Structure-function analysis of microRNA 3′-end trimming by Nibbler

ABSTRACT: Nibbler (Nbr) is a 3′-to-5′exoribonuclease whose catalytic 3′-end trimming activity impacts miRNA and piRNA biogenesis. Here, we report on structural and functional studies to decipher the contributions of Nbr’s N-terminal (NTD) and exonucleolytic (EXO) domains 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 dsRNA over ssRNA. Structure-guided functional assays in DrosophilaS2 cells confirmed a principal role of the NTD in exonucleolytic miRNA trimming, that depends on basic surface patches.Gain-of-function experiments revealed a potential role of the NTD in recruitingNbr to Argonaute-bound small RNA substrates.The EXO domain of Aedes aegypti and Drosophila melanogaster Nbr adopt a mixed a/b scaffold with a deep pocket lined by a DEDDy catalytic cleavage motif. We demonstrate that Nbr’sEXO domain exhibits Mn2+-dependent ssRNA-specific 3′-to-5′exoribonuclease activity. Modeling of a 3′ terminal Uridine in to the catalytic pocket of Nbr EXO indicates that 2′-O-methylation of the 3′-U would result in a steric clash with a Tryptophanside 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 domain.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE158054 | GEO | 2020/09/17

REPOSITORIES: GEO