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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 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 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.

SUBMITTER: Xie W

PROVIDER: S-EPMC7720153 | biostudies-literature |

REPOSITORIES: biostudies-literature

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

Project description: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.

2020-09-17 | GSE158054 | GEO

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

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

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How Complementary Targets Expose the microRNA 3' End for Tailing and Trimming during Target-Directed microRNA Degradation.

Project description:microRNAs (miRNAs) are crucial for posttranscriptional regulation of messenger RNAs. "Classical" miRNA targets predominantly interact with the miRNA seed sequence located near the miRNA 5' end. Interestingly, certain transcripts that exhibit extensive complementarity to the miRNAs 3' region, instead of being subjected to regulation, induce miRNA decay in a process termed target-directed miRNA degradation (TDMD). Here, we review recent advances in understanding the molecular mechanisms of TDMD. Specifically, we discuss how extensive miRNA complementarity to TDMD-inducing targets results in displacement of the miRNA 3' end from its protective pocket in the Argonaute protein. Unprotected miRNA 3' ends are then available for enzymatic attack by still-unidentified cellular enzymes. Identification of these cellular enzymes and discovery of additional TDMD-inducing transcripts are subjects for future research.

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The exoribonuclease Nibbler controls 3' end processing of microRNAs in Drosophila.

Project description:MicroRNAs (miRNAs) are endogenous noncoding small RNAs with important roles in many biological pathways; their generation and activity are under precise regulation [1-3]. Emerging evidence suggests that miRNA pathways are precisely modulated with controls at the level of transcription [4-8], processing [9-11], and stability [12, 13], with miRNA deregulation linked with diseases [14] and neurodegenerative disorders [15]. In the Drosophila miRNA biogenesis pathway, long primary miRNA transcripts undergo sequential cleavage [16-18] to release the embedded miRNAs. Mature miRNAs are then loaded into Argonaute1 (Ago1) within the RNA-induced silencing complex (RISC) [19, 20]. Intriguingly, we found that Drosophila miR-34 displays multiple isoforms that differ at the 3' end, suggesting a novel biogenesis mechanism involving 3' end processing. To define the cellular factors responsible, we performed an RNA interference (RNAi) screen and identified a putative 3'?5' exoribonuclease CG9247/nibbler essential for the generation of the smaller isoforms of miR-34. Nibbler (Nbr) interacts with Ago1 and processes miR-34 within RISC. Deep sequencing analysis revealed a larger set of multi-isoform miRNAs that are controlled by nibbler. These findings suggest that Nbr-mediated 3' end processing represents a critical step in miRNA maturation that impacts miRNA diversity.

| S-EPMC3255556 | biostudies-other

The Exoribonuclease Nibbler Controls 3' End Processing of MicroRNAs in Drosophila

Project description:MicroRNAs (miRNAs) are endogenous noncoding small RNAs with important roles in many biological pathways; their generation and activity are under precise regulation. Emerging evidence suggests that miRNA pathways are precisely modulated with controls at the level of transcription, processing, and stability, with miRNA deregulation linked with diseases and neurodegenerative disorders. In the Drosophila miRNA biogenesis pathway, long primary miRNA transcripts undergo sequential cleavage to release the embedded miRNAs. Mature miRNAs are then loaded into Argonaute1 (Ago1) within the RNA-induced silencing complex (RISC). Intriguingly, we found that Drosophila miR-34 displays multiple isoforms that differ at the 3′ end, suggesting a novel biogenesis mechanism involving 3′ end processing. To define the cellular factors responsible, we performed an RNA interference (RNAi) screen and identified a putative 3′→5′ exoribonuclease CG9247/nibbler essential for the generation of the smaller isoforms of miR-34. Nibbler (Nbr) interacts with Ago1 and processes miR-34 within RISC. Deep sequencing analysis revealed a larger set of multi-isoform miRNAs that are controlled by nibbler. These findings suggest that Nbr-mediated 3′ end processing represents a critical step in miRNA maturation that impacts miRNA diversity.

2011-11-17 | GSE31689 | GEO

SeqPurge: highly-sensitive adapter trimming for paired-end NGS data.

Project description:BackgroundTrimming of adapter sequences from short read data is a common preprocessing step during NGS data analysis. When performing paired-end sequencing, the overlap between forward and reverse read can be used to identify excess adapter sequences. This is exploited by several previously published adapter trimming tools. However, our evaluation on amplicon-based data shows that most of the current tools are not able to remove all adapter sequences and that adapter contamination may even lead to spurious variant calls.ResultsHere we present SeqPurge ( https://github.com/imgag/ngs-bits ), a highly-sensitive adapter trimmer that uses a probabilistic approach to detect the overlap between forward and reverse reads of Illumina sequencing data. SeqPurge can detect very short adapter sequences, even if only one base long. Compared to other adapter trimmers specifically designed for paired-end data, we found that SeqPurge achieves a higher sensitivity. The number of remaining adapter bases after trimming is reduced by up to 90 %, depending on the compared tool. In simulations with different error rates, we found that SeqPurge is also the most error-tolerant adapter trimmer in the comparison.ConclusionSeqPurge achieves a very high sensitivity and a high error-tolerance, combined with a specificity and runtime that are comparable to other state-of-the-art adapter trimmers. The very good adapter trimming performance, complemented with additional features such as quality-based trimming and basic quality control, makes SeqPurge an excellent choice for the pre-processing of paired-end NGS data.

| S-EPMC4862148 | biostudies-literature

MicroRNA turnover: a tale of tailing, trimming, and targets.

