Lin28a inhibits the biogenesis of let-7 miRNAs by triggering the polyuridylation and degradation of their precursors by terminal uridylyltransferases TUT4/7 and 3 ′ -5 ′ exoribonuclease Dis3l2, respectively. Previously, we showed that Lin28a also controls the production of neuro-specific miRNA-9 via a polyuridylation-independent mechanism. Here we reveal that the sequences and structural characteristics of pre-let-7 and pre-miRNA-9 are eliciting two distinct modes of binding to Lin28a. We present evidence that Dis3l2 controls miRNA-9 production. Finally, we show that the constitutive expression of untagged Lin28a during neuronal differentiation in vitro positively and negatively affects numerous other miRNAs. Our findings shed light on the role of Lin28a in differentiating cells and on the ways in which one RNA-binding protein can perform multiple roles in the regulation of RNA processing.

After induction of ischemia in mice, 14q32 microRNAs are regulated in three distinct temporal patterns. These expression patterns, as well as basal expression levels, are independent of the microRNA genes’ order in the 14q32 locus. This implies that posttranscriptional processing is a major determinant of 14q32 microRNA expression. Therefore, we hypothesized that RNA binding proteins (RBPs) regulate posttranscriptional processing of 14q32, and we aimed to identify these RBPs. To identify proteins responsible for this posttranscriptional regulation, we used RNA pull-down SILAC mass spectrometry (RP-SMS) on selected precursor microRNAs. We observed differential binding of cold-inducible RBP (CIRBP) and hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit beta (HADHB) to the precursors of late-upregulated miR-329-3p and unaffected miR-495-3p. Immunohistochemical staining confirmed expression of both CIRBP and HADHB in the adductor muscle of mice. Expression of both CIRBP and HADHB was upregulated after hindlimb ischemia in mice. Using RBP immunoprecipitation experiments, we showed specific binding of CIRBP to pre-miR-329 but not to pri-miR-329. Finally, using CRISPR/Cas9, we generated HADHB−/− 3T3 cells, which display reduced expression of miR-329 and miR-495 but not their precursors. These data suggest a novel role for CIRBP and HADHB in posttranscriptional regulation of 14q32 microRNAs.

MicroRNAs (miRs) play a vital role in governing cell function, with their levels tightly controlled at transcriptional and post-transcriptional levels. Different sets of RNA-binding proteins interact with primary miRs (pri-miRs) and precursor-miR transcripts (pre-miRs), controlling their biogenesis post-transcriptionally. The Hu antigen R (HuR)-mediated binding of Musashi homolog2 (MSI2) to the conserved terminal loop of pri-miR-7 regulates the levels of brain-enriched miR-7 formation in a tissue-specific manner. Here, we show that oleic acid (OA) inhibits the binding of proteins containing RNA recognition motifs (RRM) to the conserved terminal loop of pri-miR-7. Using electrophoretic mobility shift assays in HeLa cell extracts, we show that OA treatment disrupts pre-miR/protein complexes. Furthermore, OA rescues in vitro processing of pri-miR-7, which is otherwise blocked by HuR and MSI2 proteins. On the contrary, pri-miR-16 shows reduced processing in the presence of OA. This indicates that OA may inhibit the binding of other RRM-containing protein/s necessary for miR-16 processing. Finally, we demonstrate that OA induces mature miR-7 production in HeLa cells. Together, our results demonstrate that OA can regulate the processing of pri-miRs by remodeling their protein complexes. This provides a new tool to study RNA processing and a potential lead for small molecules that target the miR-7 biogenesis pathway.