RBBP6 activates the pre-mRNA 3′-end processing machinery in humans

3′-end processing of most human mRNAs is carried out by the cleavage and polyadenylation specificity factor (CPSF; CPF in yeast). Endonucleolytic cleavage of the nascent pre-mRNA defines the 3′-end of the mature transcript, which is important for mRNA localization, translation and stability. Cleavage must therefore be tightly regulated. Here, we reconstitute specific and efficient 3′-endonuclease activity of human CPSF with purified proteins. This requires the seven-subunit CPSF as well as three additional protein factors: cleavage stimulatory factor (CStF), cleavage factor IIm (CFIIm) and, importantly, the multi-domain protein RBBP6. Unlike its yeast homologue Mpe1, which is a stable subunit of CPF, RBBP6 does not copurify with CPSF and is recruited in an RNA-dependent manner. Sequence and mutational analyses suggest that RBBP6 interacts with the WDR33 and CPSF73 subunits of CPSF. Thus, it is likely that the role of RBBP6 is conserved from yeast to human. Overall, our data are consistent with CPSF endonuclease activation and site-specific pre-mRNA cleavage being highly controlled to maintain fidelity in RNA processing.

A bacterial Argonaute with efficient DNA and RNA cleavage activity guided by small DNA and RNA

ABSTRACTArgonaute proteins are widespread in prokaryotes and eukaryotes. Most prokaryotic Argonaute proteins (pAgos) use 5’P-gDNA to target complementary DNA. However, more and more studies on the properties of pAgos make their functions more diversified. Previously reported pAgos only possess several forms of high activity in all eight cleavage patterns, which limits their practical applications. Here, we described a unique pAgo from Marinitoga hydrogenitolerans (MhAgo) with eight cleavage activities. MhAgo can utilize all four types of guides (5’OH-gDNA, 5’P-gDNA, 5’OH-gRNA, and 5’P-gRNA) for ssDNA and RNA cleavage. Further studies demonstrated that MhAgo had high activities with 16-21 nt guides and no obvious preferences for the 5’-end nucleotides of 5’OH-guides. Unexpectedly, MhAgo had different preferences for the 5’-end nucleotides of 5’P-guides depending on the types of targets. Although the specificity of MhAgo was related to the types of guides, single mismatches in the central and 3’-supplementary regions of guides greatly reduced the cleavage efficiency. Additionally, the electrophoretic mobility shift assay (EMSA) demonstrated MhAgo had the weakest affinity for 5’P-gRNA:tRNA duplex, which was consistent with its cleavage efficiency. In conclusion, MhAgo is highly active under a wide range of conditions and can be used for programmable endonucleolytic cleavage of both ssDNA and RNA substrates. The abundant biochemical characteristics of MhAgo broaden our understanding of pAgos and expand the potential application in nucleic acids manipulations.

Mpe1 senses the polyadenylation signal in pre-mRNA to control cleavage and polyadenylation

Most eukaryotic messenger RNAs (mRNAs) are processed at their 3'-end by the cleavage and polyadenylation factor (CPF/CPSF). CPF mediates endonucleolytic cleavage of the pre-mRNA and addition of a polyadenosine (poly(A)) tail, which together define the 3'-end of the mature transcript. Activation of CPF is highly regulated to maintain fidelity of RNA processing. Here, using cryoEM of yeast CPF, we show that the Mpe1 subunit directly contacts the polyadenylation signal sequence in nascent pre-mRNA. This RNA-mediated link between the nuclease and polymerase modules promotes activation of the CPF endonuclease and controls polyadenylation. Mpe1 rearrangement is antagonized by another subunit, Cft2. In vivo, depletion of Mpe1 leads to widespread defects in transcription termination by RNA Polymerase II, resulting in transcription interference on neighboring genes. Together, our data suggest that Mpe1 plays a major role in selecting the cleavage site, activating CPF and ensuring timely transcription termination.

Mre11-Rad50 oligomerization promotes DNA double-strand break repair

The conserved Mre11-Rad50 (MR) complex is crucial for the detection, signaling, end tethering and processing of DNA double-strand breaks (DSBs). While it was known for decades that MR foci formation at DSBs accompanies repair, the underlying molecular assembly mechanisms and functional implications remained unclear. Combining pathway reconstitution in electron microscopy, biochemical assays and genetic studies, we show that S. cerevisiae MR oligomerizes via a conserved Rad50 beta-sheet to higher-order assemblies, which bind DNA with positive cooperativity. We designed Rad50 point mutants with enhanced or disrupted MR oligomerization, and demonstrate that MR oligomerization facilitates foci formation, DNA damage signaling and repair in vivo. MR oligomerization does not affect its exonuclease activity but drives endonucleolytic cleavage at multiple sites on the 5'-terminated DNA strand near DSBs. Interestingly, mutations in the human Rad50 beta-sheet are linked to hereditary cancer predisposition and our findings might provide new insights into their potential role in chemoresistance.

