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 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 ofAedes aegyptiNbr 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 inDrosophilaS2 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 ofA. aegyptiandDrosophila melanogasterNbr adopt a mixed α/β-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.

Endogenous piRNA-guided slicing triggers responder and trailer piRNA production from viral RNA inAedes aegyptimosquitoes

“…This analysis identified AAEL005527 as a one-to-one ortholog of DmNbr (Figure 5A ). In addition, a recent study verified that AAEL005527 exhibits Mn 2+ -dependent, ssRNA-specific 3′–5′ exonuclease activity ( 52 ). To evaluate the role of trimming for the formation of responder vpiRNA 3′ ends in Ae.…”

EpisomiR, a New Family of miRNAs, and Its Possible Roles in Human Diseases

“…The 3′–5′ exonuclease activity is required for the catalytic 3′-end trimming of miRNAs and PIWI-interacting RNA (piRNA) biogenesis [ 65 ]. The N-terminal domain of Nibbler Homolog acts as a substrate recruitment platform for exonucleolytic miRNA maturation, achieved by the trimming of the 3′-end of miRNAs [ 66 ]. The significance of Nibbler Homolog in cancer remains to be fully elucidated.…”

Isolation of Cell-Free miRNA from Biological Fluids: Influencing Factors and Methods

“…Reference 3′-uridylation [5][6][7] adenylation [5,7] N6-methyladenosine (m6A) modification [8][9][10] 3′-terminal 2′-O-methylation [5,8,11,12] 5-methylcytosine (m5C) modification [8,13] adenosine-to-inosine editing [5][6][7]9,14] pseudouridine (Ψ) modification [8] Table 2. Forms of cell-free miRNAs.…”

“…Additionally, immature miRNA species that may be present in the sample and should be excluded from quantification or analysis. Like other RNA species miRNA can carry a repertoire of base and backbone modifications, including methylation, uridinylation, adenylation, adenosine-to-inosine editing by RNA-dependent adenosine deaminase (ADAR) or inclusion of pseudouridines ( Table 1 ) [ 5 , 6 , 7 , 11 , 56 , 57 , 58 , 59 , 60 ]. These modifications can additionally affect the half-life of specific miRNAs in biofluids by giving them increased resistance to exonucleases and higher affinity to miRNA binding biomolecules.…”