 5′ isomiRs, derived from seed sequences shifted by a single nucleotide, which act by regulating different transcripts, contained in overlapping targetomes [169]. Next-generation sequencing data obtained from hESCs (CyT49 (Via-Cyte), H1, and H9 cell lines) led to the identification of some miRNAs, such as miR-302, whose stem loop generates different highly expressed isomiRs, with important roles in hESC self-renewal [170].…”

Design and Application of Mini‐libraries of miRNA Probes for an Efficient and Versatile miRNA‐mRNA Cross‐linking

“…MiRNA‐mRNA products are isolated on streptavidin beads, and targets are identified by RNA sequencing. A strength of miR‐CLIP is that non‐canonical targets, i. e. new functions of miRNAs that are difficult to identify by other means, can be identified [8c,14] …”