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tRNA splicing endonuclease (TSEN) has a well-characterized role in transfer RNA (tRNA) splicing but also other functions. For yeast TSEN, these other functions include degradation of a subset of mRNAs that encode mitochondrial proteins and an unknown essential function. In this study, we use yeast genetics to characterize the unknown tRNA-independent function(s) of TSEN. Using a high-copy suppressor screen, we found that sen2 mutants can be suppressed by overexpression of SEN54. This effect was seen both for tRNA-dependent and tRNA-independent functions indicating that SEN54 is a general suppressor of sen2, likely through structural stabilization. A spontaneous suppressor screen identified mutations in the intron-debranching enzyme, Dbr1, as tRNA splicing-independent suppressors. Transcriptome analysis showed that sen2 mutation activates the Gcn4 stress response. These Gcn4 target transcripts decreased considerably in the sen2 dbr1 double mutant. We propose that Dbr1 and TSEN may compete for a shared substrate, which TSEN normally processes into an essential RNA, while Dbr1 initiates its degradation. These data provide further insight into the essential function(s) of TSEN. Importantly, single amino acid mutations in TSEN cause the generally fatal neuronal disease pontocerebellar hypoplasia (PCH). The mechanism by which defects in TSEN cause this disease is unknown, and our results reveal new possible mechanisms.

RNA exosomopathies, a growing family of diseases, are linked to missense mutations in genes encoding structural subunits of the evolutionarily conserved, 10-subunit exoribonuclease complex, the RNA exosome. This complex consists of a three-subunit cap, a six-subunit, barrel-shaped core, and a catalytic base subunit. While a number of mutations in RNA exosome genes cause pontocerebellar hypoplasia, mutations in the cap subunit gene EXOSC2 cause an apparently distinct clinical presentation that has been defined as a novel syndrome SHRF (short stature, hearing loss, retinitis pigmentosa, and distinctive facies). We generated the first in vivo model of the SHRF pathogenic amino acid substitutions using budding yeast by modeling pathogenic EXOSC2 missense mutations (p.Gly30Val and p.Gly198Asp) in the orthologous S. cerevisiae gene RRP4. The resulting rrp4 mutant cells show defects in cell growth and RNA exosome function. Consistent with altered RNA exosome function, we detect significant transcriptomic changes in both coding and noncoding RNAs in rrp4-G226D cells that model EXOSC2 p.Gly198Asp, suggesting defects in nuclear surveillance. Biochemical and genetic analyses suggest that the Rrp4 G226D variant subunit shows impaired interactions with key RNA exosome cofactors that modulate the function of the complex. These results provide the first in vivo evidence that pathogenic missense mutations present in EXOSC2 impair the function of the RNA exosome. This study also sets the stage to compare exosomopathy models to understand how defects in RNA exosome function underlie distinct pathologies.

Faithful degradation of mRNAs is a critical step in gene expression, and eukaryotes share a major conserved mRNA decay pathway. In this major pathway, the two rate-determining steps in mRNA degradation are the initial gradual removal of the poly(A) tail, followed by removal of the cap structure. Removal of the cap structure is carried out by the decapping enzyme, containing the Dcp2 catalytic subunit. Although the mechanism and regulation of mRNA decay is well understood, the consequences of defects in mRNA degradation are less clear. Dcp2 has been reported as either essential or nonessential. Here, we clarify that Dcp2 is not absolutely required for spore germination and extremely slow growth, but in practical terms it is impossible to continuously culture dcp2∆ under laboratory conditions without suppressors arising. We show that null mutations in at least three different genes are each sufficient to restore growth to a dcp2∆, of which kap123∆ and tl(gag)g∆ appear the most specific. We show that kap123∆ and tl(gag)g∆ suppress dcp2 by mechanisms that are different from each other and from previously isolated dcp2 suppressors. The suppression mechanism for tL(GAG)G is determined by the unique GAG anticodon of this tRNA, and thus likely by translation of some CUC or CUU codons. Unlike previously reported suppressors of decapping defects, these suppressors do not detectably restore decapping or mRNA decay to normal rates, but instead allow survival while only modestly affecting RNA homeostasis. These results provide important new insight into the importance of decapping, resolve previously conflicting publications about the essentiality of DCP2, provide the first phenotype for a tl(gag)g mutant, and show that multiple distinct mechanisms can bypass Dcp2 requirement.

