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3' → 5' exoribonucleases play a critical role in many aspects of RNA metabolism. RNase R, PNPase, and RNase II are the major contributors to RNA processing, maturation, and quality control in bacteria. Bacteria don't seem to have dedicated RNA degradation machineries to process different classes of RNAs. Under different environmental and physiological conditions, their roles can be redundant and sometimes overlapping. Here, I discuss why PNPase and RNase R may have switched their physiological roles in some bacterial species to adapt to environmental conditions, despite being biochemically distinct exoribonucleases.

The yeast Saccharomyces cerevisiae and most eukaryotes carry two 5′ → 3′ exoribonuclease paralogs. In yeast, they are called Xrn1, which shuttles between the nucleus and the cytoplasm, and executes major cytoplasmic messenger RNA (mRNA) decay, and Rat1, which carries a strong nuclear localization sequence (NLS) and localizes to the nucleus. Xrn1 is 30% identical to Rat1 but has an extra ~500 amino acids C‐terminal extension. In the cytoplasm, Xrn1 can degrade decapped mRNAs during the last round of translation by ribosomes, a process referred to as "cotranslational mRNA decay." The division of labor between the two enzymes is still enigmatic and serves as a paradigm for the subfunctionalization of many other paralogs. Here we show that Rat1 is capable of functioning in cytoplasmic mRNA decay, provided that Rat1 remains cytoplasmic due to its NLS disruption (cRat1). This indicates that the physical segregation of the two paralogs plays roles in their specific functions. However, reversing segregation is not sufficient to fully complement the Xrn1 function. Specifically, cRat1 can partially restore the cell volume, mRNA stability, the proliferation rate, and 5′ → 3′ decay alterations that characterize xrn1Δ cells. Nevertheless, cotranslational decay is only slightly complemented by cRat1. The use of the AlphaFold prediction for cRat1 and its subsequent docking with the ribosome complex and the sequence conservation between cRat1 and Xrn1 suggest that the tight interaction with the ribosome observed for Xrn1 is not maintained in cRat1. Adding the Xrn1 C‐terminal domain to Rat1 does not improve phenotypes, which indicates that lack of the C‐terminal is not responsible for partial complementation. Overall, during evolution, it appears that the two paralogs have acquired specific characteristics to make functional partitioning beneficial. Take‐away: Cytoplasmic Rat1 partly restores the general physiological defects of an xrn1∆ mutant.cRat1 is very inefficient in cotranslational mRNA decay.The C‐terminal domain of Xrn1 is not involved in 5′ → 3′ cotranslational decay. [ABSTRACT FROM AUTHOR]

ERI1 is a 3-to-5 exoribonuclease involved in RNA metabolic pathways including 5.8S rRNA processing and turnover of histone mRNAs. Its biological and medical significance remain unclear. Here, we uncover a phenotypic dichotomy associated with bi-allelic ERI1 variants by reporting eight affected individuals from seven unrelated families. A severe spondyloepimetaphyseal dysplasia (SEMD) was identified in five affected individuals with missense variants but not in those with bi-allelic null variants, who showed mild intellectual disability and digital anomalies. The ERI1 missense variants cause a loss of the exoribonuclease activity, leading to defective trimming of the 5.8S rRNA 3 end and a decreased degradation of replication-dependent histone mRNAs. Affected-individual-derived induced pluripotent stem cells (iPSCs) showed impaired in vitro chondrogenesis with downregulation of genes regulating skeletal patterning. Our study establishes an entity previously unreported in OMIM and provides a model showing a more severe effect of missense alleles than null alleles within recessive genotypes, suggesting a key role of ERI1-mediated RNA metabolism in human skeletal patterning and chondrogenesis.

The regulation of gene expression in eukaryotes relies largely on the action of exoribonucleases, evolutionarily conserved enzymes that digest decapped messenger RNAs in the 5’-3’ direction. The activity of Xrn1, the major yeast exoribonuclease, is regulated by targeted changes in its cellular localisation in direct response to the cell’s metabolic state. When fermentable carbon sources are available, active Xrn1 is diffusely localised in the cytosol. Upon depletion of these sources, Xrn1 is sequestered at the plasma membrane-associated protein complex, the eisosome, and becomes inactive. Although this phenomenon has been described previously, the molecular mechanisms underlying these changes remain unknown. We report that the binding of Xrn1 to the plasma membrane is subject to glycolytic flux, rather than the availability of a fermentable carbon source, is independent of TORC1 activity and requires the core eisosomal proteins Pil1 and Lsp1. We identify the SH3-like domain of the Xrn1 protein as a putative interaction domain. In addition, we show that when expressed in Saccharomyces cerevisiae, the human orthologue of Xrn1 mirrors its yeast counterpart, i.e., it segregates to the eisosome under conditions of halted glycolysis. Our results not only advance our understanding of Xrn1 regulation but also indicate that this regulatory principle is conserved from yeast to humans.

AbstractSARS-CoV-2 non-structural protein 10 (nsp10) is essential for the stimulation of enzymatic activities of nsp14 and nsp16, acting as both an activator and scaffolding protein. Nsp14 is a bifunctional enzyme with the N-terminus containing a 3′-5′ exoribonuclease (ExoN) domain that allows the excision of nucleotide mismatches at the virus RNA 3’-end, and a C-terminal N7-methyltransferase (N7-MTase) domain. Nsp10 is required for stimulating both ExoN proofreading and the nsp16 2’-O-methyltransferase activities. This makes nsp10 a central player in both viral resistance to nucleoside-based drugs and the RNA cap methylation machinery that helps the virus evade innate immunity. We characterised the interactions between full-length nsp10 (139 residues), N- and C-termini truncated nsp10 (residues 10-133), and nsp10 with a C-terminal truncation (residues 1-133) with nsp14 using microscale thermophoresis, multi-detection SEC, and hydrogen-deuterium (H/D) exchange mass spectrometry. We describe the functional role of the C-terminal region of nsp10 for binding to nsp14 and show that full N- and C-termini of nsp10 are important for optimal binding. In addition, our H/D exchange experiments suggest an intermediary interaction of nsp10 with the N7-MTase domain of nsp14. In summary, our results suggest intermediary steps in the process of association or dissociation of the nsp10–nsp14 complex, involving contacts between the two proteins in regions not identifiable by X-ray crystallography alone. [ABSTRACT FROM AUTHOR]

High-fidelity replication of the large RNA genome of coronaviruses (CoVs) is mediated by a 3'-to-5' exoribonuclease (ExoN) in nonstructural protein 14 (nsp14), which excises nucleotides including antiviral drugs misincorporated by the low-fidelity viral RNA-dependent RNA polymerase (RdRp) and has also been implicated in viral RNA recombination and resistance to innate immunity. Here, we determined a 1.6-Å resolution crystal structure of severe acute respiratory syndrome CoV 2 (SARS-CoV-2) ExoN in complex with its essential cofactor, nsp10. The structure shows a highly basic and concave surface flanking the active site, comprising several Lys residues of nsp14 and the N-terminal amino group of nsp10. Modeling suggests that this basic patch binds to the template strand of double-stranded RNA substrates to position the 3' end of the nascent strand in the ExoN active site, which is corroborated by mutational and computational analyses. We also show that the ExoN activity can rescue a stalled RNA primer poisoned with sofosbuvir and allow RdRp to continue its extension in the presence of the chain-terminating drug, biochemically recapitulating proofreading in SARS-CoV-2 replication. Molecular dynamics simulations further show remarkable flexibility of multidomain nsp14 and suggest that nsp10 stabilizes ExoN for substrate RNA binding to support its exonuclease activity. Our high-resolution structure of the SARS-CoV-2 ExoN-nsp10 complex serves as a platform for future development of anticoronaviral drugs or strategies to attenuate the viral virulence.