DNA-RNA hybrids at DSBs interfere with repair by homologous recombination

DNA double strand breaks (DSBs) are the most harmful DNA lesions and their repair is crucial for cell viability and genome integrity. The readout of DSB repair may depend on whether DSBs occur at transcribed versus non-transcribed regions. Some studies have postulated that DNA-RNA hybrids form at DSBs to promote recombinational repair, but others have challenged this notion. To directly assess whether hybrids formed at DSBs promote or interfere with recombinational repair we have used plasmid and chromosomal-based systems for the analysis of DSB-induced recombination in Saccharomyces cerevisiae. We show that, as expected, DNA-RNA hybrid formation is stimulated at DSBs. In addition, mutations that promote DNA-RNA hybrid accumulation, such as hpr1∆ and rnh1∆ rnh201∆, cause high levels of plasmid loss when DNA breaks are induced at sites that are transcribed. Importantly, we show that high levels or unresolved DNA-RNA hybrids at the breaks interfere with their repair by homologous recombination. This interference is observed for both plasmid and chromosomal recombination and is independent of whether the DSB is generated by endonucleolytic cleavage or by DNA replication. These data support a model in which DNA-RNA hybrids form fortuitously at DNA breaks during transcription, and need to be removed to allow recombinational repair, rather than playing a positive role.

Sequence features around cleavage sites are highly conserved among different species and a critical determinant for RNA cleavage position across eukaryotes

ABSTRACTRNA degradation is critical for control of gene expression, and endonucleolytic cleavage– dependent RNA degradation is conserved among eukaryotes. Some cleavage sites are secondarily capped in the cytoplasm and identified using the CAGE method. Although uncapped cleavage sites are widespread in eukaryotes, comparatively little information has been obtained about these sites using CAGE-based degradome analysis. Previously, we developed the truncated RNA-end sequencing (TREseq) method in plant species and used it to acquire comprehensive information about uncapped cleavage sites; we observed G-rich sequences near cleavage sites. However, it remains unclear whether this finding is general to other eukaryotes. In this study, we conducted TREseq analyses in fruit flies (Drosophila melanogaster) and budding yeast (Saccharomyces cerevisiae). The results revealed specific sequence features related to RNA cleavage in D. melanogaster and S. cerevisiae that were similar to sequence patterns in Arabidopsis thaliana. Although previous studies suggest that ribosome movements are important for determining cleavage position, feature selection using a random forest classifier showed that sequences around cleavage sites were major determinant for cleaved or uncleaved sites. Together, our results suggest that sequence features around cleavage sites are critical for determining cleavage position, and that sequence-specific endonucleolytic cleavage–dependent RNA degradation is highly conserved across eukaryotes.

Comparative parallel analysis of RNA ends identifies mRNA substrates of a tRNA splicing endonuclease-initiated mRNA decay pathway

Eukaryotes share a conserved messenger RNA (mRNA) decay pathway in which bulk mRNA is degraded by exoribonucleases. In addition, it has become clear that more specialized mRNA decay pathways are initiated by endonucleolytic cleavage at particular sites. The transfer RNA (tRNA) splicing endonuclease (TSEN) has been studied for its ability to remove introns from pre-tRNAs. More recently it has been shown that single amino acid mutations in TSEN cause pontocerebellar hypoplasia. Other recent studies indicate that TSEN has other functions, but the nature of these functions has remained obscure. Here we show that yeast TSEN cleaves a specific subset of mRNAs that encode mitochondrial proteins, and that the cleavage sites are in part determined by their sequence. This provides an explanation for the counterintuitive mitochondrial localization of yeast TSEN. To identify these mRNA target sites, we developed a “comPARE” (comparative parallel analysis of RNA ends) bioinformatic approach that should be easily implemented and widely applicable to the study of endoribonucleases. The similarity of tRNA endonuclease-initiated decay to regulated IRE1-dependent decay of mRNA suggests that mRNA specificity by colocalization may be an important determinant for the degradation of localized mRNAs in a variety of eukaryotic cells.