Contains fulltext : 225143.pdf (Publisher’s version ) (Closed access) The RNA exosome is an essential ribonuclease complex required for processing and/or degradation of both coding and non-coding RNAs. We identified five patients with biallelic variants in EXOSC5, which encodes a structural subunit of the RNA exosome. The clinical features of these patients include failure to thrive, short stature, feeding difficulties, developmental delays that affect motor skills, hypotonia and esotropia. Brain MRI revealed cerebellar hypoplasia and ventriculomegaly. While we ascertained five patients, three patients with distinct variants of EXOSC5 were studied in detail. The first patient had a deletion involving exons 5-6 of EXOSC5 and a missense variant, p.Thr114Ile, that were inherited in trans, the second patient was homozygous for p.Leu206His and the third patient had paternal isodisomy for chromosome 19 and was homozygous for p.Met148Thr. The additional two patients ascertained are siblings who had an early frameshift mutation in EXOSC5 and the p.Thr114Ile missense variant that were inherited in trans. We employed three complementary approaches to explore the requirement for EXOSC5 in brain development and assess consequences of pathogenic EXOSC5 variants. Loss of function for exosc5 in zebrafish results in shortened and curved tails/bodies, reduced eye/head size and edema. We modeled pathogenic EXOSC5 variants in both budding yeast and mammalian cells. Some of these variants cause defects in RNA exosome function as well as altered interactions with other RNA exosome subunits. These findings expand the number of genes encoding RNA exosome subunits linked to human disease while also suggesting that disease mechanism varies depending on the specific pathogenic variant.

Mutations in Ski2 and Ski3 cause the intestinal and immune disorder, trichohepatoenteric syndrome (THES) by an unknown pathogenic mechanism (Bourgeois et al. 2018; Fabre et al. 2012; Hartley et al. 2010; Morton et al. 2018). The symptoms of THES vary though most, but not all, THES patient present with some degree of intractable diarrhea, inter-uterine growth retardation, woolly hair, immunodeficiency, and recurrent infections (Fabre et al. 2018; Poulton et al. 2019). Some reports suggest THES may also cause cardiac abnormalities and liver disease in some patients (Fabre et al. 2012). Why mutations in Ski2 and Ski3 only affect these specific organs/cell types is unknown as is whether specific symptoms or disease severity are linked to specific mutations.

The Dxo1/Rai1/DXO family of decapping and exonuclease enzymes can catalyze the in vitro removal of chemically diverse 5′ ends from RNA. Specifically, these enzymes act poorly on RNAs with a canonical 7mGpppN cap, but instead prefer RNAs with a triphosphate, monophosphate, hydroxyl, or nonconventional cap. In each case, these enzymes generate an RNA with a 5′ monophosphate, which is then thought to be further degraded by Rat1/Xrn1 5′ exoribonucleases. For most Dxo1/Rai1/DXO family members, it is not known which of these activities is most important in vivo. Here we describe the in vivo function of the poorly characterized cytoplasmic family member, yeast Dxo1. Using RNA-seq of 5′ monophosphate ends, we show that Dxo1 can act as a distributive exonuclease, removing a few nucleotides from endonuclease or decapping products. We also show that Dxo1 is required for the final 5′ end processing of 25S rRNA, and that this is the primary role of Dxo1. While Dxo1/Rai1/DXO members were expected to act upstream of Rat1/Xrn1, this order is reversed in 25S rRNA processing, with Dxo1 acting downstream from Rat1. Such a hand-off from a processive to a distributive exonuclease may be a general phenomenon in the precise maturation of RNA ends.

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.