The cellular environment contains numerous ribonucleases that are dedicated to process mRNA transcripts that have been targeted for degradation. Here, we review the three dimensional structures of the ribonuclease complexes (Pan2-Pan3, Ccr4-Not, Xrn1, exosome) and the mRNA decapping enzymes (Dcp2, DcpS) that are involved in mRNA turnover. Structures of major parts of these proteins have been experimentally determined. These enzymes and factors do not act in isolation, but are embedded in interaction networks which regulate enzyme activity and ensure that the appropriate substrates are recruited. The structural details of the higher order complexes that form can, in part, be accurately deduced from known structural data of sub-complexes. Interestingly, many of the ribonuclease and decapping enzymes have been observed in structurally different conformations. Together with experimental data, this highlights that structural changes are often important for enzyme function. We conclude that the known structural data of mRNA decay factors provide important functional insights, but that static structural data needs to be complemented with information regarding protein motions to complete the picture of how transcripts are turned over. In addition, we highlight multiple aspects that influence mRNA turnover rates, but that have not been structurally characterized so far. [ABSTRACT FROM AUTHOR]

Coronaviruses have a very large genome of up to 32 kb in length, almost two thirds of which encode for proteins involved in the production of the virus's own RNA. Due to the inherent mutations that occur during RNA polymerization, and the large genome size of members of the Coronaviridae family, a proofreading exoribonuclease (ExoN domain within nsp14) has evolved within these viruses to maintain high fidelity of their RNA. This proofreading exoribonuclease not only requires an enhancing protein (nsp10) to function but also performs a secondary methyltransferase of the 5′ end of produced RNA. A further 2'O-methyltransferase (nsp16) also requires the enhancing protein (nsp10) to perform the further final methylation that protects the viral RNA from the host immune system. The structural investigation of these enzymes from SARS-CoV-2 has led to a better understanding of their role in the fidelity regulation and finalization of RNA production and has enabled the development of new drugs to treat COVID-19. [ABSTRACT FROM AUTHOR]

Based on the recently added high throughput analysis data on small noncoding RNAs in modulating disease pathophysiology of malaria, we performed an integrative computational analysis for exploring the role of human-host erythrocytic microRNAs (miRNAs) and their influence on parasite survival and host homeostasis. An in silico analysis was performed on transcriptomic datasets accessed from PlasmoDB and Gene Expression Omnibus (GEO) repositories analyzed using miRanda, miRTarBase, mirDIP, and miRDB to identify the candidate miRNAs that were further subjected to network analysis using MCODE and DAVID. This was followed by immune infiltration analysis and screening for RNA degradation mechanisms. Seven erythrocytic miRNAs, miR-451a, miR-92a-3p, miR-16-5p, miR-142-3p, miR-15b-5p, miR-19b-3p, and miR-223-3p showed favourable interactions with parasite genes expressed during blood stage infection. The miR-92a-3p that targeted the virulence gene PfEMP1 showed drastic reduction during infection. Performing pathway analysis for the human-host gene targets for the miRNA identified TOB1, TOB2, CNOT4, and XRN1 genes that are associated to RNA degradation processes, with the exoribonuclease XRN1, highly enriched in the malarial samples. On evaluating the role of exoribonucleases in miRNA degradation further, the pattern of Plasmodium falciparum_XRN1 showed increased levels during infection thus suggesting a defensive role for parasite survival. This study identifies miR-92a-3p, a member of C13orf25/ miR-17-92 cluster, as a novel miRNA inhibitor of the crucial parasite genes responsible for symptomatic malaria. Evidence for a plausible link to chromosome 13q31.3 loci controlling the epigenetic disease regulation is also suggested. [ABSTRACT FROM AUTHOR]

The mitochondrial genome of the pathogenic yeast Candida albicans displays a typical organization of several (eight) primary transcription units separated by noncoding regions. Presence of genes encoding Complex I subunits and the stability of its mtDNA sequence make it an attractive model to study organellar genome expression using transcriptomic approaches. The main activity responsible for RNA degradation in mitochondria is a two-component complex (mtEXO) consisting of a 3ʹ-5ʹ exoribonuclease, in yeasts encoded by the DSS1 gene, and a conserved Suv3p helicase. In C. albicans, deletion of either DSS1 or SUV3 gene results in multiple defects in mitochondrial genome expression leading to the loss of respiratory competence. Transcriptomic analysis reveals pervasive transcription in mutants lacking the mtEXO activity, with evidence of the entire genome being transcribed, whereas in wild-type strains no RNAs corresponding to a significant fraction of the noncoding genome can be detected. Antisense ('mirror') transcripts, absent from normal mitochondria are also prominent in the mutants. The expression of multiple mature transcripts, particularly those translated from bicistronic mRNAs, as well as those that contain introns is affected in the mutants, resulting in a decreased level of proteins and reduced respiratory complex activity. The phenotype is most severe in the case of Complex IV, where a decrease of mature COX1 mRNA level to ~5% results in a complete loss of activity. These results show that RNA degradation by mtEXO is essential for shaping the mitochondrial transcriptome and is required to maintain the functional demarcation between transcription units and non-coding genome segments. [ABSTRACT FROM AUTHOR]

The rapid spread and public health impact of the novel SARS-CoV-2 variants that cause COVID-19 continue to produce major global impacts and social distress. Several vaccines were developed in record time to prevent and limit the spread of the infection, thus playing a pivotal role in controlling the pandemic. Although the repurposing of available drugs attempts to provide therapies of immediate access against COVID-19, there is still a need for developing specific treatments for this disease. Remdesivir, molnupiravir and Paxlovid remain the only evidence-supported antiviral drugs to treat COVID-19 patients, and only in severe cases. To contribute on the search of potential Covid-19 therapeutic agents, we targeted the viral RNA-dependent RNA polymerase (RdRp) and the exoribonuclease (ExoN) following two strategies. First, we modeled and analyzed nucleoside analogs sofosbuvir, remdesivir, favipiravir, ribavirin, and molnupiravir at three key binding sites on the RdRp-ExoN complex. Second, we curated and virtually screened a database containing 517 nucleotide analogs in the same binding sites. Finally, we characterized key interactions and pharmacophoric features presumably involved in viral replication halting at multiple sites. Our results highlight structural modifications that might lead to more potent SARS-CoV-2 inhibitors against an expansive range of variants and provide a collection of nucleotide analogs useful for screening campaigns.

The on-going global pandemic of COVID-19 is caused by SARS-CoV-2, which features a proofreading mechanism to facilitate the replication of its large RNA genome. The 3′-to-5′ exoribonuclease (ExoN) activity of SARS-CoV-2 non-structural protein 14 (nsp14) removes nucleotides misincorporated during RNA synthesis by the low-fidelity viral RNA-dependent RNA polymerase (RdRp) and thereby compromises the efficacy of antiviral nucleoside/nucleotide analogues. Here we show biochemically that SARS-CoV-2 nsp14 can excise the natural antiviral chain-terminating nucleotide, 3′-deoxy-3′,4′-didehydro-cytidine 5′-monophosphate (ddhCMP), incorporated by RdRp at the 3′ end of an RNA strand. Nsp14 ExoN processes an RNA strand terminated with ddhCMP more efficiently than that with a non-physiological chain terminator 3′-deoxy-cytidine monophosphate (3′-dCMP), whereas RdRp is more susceptible to chain termination by 3′-dCTP than ddhCTP. These results suggest that nsp14 ExoN could play a role in protecting SARS-CoV-2 from ddhCTP, which is produced as part of the innate immune response against viral infections, and that the SARS-CoV-2 enzymes may have adapted to minimize the antiviral effect of ddhCTP. [ABSTRACT FROM AUTHOR]

The regulation of gene expression in eukaryotes relies largely on the action of exoribonucleases, evolutionarily conserved enzymes that digest decapped messenger RNAs in the 5'-3' direction. The activity of Xrn1, the major yeast exoribonuclease, is regulated by targeted changes in its cellular localisation in direct response to the cell's metabolic state. When fermentable carbon sources are available, active Xrn1 is diffusely localised in the cytosol. Upon depletion of these sources, Xrn1 is sequestered at the plasma membrane-associated protein complex, the eisosome, and becomes inactive. Although this phenomenon has been described previously, the molecular mechanisms underlying these changes remain unknown. We report that the binding of Xrn1 to the plasma membrane is subject to glycolytic flux, rather than the availability of a fermentable carbon source, is independent of TORC1 activity and requires the core eisosomal proteins Pil1 and Lsp1. We identify the SH3-like domain of the Xrn1 protein as a putative interaction domain. In addition, we show that when expressed in Saccharomyces cerevisiae , the human orthologue of Xrn1 mirrors its yeast counterpart, i.e., it segregates to the eisosome under conditions of halted glycolysis. Our results not only advance our understanding of Xrn1 regulation but also indicate that this regulatory principle is conserved from yeast to humans., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Mitochondria are the power houses of eukaryote cells. These endosymbiotic organelles of prokaryote origin are considered as semi-autonomous since they have retained a genome and fully functional gene expression mechanisms. These pathways are particularly interesting because they combine features inherited from the bacterial ancestor of mitochondria with characteristics that appeared during eukaryote evolution. RNA biology is thus particularly diverse in mitochondria. It involves an unexpectedly vast array of factors, some of which being universal to all mitochondria and others being specific from specific eukaryote clades. Among them, ribonucleases are particularly prominent. They play pivotal functions such as the maturation of transcript ends, RNA degradation and surveillance functions that are required to attain the pool of mature RNAs required to synthesize essential mitochondrial proteins such as respiratory chain proteins. Beyond these functions, mitochondrial ribonucleases are also involved in the maintenance and replication of mitochondrial DNA, and even possibly in the biogenesis of mitochondrial ribosomes. The diversity of mitochondrial RNases is reviewed here, showing for instance how in some cases a bacterial-type enzyme was kept in some eukaryotes, while in other clades, eukaryote specific enzymes were recruited for the same function. [ABSTRACT FROM AUTHOR]

Abstract Background Coronavirus (CoV) nonstructural protein 14 (nsp14) has exoribonuclease (ExoN) activity, responsible for proofreading and contributing to replication fidelity. It has been reported that CoVs exhibit variable sensitivity to nsp14-ExoN deficiency. Betacoronavirus murine hepatitis virus (MHV) and severe acute respiratory syndrome (SARS)-CoV were viable upon nsp14-ExoN deficiency. While betacoronavirus Middle East respiratory syndrome (MERS)-CoV and SARS-CoV-2 were non-viable with disabled nsp14-ExoN. In this study, we investigated the nsp14-ExoN deficiency of alphacoronavirus porcine epidemic diarrhea virus (PEDV) in viral pathogenesis using reverse genetics. Results Eight nsp14-ExoN deficient mutants, targeting the predicted active sites and the Zinc finger or mental-coordinating sites, of PEDV were designed. Only one mutant E191A with a mutation in the Mg2+-binding site was rescued using the infectious clone of PEDV PC22A strain (icPC22A). The passage no.1–3 (P1-3) of E191A grew to very low titers in Vero cells. To evaluate the pathogenesis of the E191A, 4 or 5-day-old gnotobiotic pigs were inoculated orally with 100 TCID50/pig of the E191A-P1, icPC22A, or mock. All mock pigs did not shed virus in feces or show clinical signs. All pigs inoculated with icPC22A shed high viral RNA levels, had severe diarrhea, and died by 6 days post-inoculation (dpi). In contrast, only 3 pigs (3/4, 75%) in the E191A-P1 group shed low levels of viral RNA and 2 pigs had moderate diarrhea at acute infection phase. At 22 dpi, each pig was challenged orally with 106 plaque forming unit of virulent icPC22A. All pigs in the mock group developed severe diarrhea and 2 of the 5 pigs died. Pigs in the E191A-P1 group had less severe diarrhea and no pigs died. Sanger sequencing analysis revealed that the viral genome in the fecal sample of one E191A-P1-inoculated pig and the P4 virus passaged in vitro lost the E191A mutation, suggesting the genetic instability of the E191A mutant. Conclusion The recombinant PEDV variants carrying mutations at the essential functional sites within nsp14-ExoN were either lethal or genetically unstable. Our finding further confirmed the critical role of nsp14-ExoN in CoV life cycle, suggesting that it may be a target for the design of universal anti-CoV drugs.

Abstract Background The polynucleotide phosphorylase (PNPase) is conserved among both Gram-positive and Gram-negative bacteria. As a core part of the Escherichia coli degradosome, PNPase is involved in maintaining proper RNA levels within the bacterial cell. It plays a major role in RNA homeostasis and decay by acting as a 3′-to-5′ exoribonuclease. Furthermore, PNPase can catalyze the reverse reaction by elongating RNA molecules in 5′-to-3′ end direction which has a destabilizing effect on the prolonged RNA molecule. RNA degradation is often initiated by an endonucleolytic cleavage, followed by exoribonucleolytic decay from the new 3′ end. Results The PNPase mutant from the facultative phototrophic Rhodobacter sphaeroides exhibits several phenotypical characteristics, including diminished adaption to low temperature, reduced resistance to organic peroxide induced stress and altered growth behavior. The transcriptome composition differs in the pnp mutant strain, resulting in a decreased abundance of most tRNAs and rRNAs. In addition, PNPase has a major influence on the half-lives of several regulatory sRNAs and can have both a stabilizing or a destabilizing effect. Moreover, we globally identified and compared differential RNA 3′ ends in RNA NGS sequencing data obtained from PNPase, RNase E and RNase III mutants for the first time in a Gram-negative organism. The genome wide RNA 3′ end analysis revealed that 885 3′ ends are degraded by PNPase. A fair percentage of these RNA 3′ ends was also identified at the same genomic position in RNase E or RNase III mutant strains. Conclusion The PNPase has a major influence on RNA processing and maturation and thus modulates the transcriptome of R. sphaeroides. This includes sRNAs, emphasizing the role of PNPase in cellular homeostasis and its importance in regulatory networks. The global 3′ end analysis indicates a sequential RNA processing: 5.9% of all RNase E-dependent and 9.7% of all RNase III-dependent RNA 3′ ends are subsequently degraded by PNPase. Moreover, we provide a modular pipeline which greatly facilitates the identification of RNA 5′/3′ ends. It is publicly available on GitHub and is distributed under ICS license.

What viral RNA genomes lack in size, they make up for in intricacy. Elaborate RNA structures embedded in viral genomes can hijack essential cellular mechanisms aiding virus propagation. Exoribonuclease-resistant RNAs (xrRNAs) are an emerging class of viral elements, which resist degradation by host cellular exoribonucleases to produce viral RNAs with diverse roles during infection. Detailed three-dimensional structural studies of xrRNAs from flaviviruses and a subset of plant viruses led to a mechanistic model in which xrRNAs block enzymatic digestion using a ring-like structure that encircles the 5′ end of the resistant structure. In this mini-review, we describe the state of our understanding of the phylogenetic distribution of xrRNAs, their structures, and their conformational dynamics. Because xrRNAs have now been found in several major superfamilies of RNA viruses, they may represent a more widely used strategy than currently appreciated. Could xrRNAs represent a ‘molecular clock’ that would help us understand virus evolution and pathogenicity? The more we study xrRNAs in viruses, the closer we get to finding xrRNAs within cellular RNAs.

Abstract Background The archaeal exosome is an exoribonucleolytic multiprotein complex, which degrades single-stranded RNA in 3′ to 5′ direction phosphorolytically. In a reverse reaction, it can add A-rich tails to the 3′-end of RNA. The catalytic center of the exosome is in the aRrp41 subunit of its hexameric core. Its RNA-binding subunits aRrp4 and aDnaG confer poly(A) preference to the complex. The archaeal exosome was intensely characterized in vitro, but still little is known about its interaction with natural substrates in the cell, particularly because analysis of the transcriptome-wide interaction of an exoribonuclease with RNA is challenging. Results To determine binding sites of the exosome to RNA on a global scale, we performed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) analysis with antibodies directed against aRrp4 and aRrp41 of the chrenarchaeon Sulfolobus solfataricus. A relatively high proportion (17–19%) of the obtained cDNA reads could not be mapped to the genome. Instead, they corresponded to adenine-rich RNA tails, which are post-transcriptionally synthesized by the exosome, and to circular RNAs (circRNAs). We identified novel circRNAs corresponding to 5′ parts of two homologous, transposase-related mRNAs. To detect preferred substrates of the exosome, the iCLIP reads were compared to the transcript abundance using RNA-Seq data. Among the strongly enriched exosome substrates were RNAs antisense to tRNAs, overlapping 3′-UTRs and RNAs containing poly(A) stretches. The majority of the read counts and crosslink sites mapped in mRNAs. Furthermore, unexpected crosslink sites clustering at 5′-ends of RNAs was detected. Conclusions In this study, RNA targets of an exoribonuclease were analyzed by iCLIP. The data documents the role of the archaeal exosome as an exoribonuclease and RNA-tailing enzyme interacting with all RNA classes, and underlines its role in mRNA turnover, which is important for adaptation of prokaryotic cells to changing environmental conditions. The clustering of crosslink sites near 5′-ends of genes suggests simultaneous binding of both RNA ends by the S. solfataricus exosome. This may serve to prevent translation of mRNAs dedicated to degradation in 3′-5′ direction.

One of the many challenges faced by RNA viruses is the maintenance of their genomes during infections of host cells. Members of the family Tombusviridae are plus-strand RNA viruses with unmodified triphosphorylated genomic 59 termini. The tombusvirus Carnation Italian ringspot virus was used to investigate how it protects its RNA genome from attack by 59-end-targeting degradation enzymes. In vivo and in vitro assays were employed to determine the role of genomic RNA structure in conferring protection from the 59-to-39 exoribonuclease Xrn. The results revealed that (i) the CIRV RNA genome is more resistant to Xrn than its sg mRNAs, (ii) the genomic 59-untranslated region (UTR) folds into a compact RNA structure that effectively and independently prevents Xrn access, (iii) the RNA structure limiting 59 access is formed by secondary and tertiary interactions that function cooperatively, (iv) the structure is also able to block access of RNA pyrophosphohydrolase to the genomic 59 terminus, and (v) the RNA structure does not stall an actively digesting Xrn. Based on its proficiency at impeding Xrn 59 access, we have termed this 59-terminal structure an Xrn-evading RNA, or xeRNA. These and other findings demonstrate that the 59UTR of the CIRV RNA genome folds into a complex structural conformation that helps to protect its unmodified 59 terminus from enzymatic decay during infections. [ABSTRACT FROM AUTHOR]

The mitochondrial genome of the pathogenic yeast Candida albicans displays a typical organization of several (eight) primary transcription units separated by noncoding regions. Presence of genes encoding Complex I subunits and the stability of its mtDNA sequence make it an attractive model to study organellar genome expression using transcriptomic approaches. The main activity responsible for RNA degradation in mitochondria is a two-component complex (mtEXO) consisting of a 3ʹ-5ʹ exoribonuclease, in yeasts encoded by the DSS1 gene, and a conserved Suv3p helicase. In C. albicans, deletion of either DSS1 or SUV3 gene results in multiple defects in mitochondrial genome expression leading to the loss of respiratory competence. Transcriptomic analysis reveals pervasive transcription in mutants lacking the mtEXO activity, with evidence of the entire genome being transcribed, whereas in wild-type strains no RNAs corresponding to a significant fraction of the noncoding genome can be detected. Antisense ('mirror') transcripts, absent from normal mitochondria are also prominent in the mutants. The expression of multiple mature transcripts, particularly those translated from bicistronic mRNAs, as well as those that contain introns is affected in the mutants, resulting in a decreased level of proteins and reduced respiratory complex activity. The phenotype is most severe in the case of Complex IV, where a decrease of mature COX1 mRNA level to ~5% results in a complete loss of activity. These results show that RNA degradation by mtEXO is essential for shaping the mitochondrial transcriptome and is required to maintain the functional demarcation between transcription units and non-coding genome segments. [ABSTRACT FROM AUTHOR]

As key players of gene regulation in many bacteria, small regulatory RNAs (sRNAs) associated with the RNA chaperone Hfq shape numerous phenotypic traits, including metabolism, stress response and adaptation, as well as virulence. sRNAs can alter target messenger RNA (mRNA) translation and stability via base pairing. sRNA synthesis is generally under tight transcriptional regulation, but other levels of regulation of sRNA signaling are less well understood. Here we used a fluorescence-based functional screen to identify regulators that can quench sRNA signaling of the iron-responsive sRNA RyhB in Escherichia coli. The identified regulators fell into two classes, general regulators (affecting signaling by many sRNAs) and RyhB-specific regulators; we focused on the specific ones here. General regulators include three Hfq-interacting sRNAs, CyaR, ChiX, and McaS, previously found to act through Hfq competition, RNase T, a 3' to 5' exonuclease not previously implicated in sRNA degradation, and YhbS, a putative GCN5-related N-acetyltransferase (GNAT). Two specific regulators were identified. AspX, a 3'end-derived small RNA, specifically represses RyhB signaling via an RNA sponging mechanism. YicC, a previously uncharacterized but widely conserved protein, triggers rapid RyhB degradation via collaboration with the exoribonuclease PNPase. These findings greatly expand our knowledge of regulation of bacterial sRNA signaling and suggest complex regulatory networks for controlling iron homeostasis in bacteria. The fluorescence-based genetic screen system described here is a powerful tool expected to accelerate the discovery of novel regulators of sRNA signaling in many bacteria. [ABSTRACT FROM AUTHOR]

