Posttranscriptional Regulation of 14q32 microRNAs by RNA Binding Proteins CIRBP and HADHB during Vascular Regeneration after Ischemia: Posttranscriptional regulation of 14q32 microRNAs

Abstract
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 miR 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. In order 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 RNA Binding Protein (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 hind limb 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.