RNA exosomopathies, a growing family of diseases, are linked to missense mutations in genes encoding structural subunits of the evolutionarily conserved, 10-subunit exoribonuclease complex, the RNA exosome. This complex consists of a three-subunit cap, a six-subunit, barrel-shaped core, and a catalytic base subunit. While a number of mutations in RNA exosome genes cause pontocerebellar hypoplasia, mutations in the cap subunit gene EXOSC2 cause an apparently distinct clinical presentation that has been defined as a novel syndrome SHRF (short stature, hearing loss, retinitis pigmentosa, and distinctive facies). We generated the first in vivo model of the SHRF pathogenic amino acid substitutions using budding yeast by modeling pathogenic EXOSC2 missense mutations (p.Gly30Val and p.Gly198Asp) in the orthologous S. cerevisiae gene RRP4. The resulting rrp4 mutant cells show defects in cell growth and RNA exosome function. Consistent with altered RNA exosome function, we detect significant transcriptomic changes in both coding and noncoding RNAs in rrp4-G226D cells that model EXOSC2 p.Gly198Asp, suggesting defects in nuclear surveillance. Biochemical and genetic analyses suggest that the Rrp4 G226D variant subunit shows impaired interactions with key RNA exosome cofactors that modulate the function of the complex. These results provide the first in vivo evidence that pathogenic missense mutations present in EXOSC2 impair the function of the RNA exosome. This study also sets the stage to compare exosomopathy models to understand how defects in RNA exosome function underlie distinct pathologies.

RNA exosomopathies, a growing family of tissue-specific diseases, are linked to missense mutations in genes encoding the structural subunits of the conserved 10-subunit exoribonuclease complex, the RNA exosome. Such mutations in the cap subunit gene EXOSC2 cause the novel syndrome SHRF (Short stature, Hearing loss, Retinitis pigmentosa and distinctive Facies). In contrast, exosomopathy mutations in the cap subunit gene EXOSC3 cause pontocerebellar hypoplasia type 1b (PCH1b). Though having strikingly different disease pathologies, EXOSC2 and EXOSC3 exosomopathy mutations result in amino acid substitutions in similar, conserved domains of the cap subunits, suggesting that these exosomopathy mutations have distinct consequences for RNA exosome function. We generated the first in vivo model of the SHRF pathogenic amino acid substitutions using budding yeast by introducing the EXOSC2 mutations in the orthologous S. cerevisiae gene RRP4. The resulting rrp4 mutant cells have defects in cell growth and RNA exosome function. We detect significant transcriptomic changes in both coding and non-coding RNAs in the rrp4 variant, rrp4-G226D, which models EXOSC2 p.Gly198Asp. Comparing this rrp4-G226D mutant to the previously studied S. cerevisiae model of EXOSC3 PCH1b mutation, rrp40-W195R, reveals that these mutants have disparate effects on certain RNA targets, providing the first evidence for different mechanistic consequences of these exosomopathy mutations. Congruently, we detect specific negative genetic interactions between RNA exosome cofactor mutants and rrp4-G226D but not rrp40-W195R. These data provide insight into how SHRF mutations could alter the function of the RNA exosome and allow the first direct comparison of exosomopathy mutations that cause distinct pathologies.

Small regulatory RNAs (sRNAs) are short transcripts that base-pair to mRNA targets or interact with regulatory proteins. sRNA function has been studied extensively in Gram-negative bacteria; comparatively less is known about sRNAs in Firmicutes. Here we investigate two sRNAs encoded by virulence plasmid pXO1 of Bacillus anthracis, the causative agent of anthrax. The sRNAs, named “XrrA and XrrB” (for pXO1-encoded regulatory RNA) are abundant and highly stable primary transcripts, whose expression is dependent upon AtxA, the master virulence regulator of B. anthracis. sRNA levels are highest during culture conditions that promote AtxA expression and activity, and sRNA levels are unaltered in Hfq RNA chaperone null-mutants. Comparison of the transcriptome of a virulent Ames-derived strain to the transcriptome of isogenic sRNA-null mutants revealed multiple 4.0- to >100-fold differences in gene expression. Most regulatory effects were associated with XrrA, although regulation of some transcripts suggests functional overlap between the XrrA and XrrB. Many sRNA-regulated targets were chromosome genes associated with branched-chain amino acid metabolism, proteolysis, and transmembrane transport. Finally, in a mouse model for systemic anthrax, the lungs and livers of animals infected with xrrA-null mutants had a small reduction in bacterial burden, suggesting a role for XrrA in B. anthracis pathogenesis.