The recent coronavirus disease 2019 (COVID‐19), causing a global pandemic with devastating effects on healthcare and social‐economic systems, has no special antiviral therapies available for human coronaviruses (CoVs). The severe acute respiratory syndrome coronavirus 2 (SARS‐Cov‐2) possesses a nonstructural protein (nsp14), with amino‐terminal domain coding for proofreading exoribonuclease (ExoN) that is required for high‐fidelity replication. The ability of CoVs during genome replication and transcription to proofread and exclude mismatched nucleotides has long hindered the development of anti‐CoV drugs. The resistance of SARS‐CoV‐2 to antivirals, especially nucleoside analogs (NAs), shows the need to identify new CoV inhibition targets. Therefore, this review highlights the importance of nsp14‐ExoN as a target for inhibition. Also, nucleoside analogs could be used in combination with existing anti‐CoV therapeutics to target the proofreading mechanism. [ABSTRACT FROM AUTHOR]

Background: Coronavirus (CoV) nonstructural protein 14 (nsp14) has exoribonuclease (ExoN) activity, responsible for proofreading and contributing to replication fidelity. It has been reported that CoVs exhibit variable sensitivity to nsp14-ExoN deficiency. Betacoronavirus murine hepatitis virus (MHV) and severe acute respiratory syndrome (SARS)-CoV were viable upon nsp14-ExoN deficiency. While betacoronavirus Middle East respiratory syndrome (MERS)-CoV and SARS-CoV-2 were non-viable with disabled nsp14-ExoN. In this study, we investigated the nsp14-ExoN deficiency of alphacoronavirus porcine epidemic diarrhea virus (PEDV) in viral pathogenesis using reverse genetics. Results: Eight nsp14-ExoN deficient mutants, targeting the predicted active sites and the Zinc finger or mental-coordinating sites, of PEDV were designed. Only one mutant E191A with a mutation in the Mg2+-binding site was rescued using the infectious clone of PEDV PC22A strain (icPC22A). The passage no.1–3 (P1-3) of E191A grew to very low titers in Vero cells. To evaluate the pathogenesis of the E191A, 4 or 5-day-old gnotobiotic pigs were inoculated orally with 100 TCID50/pig of the E191A-P1, icPC22A, or mock. All mock pigs did not shed virus in feces or show clinical signs. All pigs inoculated with icPC22A shed high viral RNA levels, had severe diarrhea, and died by 6 days post-inoculation (dpi). In contrast, only 3 pigs (3/4, 75%) in the E191A-P1 group shed low levels of viral RNA and 2 pigs had moderate diarrhea at acute infection phase. At 22 dpi, each pig was challenged orally with 106 plaque forming unit of virulent icPC22A. All pigs in the mock group developed severe diarrhea and 2 of the 5 pigs died. Pigs in the E191A-P1 group had less severe diarrhea and no pigs died. Sanger sequencing analysis revealed that the viral genome in the fecal sample of one E191A-P1-inoculated pig and the P4 virus passaged in vitro lost the E191A mutation, suggesting the genetic instability of the E191A mutant. Conclusion: The recombinant PEDV variants carrying mutations at the essential functional sites within nsp14-ExoN were either lethal or genetically unstable. Our finding further confirmed the critical role of nsp14-ExoN in CoV life cycle, suggesting that it may be a target for the design of universal anti-CoV drugs. [ABSTRACT FROM AUTHOR]

Mitochondria are the power houses of eukaryote cells. These endosymbiotic organelles of prokaryote origin are considered as semi-autonomous since they have retained a genome and fully functional gene expression mechanisms. These pathways are particularly interesting because they combine features inherited from the bacterial ancestor of mitochondria with characteristics that appeared during eukaryote evolution. RNA biology is thus particularly diverse in mitochondria. It involves an unexpectedly vast array of factors, some of which being universal to all mitochondria and others being specific from specific eukaryote clades. Among them, ribonucleases are particularly prominent. They play pivotal functions such as the maturation of transcript ends, RNA degradation and surveillance functions that are required to attain the pool of mature RNAs required to synthesize essential mitochondrial proteins such as respiratory chain proteins. Beyond these functions, mitochondrial ribonucleases are also involved in the maintenance and replication of mitochondrial DNA, and even possibly in the biogenesis of mitochondrial ribosomes. The diversity of mitochondrial RNases is reviewed here, showing for instance how in some cases a bacterial-type enzyme was kept in some eukaryotes, while in other clades, eukaryote specific enzymes were recruited for the same function.

Background: The polynucleotide phosphorylase (PNPase) is conserved among both Gram-positive and Gram-negative bacteria. As a core part of the Escherichia coli degradosome, PNPase is involved in maintaining proper RNA levels within the bacterial cell. It plays a major role in RNA homeostasis and decay by acting as a 3′-to-5′ exoribonuclease. Furthermore, PNPase can catalyze the reverse reaction by elongating RNA molecules in 5′-to-3′ end direction which has a destabilizing effect on the prolonged RNA molecule. RNA degradation is often initiated by an endonucleolytic cleavage, followed by exoribonucleolytic decay from the new 3′ end. Results: The PNPase mutant from the facultative phototrophic Rhodobacter sphaeroides exhibits several phenotypical characteristics, including diminished adaption to low temperature, reduced resistance to organic peroxide induced stress and altered growth behavior. The transcriptome composition differs in the pnp mutant strain, resulting in a decreased abundance of most tRNAs and rRNAs. In addition, PNPase has a major influence on the half-lives of several regulatory sRNAs and can have both a stabilizing or a destabilizing effect. Moreover, we globally identified and compared differential RNA 3′ ends in RNA NGS sequencing data obtained from PNPase, RNase E and RNase III mutants for the first time in a Gram-negative organism. The genome wide RNA 3′ end analysis revealed that 885 3′ ends are degraded by PNPase. A fair percentage of these RNA 3′ ends was also identified at the same genomic position in RNase E or RNase III mutant strains. Conclusion: The PNPase has a major influence on RNA processing and maturation and thus modulates the transcriptome of R. sphaeroides. This includes sRNAs, emphasizing the role of PNPase in cellular homeostasis and its importance in regulatory networks. The global 3′ end analysis indicates a sequential RNA processing: 5.9% of all RNase E-dependent and 9.7% of all RNase III-dependent RNA 3′ ends are subsequently degraded by PNPase. Moreover, we provide a modular pipeline which greatly facilitates the identification of RNA 5′/3′ ends. It is publicly available on GitHub and is distributed under ICS license. [ABSTRACT FROM AUTHOR]

We report a molecular-docking and virtual-screening-based identification and characterization of interactions of lead molecules with exoribonuclease (ExoN) enzyme in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). From previously identified DEDDh/DEEDh subfamily nuclease inhibitors, our results revealed strong binding of pontacyl violet 6R (PV6R) at the catalytic active site of ExoN. The binding was found to be stabilized via two hydrogen bonds and hydrophobic interactions. Molecular dynamics simulations further confirmed the stability of PV6R at the active site showing a shift in ligand to reach a more stabilized binding. Using PV6R as the lead molecule, we employed virtual screening to identify potential molecular candidates that form strong interactions at the ExoN active site. Our study paves ways for evaluating the ExoN as a novel drug target for antiviral treatment against SARS-CoV-2.

