Abstract:

<p>Nibbler (Nbr) is a 3&#39;-to-5&#39; exoribonuclease whose catalytic 3&#39;-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&#39;s N-terminal domain (NTD) and exonucleolytic domain (EXO) in miRNA 3&#39;-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 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 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 and Nbr adopt a mixed α/β-scaffold with a deep pocket lined by a DEDDy catalytic cleavage motif. We demonstrate that Nbr&#39;s EXO domain exhibits Mn-dependent ssRNA-specific 3&#39;-to-5&#39; exoribonuclease activity. Modeling of a 3&#39; terminal Uridine into the catalytic pocket of Nbr EXO indicates that 2&#39;--methylation of the 3&#39;-U would result in a steric clash with a tryptophan side chain, suggesting that 2&#39;--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.</p>