Faithful degradation of mRNAs is a critical step in gene expression, and eukaryotes share a major conserved mRNA decay pathway. In this major pathway, the two rate determining steps in mRNA degradation are the initial gradual removal of the poly(A) tail, followed by removal of the cap structure. Removal of the cap structure is carried out by the decapping enzyme, containing the Dcp2 catalytic subunit. While the mechanism and regulation of mRNA decay is well-understood, the consequences of defects in mRNA degradation are less clear. Dcp2 has been reported as either essential or nonessential. Here we clarify that Dcp2 is essential for continuous growth and use experimental evolution to identify suppressors of this essentiality. We show that null mutations in at least three different are each sufficient to restore viability to adcp2Δ, of whichkap123Δ andtl(gag)gΔ appear the most specific. Unlike previously reported suppressors of decapping defects, these suppressor do not restore decapping or mRNA decay to normal rates, but instead allow survival while only modestly affecting transcriptome homeostasis. These effects are not limited to mRNAs, but extend to ncRNAs including snoRNAs and XUTs. These results provide important new insight into the importance of decapping and resolves previously conflicting publications about the essentiality ofDCP2.

Pontocerebellar hypoplasia type 1b (PCH1b) is an autosomal recessive disorder that causes cerebellar hypoplasia and spinal motor neuron degeneration, leading to mortality in early childhood. PCH1b is caused by mutations in the RNA exosome subunit gene, EXOSC3. The RNA exosome is an evolutionarily conserved complex, consisting of nine different core subunits, and one or two 3′-5′ exoribonuclease subunits, that mediates several RNA degradation and processing steps. The goal of this study is to assess the functional consequences of the amino acid substitutions that have been identified in EXOSC3 in PCH1b patients. To analyze these EXOSC3 substitutions, we generated the corresponding amino acid substitutions in the Saccharomyces cerevisiae ortholog of EXOSC3, Rrp40. We find that the rrp40 variants corresponding to EXOSC3-G31A and -D132A do not affect yeast function when expressed as the sole copy of the essential Rrp40 protein. In contrast, the rrp40-W195R variant, corresponding to EXOSC3-W238R in PCH1b patients, impacts cell growth and RNA exosome function when expressed as the sole copy of Rrp40. The rrp40-W195R protein is unstable, and does not associate efficiently with the RNA exosome in cells that also express wild-type Rrp40. Consistent with these findings in yeast, the levels of mouse EXOSC3 variants are reduced compared to wild-type EXOSC3 in a neuronal cell line. These data suggest that cells possess a mechanism for optimal assembly of functional RNA exosome complex that can discriminate between wild-type and variant exosome subunits. Budding yeast can therefore serve as a useful tool to understand the molecular defects in the RNA exosome caused by PCH1b-associated amino acid substitutions in EXOSC3, and potentially extending to disease-associated substitutions in other exosome subunits.

Summary The RNA exosome is a 3′–5′ ribonuclease complex that is composed of nine core subunits and an essential catalytic subunit, Rrp44. Two distinct conformations of Rrp44 were revealed in previous structural studies, suggesting that Rrp44 may change its conformation to exert its function. In the channeling conformation, (Rrp44 ch ), RNA accesses the active site after traversing the central channel of the RNA exosome, whereas in the other conformation, (Rrp44 da ), RNA gains direct access to the active site. Here, we show that the Rrp44 da exosome is important for nuclear function of the RNA exosome. Defects caused by disrupting the direct access conformation are distinct from those caused by channel-occluding mutations, indicating specific functions for each conformation. Our genetic analyses provide in vivo evidence that the RNA exosome employs a direct-access route to recruit specific substrates, indicating that the RNA exosome uses alternative conformations to act on different RNA substrates.

