Microprocessor, composed of DROSHA and its cofactor DGCR8, initiates microRNA (miRNA) biogenesis by processing the primary transcripts of miRNA (pri-miRNAs). Here we investigate the mechanism by which Microprocessor selects the cleavage site with single-nucleotide precision, which is crucial for the specificity and functionality of miRNAs. By testing ∼40,000 pri-miRNA variants, we find that for some pri-miRNAs the cleavage site is dictated mainly by the mGHG motif embedded in the lower stem region of pri-miRNA. Structural modeling and deep-sequencing-based complementation experiments show that the double-stranded RNA-binding domain (dsRBD) of DROSHA recognizes mGHG to place the catalytic center in the appropriate position. The mGHG motif as well as the mGHG-recognizing residues in DROSHA dsRBD are conserved across eumetazoans, suggesting that this mechanism emerged in an early ancestor of the animal lineage. Our findings provide a basis for the understanding of miRNA biogenesis and rational design of accurate small-RNA-based gene silencing.
Bibliographical noteFunding Information:
We thank Hyeshik Chang and Yoonseok Jung for the bioinformatical supervision, Haedong Kim and Jimi Kim for sharing the optimized small RNA sequencing protocol, Kijun Kim for sharing unpublished results, Baekgyu Kim for sharing the HA-tagged DROSHA plasmid, and Seungmi Ji, Daeun Choi, Eunji Kim, and Young Yoon Lee for technical help. This research was supported by IBS-R008-D1 of the Institute for Basic Science funded by the Ministry of Science and ICT .
© 2018 Elsevier Inc.
- RNase III
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology