Single cut products of Microprocessor on pri-miRNAs

The human Microprocessor complex cleaves primary microRNA (miRNA) transcripts (pri-miRNAs) to initiate miRNA synthesis. Microprocessor consists of DROSHA (an RNase III enzyme), and DGCR8. DROSHA has two conserved RNase III domains, which make double cuts on each of pri-miRNA strands. In this study, we show that Microprocessor has an unexpected single-cut activity, which creates a single cut on just one of the pri-miRNA strands using one of the two RNase III domains of DROSHA. This cleavage does not lead to the production of miRNA but instead it downregulates miRNA expression. We also demonstrate that certain RNA elements facilitate the single-cut activity of Microprocessor, and by manipulating these elements, we can regulate the ratio of single-cut to double-cut activities, thus controlling miRNA production both in vitro and in vivo. Overall design: The 2 samples were generated from two experiments. 1) The single cut products of pri-mir-92a-1 in in vitro cleavage assay of Microprocessor were confirmed by sequencing. 2) The single cut products of some pri-miRNAs, which contain a large asymmetric internal loop in their lower stem in in vitro cleavage assay of Microprocessor were also confirmed by sequencing.