Eukaryotes maintain fidelity of gene expression by preferential degradation of aberrant mRNAs that arise by errors in RNA processing reactions. In Saccharomyces cerevisiae, Ski7 plays an important role in this mRNA quality control by mediating mRNA degradation by the RNA exosome. Ski7 was initially thought to be restricted to Saccharomyces cerevisiae and close relatives because the SKI7 gene and its paralog HBS1 arose by whole genome duplication (WGD) in a recent ancestor. We have recently shown that the preduplication gene was alternatively spliced and that Ski7 function predates WGD. Here, we use transcriptome analysis of diverse eukaryotes to show that diverse eukaryotes use alternative splicing of SKI7/HBS1 to encode two proteins. Although alternative splicing affects the same intrinsically disordered region of the protein, the pattern of splice site usage varies. This alternative splicing event arose in an early eukaryote that is a common ancestor of plants, animals, and fungi. Remarkably, through changes in alternative splicing and gene duplication, the Ski7 protein has diversified such that different species express one of four distinct Ski7-like proteins. We also show experimentally that the Saccharomyces cerevisiae SKI7 gene has undergone multiple changes that are incompatible with the Hbs1 function and may also have undergone additional changes to optimize mRNA quality control. The combination of transcriptome analysis in diverse eukaryotes and genetic analysis in yeast clarifies the mechanism by which a Ski7-like protein is expressed across eukaryotes and provides a unique view of changes in alternative splicing patterns of one gene over long evolutionary time.

Mtr4 is a conserved Ski2-like RNA helicase and a subunit of the TRAMP complex that activates exosome-mediated 3'-5' turnover in nuclear RNA surveillance and processing pathways. Prominent features of the Mtr4 structure include a four-domain ring-like helicase core and a large arch domain that spans the core. The 'ratchet helix' is positioned to interact with RNA substrates as they move through the helicase. However, the contribution of the ratchet helix in Mtr4 activity is poorly understood. Here we show that strict conservation along the ratchet helix is particularly extensive for Ski2-like RNA helicases compared to related helicases. Mutation of residues along the ratchet helix alters in vitro activity in Mtr4 and TRAMP and causes slow growth phenotypes in vivo. We also identify a residue on the ratchet helix that influences Mtr4 affinity for polyadenylated substrates. Previous work indicated that deletion of the arch domain has minimal effect on Mtr4 unwinding activity. We now show that combining the arch deletion with ratchet helix mutations abolishes helicase activity and produces a lethal in vivo phenotype. These studies demonstrate that the ratchet helix modulates helicase activity and suggest that the arch domain plays a previously unrecognized role in unwinding substrates.

RNA degradation plays important roles for maintaining temporal control and fidelity of gene expression, as well as processing of transcripts. In Saccharomyces cerevisiae the RNA exosome is a major 3′-to-5′ exoribonuclease and also has an endonuclease domain of unknown function. Here we report a physiological role for the exosome in response to a stimulus. We show that inactivating the exoribonuclease active site of Rrp44 up-regulates the iron uptake regulon. This up-regulation is caused by increased levels of reactive oxygen species (ROS) in the mutant. Elevated ROS also causes hypersensitivity to H2O2, which can be reduced by the addition of iron to H2O2 stressed cells. Finally, we show that the previously characterized slow growth phenotype of rrp44-exo− is largely ameliorated during fermentative growth. While the molecular functions of Rrp44 and the RNA exosome have been extensively characterized, our studies characterize how this molecular function affects the physiology of the organism.

