A novel deafness-associated mutation within the microRNA MIR96 gene alters pre-miRNA folding and processing

MiR-96 belongs to the conserved miR-183 family, which plays essential roles in the vertebrate inner ear. Point mutations within this microRNA (miRNA) were recently reported to cause autosomal dominant nonsyndromic sensorineural hearing loss (AD-NSHL) in two Spanish families. We screened a large NSHL case-control cohort and identified one putative novel mutation within the MIR96 gene in an Italian AD-NSHL family. This variant replaces a highly conserved nucleotide within the miR-96* sequence, which is processed from the opposite strand of the miR-96 precursor, and is predicted to reduce the stability of the pre-miRNA. Therefore, we evaluated the effect of the novel mutation on miR-96 maturation by transient expression in HeLa cells and real-time RT-PCR. We found that both miR-96 and miR-96* levels were significantly reduced in the mutant, whereas the precursor levels were unaffected. Moreover, miR-96 and miR-96* expression was restored by a compensatory mutation that reconstitutes the folding of the pre-miR-96 hairpin, demonstrating that this mutation impairs precursor processing. Finally, we evaluated the effect of the novel mutation on the regulation of miR-96/miR-96* putative targets by luciferase reporter assays. The expression of the mutant pre-miR-96 led to a significantly reduced silencing of three miR-96/miR-96* predicted targets. In particular, for one of these genes, MYRIP, we demonstrated by site-specific mutagenesis that miRNA-mediated regulation is strictly dependent on the integrity of the miR-96 target site. Moreover, miR-96-mediated downregulation of MYRIP could be restored by expressing the pre-miR-96 double mutant, which has a wild-type secondary structure, indicating that the altered precursor processing can significantly impact on the normal regulation of miR-96 targets. In conclusion, we provide further evidence of the involvement of miR-96 mutations in human deafness and demonstrate that a quantitative defect in this miRNA contributes to NSHL.