microRNA (miRNA) synthesis is initiated by the cleavage event of the human Microprocessor complex on primary microRNA transcripts (pri-miRNAs). Microprocessor is constituted by DROSHA, which is an RNase III enzyme, and its obligate RNA-binding partner DGCR8. DROSHA harbors two RNase III domains, RIIIDa and RIIIDb, which concertedly execute the cleavage on the 3p- and 5p-strands of pri-miRNAs, respectively. In our study, we demonstrate that the internal loop located in the lower stem of multiple pri-miRNAs selectively restrains the cleavage of Microprocessor on their 3p-strand, thereby, stimulating the single cleavage on their 5p-strand. This single cut product fails to generate miRNA and thereby downregulates miRNA expression. We can manipulate the ratio of single-c...[ Read more ]

microRNA (miRNA) synthesis is initiated by the cleavage event of the human Microprocessor complex on primary microRNA transcripts (pri-miRNAs). Microprocessor is constituted by DROSHA, which is an RNase III enzyme, and its obligate RNA-binding partner DGCR8. DROSHA harbors two RNase III domains, RIIIDa and RIIIDb, which concertedly execute the cleavage on the 3p- and 5p-strands of pri-miRNAs, respectively. In our study, we demonstrate that the internal loop located in the lower stem of multiple pri-miRNAs selectively restrains the cleavage of Microprocessor on their 3p-strand, thereby, stimulating the single cleavage on their 5p-strand. This single cut product fails to generate miRNA and thereby downregulates miRNA expression. We can manipulate the ratio of single-cleavage to double-cleavage products from Microprocessor catalysis on multiple pri-miRNAs by engineering the size of the internal loop in their lower stem. We successfully alter miRNA production in the in vitro pri-miRNA processing assays and in human cells. Therefore, the oscillating level of the single cleavage implies another layer of regulating miRNA expression and offers an alternative approach to knockdown miRNA level without interfering the mature miRNA sequences.