Project description:MicroRNAs (miRNAs) post-transcriptionally repress gene expression by guiding Argonaute (AGO) proteins to target mRNAs. While much is known about the regulation of miRNA biogenesis, miRNA degradation pathways are comparatively poorly understood. Although miRNAs generally exhibit slow turnover, they can be rapidly degraded through regulated mechanisms that act in a context- or sequence-specific manner. Recent work has revealed a particularly important role for specialized target interactions in controlling rates of miRNA degradation. Engagement of these targets is associated with the addition and removal of nucleotides from the 3' ends of miRNAs, a process known as tailing and trimming. Here we review these mechanisms of miRNA modification and turnover, highlighting the contexts in which they impact miRNA stability and discussing important questions that remain unanswered.

| S-EPMC9789169 | biostudies-literature

The Exoribonuclease Nibbler Controls 3' End Processing of MicroRNAs in Drosophila

Project description:MicroRNAs (miRNAs) are endogenous noncoding small RNAs with important roles in many biological pathways; their generation and activity are under precise regulation. Emerging evidence suggests that miRNA pathways are precisely modulated with controls at the level of transcription, processing, and stability, with miRNA deregulation linked with diseases and neurodegenerative disorders. In the Drosophila miRNA biogenesis pathway, long primary miRNA transcripts undergo sequential cleavage to release the embedded miRNAs. Mature miRNAs are then loaded into Argonaute1 (Ago1) within the RNA-induced silencing complex (RISC). Intriguingly, we found that Drosophila miR-34 displays multiple isoforms that differ at the 3M-bM-^@M-2 end, suggesting a novel biogenesis mechanism involving 3M-bM-^@M-2 end processing. To define the cellular factors responsible, we performed an RNA interference (RNAi) screen and identified a putative 3M-bM-^@M-2M-bM-^FM-^R5M-bM-^@M-2 exoribonuclease CG9247/nibbler essential for the generation of the smaller isoforms of miR-34. Nibbler (Nbr) interacts with Ago1 and processes miR-34 within RISC. Deep sequencing analysis revealed a larger set of multi-isoform miRNAs that are controlled by nibbler. These findings suggest that Nbr-mediated 3M-bM-^@M-2 end processing represents a critical step in miRNA maturation that impacts miRNA diversity. Total RNA was isolated using mirVana (Ambion) and size-fractionated by PAGE into 18-30nt. These were independently processed and sequenced using the Illumina GAII platform. In total, four libraries were analyzed.

2011-11-17 | E-GEOD-31689 | biostudies-arrayexpress

PEAT: an intelligent and efficient paired-end sequencing adapter trimming algorithm.

Project description:BACKGROUND: In modern paired-end sequencing protocols short DNA fragments lead to adapter-appended reads. Current paired-end adapter removal approaches trim adapter by scanning the fragment of adapter on the 3' end of the reads, which are not competent in some applications. RESULTS: Here, we propose a fast and highly accurate adapter-trimming algorithm, PEAT, designed specifically for paired-end sequencing. PEAT requires no a priori adaptor sequence, which is convenient for large-scale meta-analyses. We assessed the performance of PEAT with many adapter trimmers in both simulated and real life paired-end sequencing libraries. The importance of adapter trimming was exemplified by the influence of the downstream analyses on RNA-seq, ChIP-seq and MNase-seq. Several useful guidelines of applying adapter trimmers with aligners were suggested. CONCLUSIONS: PEAT can be easily included in the routine paired-end sequencing pipeline. The executable binaries and the standalone C++ source code package of PEAT are freely available online.

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MicroRNA biogenesis via splicing and exosome-mediated trimming in Drosophila.

Project description:microRNAs (miRNAs) are approximately 22 nucleotide regulatory RNAs derived from hairpins generated either by Drosha cleavage (canonical substrates) or by splicing and debranching of short introns (mirtrons). The 5' end of the highly conserved Drosophila mirtron-like locus mir-1017 is coincident with the splice donor, but a substantial "tail" separates its hairpin from the 3'splice acceptor. Genetic and biochemical studies define a biogenesis pathway involving splicing, lariat debranching, and RNA exosome-mediated "trimming," followed by conventional dicing and loading into AGO1 to yield a miRNA that can repress seed-matched targets. Analysis of cloned small RNAs yielded six additional candidate 3' tailed mirtrons in D. melanogaster. Altogether, these data reveal an unexpected role for the exosome in the biogenesis of miRNAs from hybrid mirtron substrates.

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