Heme-containing peroxidases are important components of innate immunity. Many of them functionally associate with NADPH oxidase (NOX)/dual oxidase (DUOX) enzymes by using the hydrogen peroxide they generate in downstream reactions. Caenorhabditis elegans encodes for several heme peroxidases, and in a previous study we identified the ShkT-containing peroxidase, SKPO-1, as necessary for pathogen resistance. Here, we demonstrated that another peroxidase, HPX-2 (Heme-PeroXidase 2), is required for resistance against some, but not all pathogens. Tissue specific RNA interference (RNAi) revealed that HPX-2 functionally localizes to the hypodermis of the worm. In congruence with this observation, hpx-2 mutant animals possessed a weaker cuticle structure, indicated by higher permeability to a DNA dye, but exhibited no obvious morphological defects. In addition, fluorescent labeling of HPX-2 revealed its expression in the pharynx, an organ in which BLI-3 is also present. Interestingly, loss of HPX-2 increased intestinal colonization of E. faecalis, suggesting its role in the pharynx may limit intestinal colonization. Moreover, disruption of a catalytic residue in the peroxidase domain of HPX-2 resulted in decreased survival on E. faecalis, indicating its peroxidase activity is required for pathogen resistance. Finally, RNA-seq analysis of an hpx-2 mutant revealed changes in genes encoding for cuticle structural components under the non-pathogenic conditions. Under pathogenic conditions, genes involved in infection response were differentially regulated to a greater degree, likely due to increased microbial burden. In conclusion, the characterization of the heme-peroxidase, HPX-2, revealed that it contributes to C. elegans pathogen resistance through a role in generating cuticle material in the hypodermis and pharynx., Author summary Reactive oxygen species (ROS) production by the host tissues is one of the first lines of defense when microbial infection occurs. ROS has been shown to be involved in multiple protective pathways in innate immunity. However, given the complexity of mammalian systems, the exact manner in which ROS are used for host defense remains incompletely understood. In this study, we use Caenorhabditis elegans as a simplified model system to decipher the protective functions of ROS in innate immunity. We describe a peroxidase, HPX-2, that protects C. elegans from multiple infectious microbes by strengthening barrier tissue. This finding brings insight into the mechanisms by which peroxidases utilizes ROS to contribute to innate immunity. With infectious diseases being one of the most important causes of morbidity and mortality around the world, understanding ROS production and its function in pathogen resistance will provide us with important information in developing new therapies against pathogens.

Nonstop decay is the mechanism of identifying and disposing aberrant transcripts that lack in-frame stop codons. It is hypothesized that these transcripts are identified during translation when the ribosome arrives at the 3' end of the mRNA and stalls. Presumably, the ribosome stalling recruits additional cofactors, Ski7 and the exosome complex. The exosome degrades the transcript using either one of its ribonucleolytic activities, and the ribosome and the peptide are both released. Additional precautionary measures by the nonstop decay pathway may include translational repression of the nonstop transcript after translation, and proteolysis of the released peptide by the proteasome. This surveillance mechanism protects the cells from potentially harmful truncated proteins, but it may also be involved in mediating critical cellular functions of transcripts that are prone to stop codon read-through. Important advances have been made in the past decade as we learn that nonstop decay may have implications in human disease.

For most protein coding genes, termination of transcription by RNA polymerase II is preceded by an endonucleolytic cleavage of the nascent transcript. The 3' product of this cleavage is rapidly degraded via the 5' exoribonuclease Rat1p which is thought to destabilize the RNA polymerase II complex. It is not clear whether RNA cleavage is sufficient to trigger nuclear RNA degradation and transcription termination or whether the fate of the RNA depends on additional elements. For most mRNAs, this cleavage is mediated by the cleavage and polyadenylation machinery, but it can also be mediated by Rnt1p. We show that Rnt1p cleavage of an mRNA is not sufficient to trigger nuclear degradation or transcription termination. Insertion of an Rnt1p target site into a reporter mRNA did not block transcription downstream of the cleavage site, but instead produced two unstable cleavage products, neither of which were stabilized by inactivation of Rat1p. In contrast, the 3' and 5' cleavage products were stabilized by the deletion of the cytoplasmic 5' exoribonuclease (Xrn1p) or by inactivation of the cytoplasmic RNA exosome. These data indicate that transcription termination and nuclear degradation is not the default fate of cleaved RNAs and that specific promoter and/or sequence elements are required to determine the fate of the cleavage products.