Mapping yeast mitotic 5’ resection at base resolution reveals the sequence and positional dependence of nucleases in vivo

Resection of the 5’-terminated strand at DNA double strand breaks (DSBs) is the critical regulated step in the transition to homologous recombination. Biochemical and genetic studies have led to a multi-step model of DSB resection in which endonucleolytic cleavage mediated by Mre11 in partnership with Sae2 is coupled with exonucleolytic cleavage mediated by redundant pathways catalyzed by Exo1 and Sgs1/Dna2. These models have not been well tested at mitotic DSBs in vivo because most methods commonly used to monitor resection cannot precisely map early cleavage events. Here we report resection monitoring with next-generation sequencing in which unique molecular identifiers allow exact counting of cleaved 5’ ends at base pair resolution. Mutant strains, including exo1Δ, mre11-H125N, exo1Δ and exo1Δ sgs1Δ, revealed a major Mre11-dependent cleavage position 60 to 70 bp from the DSB end whose exact position depended on local sequence and tracked an apparent motif. They further revealed an Exo1-dependent pause point approximately 200 bp from the DSB. Suppressing resection extension in exo1Δ sgs1Δ yeast exposed a footprint of regions where cleavage was restricted within 119 bp of the DSB and near the Exo1 pause point and where it was much less restrained. These results provide detailed in vivo support of prevailing models of DSB resection and extend them to show the combined influence of sequence specificity and access restrictions on Mre11 and Exo1 nucleases.

High-pressure sprayed siRNA triggers influence the efficiency but not the profile of transitive silencing

ABSTRACTIn plants, small interfering RNAs (siRNAs) are a quintessential class of RNA interference (RNAi)-inducing molecules produced by the endonucleolytic cleavage of double stranded RNAs (dsRNAs). In order to ensure robust RNAi, the siRNAs are amplified through a positive feedback mechanism called transitivity. Transitivity relies on RNA-DIRECTED-RNA POLYMERASE 6 (RDR6)-mediated dsRNA synthesis using siRNA-targeted RNA. This secondary dsRNA is subsequently cleaved into secondary, mainly phased, siRNAs (phasiRNAs) by DICER-LIKE (DCL) endonucleases. As primary siRNAs, secondary siRNAs are also loaded into ARGONAUTE proteins (AGOs) to form an RNA-induced silencing complex (RISC) reinforcing cleavage of the target RNA. Although the molecular players underlying transitivity are well established, the mode of action of transitivity remains elusive. In this study, we investigated the influence of primary target sites on transgene silencing and transitivity using the GFP-expressing Nicotiana benthamiana 16C line, high pressure spraying protocol (HPSP), and synthetic 22-nucleotide (nt) long siRNAs. We found that the siRNA targeting the 3’ of the GFP transgene was less efficient in inducing silencing when compared to the siRNAs targeting the 5’ and middle region of the GFP. Moreover, sRNA sequencing of locally silenced leaves showed that the amount but not the profile of secondary RNAs are shaped by the occupancy of the primary siRNA triggers on the target RNA. Our findings suggest that RDR6-mediated dsRNA synthesis is not primed by primary siRNAs and that dsRNA synthesis appears to be generally initiated at the 3’ end of the target RNA.

Readthrough of stop codons under limiting ABCE1 concentration involves frameshifting and inhibits nonsense-mediated mRNA decay

To gain insight into the mechanistic link between translation termination and nonsense-mediated mRNA decay (NMD), we depleted the ribosome recycling factor ABCE1 in human cells, resulting in an upregulation of NMD-sensitive mRNAs. Suppression of NMD on these mRNAs occurs prior to their SMG6-mediated endonucleolytic cleavage. ABCE1 depletion caused ribosome stalling at termination codons (TCs) and increased ribosome occupancy in 3′ UTRs, implying enhanced TC readthrough. ABCE1 knockdown indeed increased the rate of readthrough and continuation of translation in different reading frames, providing a possible explanation for the observed NMD inhibition, since enhanced readthrough displaces NMD activating proteins from the 3′ UTR. Our results indicate that stalling at TCs triggers ribosome collisions and activates ribosome quality control. Collectively, we show that improper translation termination can lead to readthrough of the TC, presumably due to ribosome collisions pushing the stalled ribosomes into the 3′ UTR, where it can resume translation in-frame as well as out-of-frame.