SARS-CoV-2is the causative agent for the ongoing COVID19 pandemic, and this virus belongs to the Coronaviridae family. The nsp14 protein of SARS-CoV-2 houses a 3′ to 5′ exoribonuclease activity responsible for removing mismatches that arise during genome duplication. A homology model of nsp10-nsp14 complex was used to carry out in silico screening to identify molecules among natural products, or FDA approved drugs that can potentially inhibit the activity of nsp14. This exercise showed that ritonavir might bind to the exoribonuclease active site of the nsp14 protein. A model of the SARS-CoV-2-nsp10-nsp14 complex bound to substrate RNA showed that the ritonavir binding site overlaps with that of the 3′ nucleotide of substrate RNA. A comparison of the calculated energies of binding for RNA and ritonavir suggested that the drug may bind to the active site of nsp14 with significant affinity. It is, therefore, possible that ritonavir may prevent association with substrate RNA and thus inhibit the exoribonuclease activity of nsp14. Overall, our computational studies suggest that ritonavir may serve as an effective inhibitor of the nsp14 protein. nsp14 is known to attenuate the inhibitory effect of drugs that function through premature termination of viral genome replication. Hence, ritonavir may potentiate the therapeutic properties of drugs such as remdesivir, favipiravir and ribavirin. [ABSTRACT FROM AUTHOR]

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 singlestrand 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 a/ß-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. [ABSTRACT FROM AUTHOR]

The onco-suppressor p53 protein plays also an important role in the control of various aspects of health and disease. p53 levels are low in normal cells and elevated under stress conditions. While low levels of p53 promote tumor formation, overactive p53 leads to premature aging and cell death. RNA degradation is a critical level of regulation contributing to the control of gene expression. p53, as an RNA-binding protein, exerts 3′ → 5′ exoribonuclease activity, mediating degradation of adenylate/uridylate-rich elements (ARE)–containing ssRNAs. The 3′-UTR of p53-mRNA, which is a target of p53 itself, harbors cis-acting AREs. Our results suggest that p53 controls its own expression through murine double-minute 2 (mdm2)–independent "RNA decay" function in cytoplasm. We demonstrate that p53 expresses an exoribonuclease activity through the binding to ARE sequences of p53-mRNA via translation-independent and translation-dependent polysome-associated pathways. Antagonistic interplay was detected between p53 levels and execution of its exoribonuclease function mirrored in low p53 levels in normal cells, due to the efficient exoribonuclease activity, and in the accumulation of p53 in cells exposed to p53-activating drugs in accordance with the reduced exoribonuclease activity. Apparently, p53, via control of its own mRNA stability and/or translation in cytoplasm, might act as a negative regulator of p53-mRNA levels. The observed connection between exoribonuclease activity and p53 abundance highlights the importance of this function affecting p53 expression, imperative for multiple functions, with implications for the steady-state levels of protein and for the p53 stress response. The modulation in expression of exoribonuclease activity would be translated into the alterations in p53 level. Key messages: p53 controls its own expression through mdm2-independent "RNA decay" function in cytoplasm. p53 expresses an exoribonuclease activity through the binding to ARE sequences of p53-mRNA. Antagonistic interplay exists between stress-induced p53 and execution of its exoribonuclease function. [ABSTRACT FROM AUTHOR]

Among RNA viruses, the order Nidovirales stands out for including viruses with the largest RNA genomes currently known. Nidoviruses employ a complex RNA-synthesizing machinery comprising a variety of non-structural proteins (nsps). One of the postulated drivers of the expansion of nidovirus genomes is the presence of a proofreading 3′-to-5′ exoribonuclease (ExoN) belonging to the DEDDh family. ExoN may enhance the fidelity of RNA synthesis by correcting nucleotide incorporation errors made by the RNA-dependent RNA polymerase. Here, we review our current understanding of ExoN evolution, structure, and function. Most experimental data are derived from studies of the ExoN domain of coronaviruses (CoVs), which were triggered by the bioinformatics-based identification of ExoN in the genome of severe acute respiratory syndrome coronavirus (SARS-CoV) and its relatives in 2003. Although convincing data supporting the proofreading hypothesis have been obtained, from biochemical assays and studies with CoV mutants lacking ExoN functionality, the features of ExoN from most other nidovirus families remain to be characterized. Remarkably, viable ExoN knockout mutants were obtained only for two CoVs, mouse hepatitis virus (MHV) and SARS-CoV, whose RNA synthesis and replication kinetics were mildly affected by the lack of ExoN function. In several other CoV species, ExoN inactivation was not tolerated, and knockout mutants could not be rescued when launched using a reverse genetics system. This suggests that ExoN is also critical for primary viral RNA synthesis, a property that poorly matches the profile of an enzyme that would merely boost long-term replication fidelity. In CoVs, ExoN resides in a bifunctional replicase subunit (nsp14) whose C-terminal part has (N7-guanine)-methyltransferase activity. The crystal structure of SARS-CoV nsp14 has shed light on the interplay between these two domains, and on nsp14’s interactions with nsp10, a co-factor that strongly enhances ExoN activity in vitro assays. Further elucidation of the structure-function relationships of ExoN and its interactions with other (viral and/or host) members of the CoV replication machinery will be key to understanding the enzyme’s role in viral RNA synthesis and pathogenesis, and may contribute to the design of new approaches to combat emerging nidoviruses.

The 3'-to-5' exoribonuclease in coronavirus (CoV) nonstructural protein 14 (nsp14-ExoN) mediates RNA proofreading during genome replication. ExoN catalytic residues are arranged in three motifs: I (DE), II (E), and III (D). Alanine replacement of the motif I residues (AA-E-D; four nucleotide substitutions) in murine hepatitis virus (MHV) and severe acute respiratory syndrome (SARS)-CoV yields viable mutants with impaired replication and fitness, increased mutation rates, and attenuated virulence in vivo. Despite these impairments, MHV- and SARS-CoV ExoN motif I AA mutants (ExoN-AA) have not reverted at motif I in diverse in vitro and in vivo environments, suggesting that profound fitness barriers prevent motif I reversion. To test this hypothesis, we engineered MHV-ExoN-AA with 1, 2, or 3 nucleotide mutations along genetic pathways to AA-to-DE reversion. We show that engineered intermediate revertants were viable but had no increased replication or competitive fitness compared to that of MHV-ExoN-AA. In contrast, a low-passage-number (passage 10 [P10]) MHVExoN- AA showed increased replication and competitive fitness without reversion of ExoN-AA. Finally, engineered reversion of ExoN-AA to ExoN-DE in the presence of ExoN-AA passage-adaptive mutations resulted in significant fitness loss. These results demonstrate that while reversion is possible, at least one alternative adaptive pathway is more rapidly advantageous than intermediate revertants and may alter the genetic background to render reversion detrimental to fitness. Our results provide an evolutionary rationale for lack of ExoN-AA reversion, illuminate potential multiprotein replicase interactions and coevolution, and support future studies aimed at stabilizing attenuated CoV ExoN-AA mutants. [ABSTRACT FROM AUTHOR]

