In vivo structure-function analysis of human Dicer reveals directional processing of precursor miRNAs. Mus musculus

Dicer is an RNase III-family endoribonuclease and haploinsufficient tumor suppressor that is required for the biogenesis of miRNAs, yet in vivo structure-function characterization of its RNase IIIA and IIIB domains have not been reported. In murine Dicer knockout fibroblasts, we expressed human Dicer with point mutations in the RNase III, helicase, and PAZ domains and characterized miRNA expression by Northern blot and massively parallel sequencing of small RNAs. Inactivation of the RNase IIIA or IIIB domain blocked maturation of miRNAs derived from the 3’ or 5’ arms of miRNA precursors, respectively, and resulted in altered miRNA expression profiles. Overall design: Small RNAs from murine mesenchymal stem cells (MScs) with and without Dicer (WT:Dicer f/f, KO:Dicer -/-, KO transfected with various hsDicer point mutants) were analyzed.