期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 117, 期 48, 页码 30370-30379出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.2018156117
关键词
Nibbler; exoribonuclease; microRNA trimming; HEAT repeats
资金
- European Research Council under the European Union's Horizon 2020 research and innovation programme [ERC-CoG-866166]
- NIH National Institute of General Medical Sciences [P30 GM124165]
- NIH-ORIP HEI Grant [S10 RR029205]
- DOE Office of Science [DE-AC02-06CH11357]
Nibbler (Nbr) is a 3 '-to-5 ' exoribonuclease whose catalytic 3 '-end trimming activity impacts microRNA (miRNA) and PIWI-interacting RNA (piRNA) biogenesis. Here, we report on structural and functional studies to decipher the contributions of Nbr's N-terminal domain (NTD) and exonucleolytic domain (EXO) in miRNA 3 '-end trimming. We have solved the crystal structures of the NTD core and EXO domains of Nbr, both in the apo-state. The NTD-core domain of Aedes aegypti Nbr adopts a HEAT-like repeat scaffold with basic patches constituting an RNA-binding surface exhibiting a preference for binding double-strand RNA (dsRNA) over single strand RNA (ssRNA). Structure-guided functional assays in Drosophila S2 cells confirmed a principal role of the NTD in exonucleolytic miRNA trimming, which depends on basic surface patches. Gain-of-function experiments revealed a potential role of the NTD in recruiting Nbr to Argonaute-bound small RNA substrates. The EXO domain of A. aegypti and Drosophila melanogaster Nbr adopt a mixed alpha/beta-scaffold with a deep pocket lined by a DEDDy catalytic cleavage motif. We demonstrate that Nbr's EXO domain exhibits Mn2*-dependent ssRNA-specific 3 '-to-5 ' exoribonuclease activity. Modeling of a 3 ' terminal Uridine into the catalytic pocket of Nbr EXO indicates that 2 '-O-methylation of the 3 '-U would result in a steric clash with a tryptophan side chain, suggesting that 2 '-O-methylation protects small RNAs from Nbr-mediated trimming. Overall, our data establish that Nbr requires its NTD as a substrate recruitment platform to execute exonucleolytic miRNA maturation, catalyzed by the ribonuclease EXO domain.
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