Among RNA viruses, the order Nidovirales stands out for including viruses with the largest RNA genomes currently known. Nidoviruses employ a complex RNA-synthesizing machinery comprising a variety of non-structural proteins (nsps). One of the postulated drivers of the expansion of nidovirus genomes is the presence of a proofreading 3′-to-5′ exoribonuclease (ExoN) belonging to the DEDDh family. ExoN may enhance the fidelity of RNA synthesis by correcting nucleotide incorporation errors made by the RNA-dependent RNA polymerase. Here, we review our current understanding of ExoN evolution, structure, and function. Most experimental data are derived from studies of the ExoN domain of coronaviruses (CoVs), which were triggered by the bioinformatics-based identification of ExoN in the genome of severe acute respiratory syndrome coronavirus (SARS-CoV) and its relatives in 2003. Although convincing data supporting the proofreading hypothesis have been obtained, from biochemical assays and studies with CoV mutants lacking ExoN functionality, the features of ExoN from most other nidovirus families remain to be characterized. Remarkably, viable ExoN knockout mutants were obtained only for two CoVs, mouse hepatitis virus (MHV) and SARS-CoV, whose RNA synthesis and replication kinetics were mildly affected by the lack of ExoN function. In several other CoV species, ExoN inactivation was not tolerated, and knockout mutants could not be rescued when launched using a reverse genetics system. This suggests that ExoN is also critical for primary viral RNA synthesis, a property that poorly matches the profile of an enzyme that would merely boost long-term replication fidelity. In CoVs, ExoN resides in a bifunctional replicase subunit (nsp14) whose C-terminal part has (N7-guanine)-methyltransferase activity. The crystal structure of SARS-CoV nsp14 has shed light on the interplay between these two domains, and on nsp14's interactions with nsp10, a co-factor that strongly enhances ExoN activity in vitro assays. Further elucidation of the structure-function relationships of ExoN and its interactions with other (viral and/or host) members of the CoV replication machinery will be key to understanding the enzyme's role in viral RNA synthesis and pathogenesis, and may contribute to the design of new approaches to combat emerging nidoviruses. [ABSTRACT FROM AUTHOR]

ABSTRACT The coronavirus (CoV) RNA genome is the largest among the single-stranded positive-sense RNA viruses. CoVs encode a proofreading 3′-to-5′ exoribonuclease within nonstructural protein 14 (nsp14-ExoN) that is responsible for CoV high-fidelity replication. Alanine substitution of ExoN catalytic residues [ExoN(-)] in severe acute respiratory syndrome-associated coronavirus (SARS-CoV) and murine hepatitis virus (MHV) disrupts ExoN activity, yielding viable mutant viruses with defective replication, up to 20-fold-decreased fidelity, and increased susceptibility to nucleoside analogues. To test the stability of the ExoN(-) genotype and phenotype, we passaged MHV-ExoN(-) 250 times in cultured cells (P250), in parallel with wild-type MHV (WT-MHV). Compared to MHV-ExoN(-) P3, MHV-ExoN(-) P250 demonstrated enhanced replication and increased competitive fitness without reversion at the ExoN(-) active site. Furthermore, MHV-ExoN(-) P250 was less susceptible than MHV-ExoN(-) P3 to multiple nucleoside analogues, suggesting that MHV-ExoN(-) was under selection for increased replication fidelity. We subsequently identified novel amino acid changes within the RNA-dependent RNA polymerase and nsp14 of MHV-ExoN(-) P250 that partially accounted for the reduced susceptibility to nucleoside analogues. Our results suggest that increased replication fidelity is selected in ExoN(-) CoVs and that there may be a significant barrier to ExoN(-) reversion. These results also support the hypothesis that high-fidelity replication is linked to CoV fitness and indicate that multiple replicase proteins could compensate for ExoN functions during replication. IMPORTANCE Uniquely among RNA viruses, CoVs encode a proofreading exoribonuclease (ExoN) in nsp14 that mediates high-fidelity RNA genome replication. Proofreading-deficient CoVs with disrupted ExoN activity [ExoN(-)] either are nonviable or have significant defects in replication, RNA synthesis, fidelity, fitness, and virulence. In this study, we showed that ExoN(-) murine hepatitis virus can adapt during long-term passage for increased replication and fitness without reverting the ExoN-inactivating mutations. Passage-adapted ExoN(-) mutants also demonstrate increasing resistance to nucleoside analogues that is explained only partially by secondary mutations in nsp12 and nsp14. These data suggest that enhanced resistance to nucleoside analogues is mediated by the interplay of multiple replicase proteins and support the proposed link between CoV fidelity and fitness.

DIS3 is a conserved exoribonuclease and catalytic subunit of the exosome, a protein complex involved in the 3' to 5' degradation and processing of both nuclear and cytoplasmic RNA species. Recently, aberrant expression of DIS3 has been found to be implicated in a range of different cancers. Perhaps most striking is the finding that DIS3 is recurrently mutated in 11% of multiple myeloma patients. Much work has been done to elucidate the structural and biochemical characteristics of DIS3, including the mechanistic details of its role as an effector of RNA decay pathways. Nevertheless, we do not understand how DIS3 mutations can lead to cancer. There are a number of studies that pertain to the function of DIS3 at the organismal level. Mutant phenotypes in S. pombe, S. cerevisiae and Drosophila suggest DIS3 homologues have a common role in cell-cycle progression and microtubule assembly. DIS3 has also recently been implicated in antibody diversification of mouse B-cells. This article aims to review current knowledge of the structure, mechanisms and functions of DIS3 as well as highlighting the genetic patterns observed within myeloma patients, in order to yield insight into the putative role of DIS3 mutations in oncogenesis.

Precursor tRNAs are transcribed with flanking and intervening sequences known to be processed by specific ribonucleases. Here, we show that transcription complexes of RNA polymerase III assembled on tRNA genes comprise RNase P that cleaves precursor tRNA and subsequently degrades the excised 5' leader. Degradation is based on a 3'-5' exoribonucleolytic activity carried out by the protein subunit Rpp14, as determined by biochemical and reverse genetic analyses. Neither reconstituted nor purified RNase P displays this magnesium ion-dependent, processive exoribonucleolytic activity. Markedly, knockdown of Rpp14 by RNA interference leads to a wide-ranging inhibition of cleavage of flanking and intervening sequences of various precursor tRNAs in extracts and cells. This study reveals that RNase P controls tRNA splicing complex and RNase Z for ordered maturation of nascent precursor tRNAs by transcription complexes.

The 3' end methylation catalyzed by HUA Enhancer 1 (HEN1) is a crucial step of small RNA stabilization in plants, yet how unmethylated small RNAs undergo degradation remains largely unknown. Using a reverse genetic approach, we here show that Atrimmer 2 (ATRM2), a DEDDy-type 3' to 5' exoribonuclease, acts in the degradation of unmethylated miRNAs and miRNA*s in Arabidopsis. Lossof- function mutations in ATRM2 partially suppress the morphological defects caused by HEN1 malfunction, with restored levels of a subset of miRNAs and receded expression of corresponding miRNA targets. Dysfunction of ATRM2 has negligible effect on miRNA trimming, and further increase the fertility of hen1 heso1 urt1, a mutant with an almost complete abolishment of miRNA uridylation, indicating that ATRM2 may neither be involved in 3' to 5' trimming nor be the enzyme that specifically degrades uridylated miRNAs. Notably, the fold changes of miRNAs and their corresponding miRNA*s were significantly correlated in hen1 atrm2 versus hen1. Unexpectedly, we observed a marked increase of 3' to 5' trimming of several miRNA*s but not miRNAs in ATRM2 compromised backgrounds. These data suggest an action of ATRM2 on miRNA/miRNA* duplexes, and the existence of an unknown exoribonuclease for specific trimming of miRNA*. This asymmetric effect on miRNA/miRNA* is likely related to Argonaute (AGO) proteins, which can distinguish miRNAs from miRNA*s. Finally, we show that ATRM2 colocalizes and physically interacts with Argonaute 1 (AGO1). Taken together, our results suggest that ATRM2 may be involved in the surveillance of unmethylated miRNA/miRNA* duplexes during the initiation step of RNA-induced silencing complex assembly. [ABSTRACT FROM AUTHOR]

Key message: Chlamydomonas RNase J is the first member of this enzyme family that has endo- but no intrinsic 5′ exoribonucleolytic activity. This questions its proposed role in chloroplast mRNA maturation.Abstract: RNA maturation and stability in the chloroplast are controlled by nuclear-encoded ribonucleases and RNA binding proteins. Notably, mRNA 5′ end maturation is thought to be achieved by the combined action of a 5′ exoribonuclease and specific pentatricopeptide repeat proteins (PPR) that block the progression of the nuclease. In Arabidopsis the 5′ exo- and endoribonuclease RNase J has been implicated in this process. Here, we verified the chloroplast localization of the orthologous Chlamydomonas (Cr) RNase J and studied its activity, both in vitro and in vivo in a heterologous B. subtilis system. Our data show that Cr RNase J has endo- but no significant intrinsic 5′ exonuclease activity that would be compatible with its proposed role in mRNA maturation. This is the first example of an RNase J ortholog that does not possess a 5′ exonuclease activity. A yeast two-hybrid screen revealed a number of potential interaction partners but three of the most promising candidates tested, failed to induce the latent exonuclease activity of Cr RNase J. We still favor the hypothesis that Cr RNase J plays an important role in RNA metabolism, but our findings suggest that it rather acts as an endoribonuclease in the chloroplast. [ABSTRACT FROM AUTHOR]

The RNA3 species of the beet necrotic yellow vein virus (BNYVV), a multipartite positive-stranded RNA phytovirus, contains the 'core' nucleotide sequence required for its systemic movement in Beta macrocarpa. Within this 'core' sequence resides a conserved "coremin" motif of 20 nucleotides that is absolutely essential for long-distance movement. RNA3 undergoes processing steps to yield a noncoding RNA3 (ncRNA3) possessing "coremin" at its 5' end, a mandatory element for ncRNA3 accumulation. Expression of wild-type (wt) or mutated RNA3 in Saccharomyces cerevisiae allows for the accumulation of ncRNA3 species. Screening of S. cerevisiae ribonuclease mutants identified the 5'-to-3' exoribonuclease Xrn1 as a key enzyme in RNA3 processing that was recapitulated both in vitro and in insect cell extracts. Xrn1 stalled on ncRNA3-containing RNA substrates in these decay assays in a similar fashion as the flavivirus Xrn1-resistant structure (sfRNA). Substitution of the BNYVV-RNA3 'core' sequence by the sfRNA sequence led to the accumulation of an ncRNA species in yeast in vitro but not in planta and no viral long distance occurred. Interestingly, XRN4 knockdown reduced BNYVV RNA accumulation suggesting a dual role for the ribonuclease in the viral cycle. [ABSTRACT FROM AUTHOR]

mRNA turnover controls gene expression under various conditions in aging and neurodegenerative diseases. Polyglutamine (polyQ) diseases and intranuclear inclusion body disease (INIBD) are neurodegenerative diseases characterized by the formation of nuclear inclusions. These inclusions are immunopositive for several proteins associated with amyotrophic lateral sclerosis. In amyotrophic lateral sclerosis, the processing of RNA from gene transcription through degradation (RNA metabolism) has been reported to be altered. We hypothesized that the proteins associated with RNA metabolism are also involved in the formation of nuclear inclusions in polyQ diseases and INIBD. 3′-5′ exoribonuclease DIS3L2 and 5′-3′ exoribonuclease XRN1 play critical roles in mRNA decay. Using immunohistochemistry with antibodies against DIS3L2 and XRN1, we examined the brains of patients with polyQ diseases and INIBD and normal control subjects. In controls, immunoreactivity for DIS3L2 and XRN1 was found in the neuronal cytoplasm and neuropil, respectively. In INIBD, immunoreactivity for DIS3L2 and XRN1 was present in neuronal and glial nuclear inclusions. Co-localization of ubiquitin and DIS3L2 or XRN1 was demonstrated in these inclusions. In polyQ diseases, however, nuclear inclusions were immunonegative for DIS3L2 and XRN1. These findings suggest that sequestration of exoribonucleases to nuclear inclusions may be related to the pathogenesis of INIBD. [ABSTRACT FROM AUTHOR]

Coronaviruses (CoVs) are positive-sense RNA viruses that infect numerous mammalian and avian species and are capable of causing severe and lethal disease in humans. CoVs encode several innate immune antagonists that counteract the host innate immune response to facilitate efficient viral replication. CoV nonstructural protein 14 (nsp14) encodes 3=-to-5= exoribonuclease activity (ExoN), which performs a proofreading function and is required for high-fidelity replication. Outside of the order Nidovirales, arenaviruses are the only RNA viruses that encode an ExoN, which functions to degrade double-stranded RNA (dsRNA) replication intermediates. In this study, we tested the hypothesis that CoV ExoN also functions to antagonize the innate immune response. We demonstrate that viruses lacking ExoN activity [ExoN(-)] are sensitive to cellular pretreatment with interferon beta (IFN-β) in a dose-dependent manner. In addition, ExoN(-) virus replication was attenuated in wild-type bone marrow-derived macrophages (BMMs) and partially restored in interferon alpha/beta receptor-deficient (IFNAR-/-) BMMs. ExoN(-) virus replication did not result in IFN-β gene expression, and in the presence of an IFN-β-mediated antiviral state, ExoN(-) viral RNA levels were not substantially reduced relative to those of untreated samples. However, ExoN(-) virus generated from IFN--pretreated cells had reduced specific infectivity and decreased relative fitness, suggesting that ExoN(-) virus generated during an antiviral state is less viable to establish a subsequent infection. Overall, our data suggest murine hepatitis virus (MHV) ExoN activity is required for resistance to the innate immune response, and antiviral mechanisms affecting the viral RNA sequence and/or an RNA modification act on viruses lacking ExoN activity. [ABSTRACT FROM AUTHOR]