Two general pathways of mRNA decay have been characterized in yeast. In one pathway, the mRNA is degraded by the cytoplasmic form of the exosome. The exosome has both 3′ to 5′ exoribonuclease and endoribonuclease activity, and the available evidence suggests that the exonuclease activity is required for the degradation of mRNAs. We confirm here that this is true for normal mRNAs, but that aberrant mRNAs that lack a stop codon can be efficiently degraded in the absence of the exonuclease activity of the exosome. Specifically, we show that the endo- and exonuclease activities of the exosome are both capable of rapidly degrading nonstop mRNAs and ribozyme-cleaved mRNAs. Additionally, the endonuclease activity of the exosome is not required for endonucleolytic cleavage in no-go decay. In vitro, the endonuclease domain of the exosome is active only under nonphysiological conditions, but our findings show that the in vivo activity is sufficient for the rapid degradation of nonstop mRNAs. Thus, whereas normal mRNAs are degraded by two exonucleases (Xrn1p and Rrp44p), several endonucleases contribute to the decay of many aberrant mRNAs, including transcripts subject to nonstop and no-go decay. Our findings suggest that the nuclease requirements for general and nonstop mRNA decay are different, and describe a molecular function of the core exosome that is not disrupted by inactivating its exonuclease activity.

Pili in Gram-positive bacteria play a major role in the colonization of host tissue and in the development of biofilms. They are promising candidates for vaccines or drug targets since they are highly immunogenic and share common structural and functional features among various Gram-positive pathogens. Numerous publications have helped build a detailed understanding of pilus surface assembly, yet regulation of pilin gene expression has not been well defined. Utilizing a monoclonal antibody developed against the Enterococcus faecalis major pilus protein EbpC, we identified mutants from a transposon (Tn) insertion library which lack surface-exposed Ebp pili. In addition to insertions in the ebp regulon, an insertion in ef1184 ( dapA ) significantly reduced levels of EbpC. Analysis of in-frame dapA deletion mutants and mutants with the downstream gene rnjB deleted further demonstrated that rnjB was responsible for the deficiency of EbpC. Sequence analysis revealed that rnjB encodes a putative RNase J2. Subsequent quantitative real-time PCR (qRT-PCR) and Northern blotting demonstrated that the ebpABC mRNA transcript level was significantly decreased in the rnjB deletion mutant. In addition, using a reporter gene assay, we confirmed that rnjB affects the expression of the ebpABC operon. Functionally, the rnjB deletion mutant was attenuated in its ability to produce biofilm, similar to that of an ebpABC deletion mutant which lacks Ebp pili. Together, these results demonstrate the involvement of rnjB in E. faecalis pilin gene expression and provide insight into a novel mechanism of regulation of pilus production in Gram-positive pathogens.

Eukaryotic gene expression is a complex, multistep process that needs to be executed with high fidelity and two general methods help achieve the overall accuracy of this process. Maximizing accuracy in each step in gene expression increases the fraction of correct mRNAs made. Fidelity is further improved by mRNA surveillance mechanisms that degrade incorrect or aberrant mRNAs that are made when a step is not perfectly executed[MP2]. Here, we review how cytoplasmic mRNA surveillance mechanisms selectively recognize and degrade a surprisingly wide variety of aberrant mRNAs that are exported from the nucleus into the cytoplasm.

Eukaryotic mRNAs harboring premature translation termination codons are recognized and rapidly degraded by the nonsense-mediated mRNA decay (NMD) pathway. The mechanism for discriminating between mRNAs that terminate translation prematurely and those subject to termination at natural stop codons remains unclear. Studies in multiple organisms indicate that proximity of the termination codon to the 3' poly(A) tail and the poly(A) RNA-binding protein, PAB1, constitute the critical determinant in NMD substrate recognition. We demonstrate that mRNA in yeast lacking a poly(A) tail can be destabilized by introduction of a premature termination codon and, importantly, that this mRNA is a substrate of the NMD machinery. We further show that, in cells lacking Pab1p, mRNA substrate recognition and destabilization by NMD are intact. These results establish that neither the poly(A) tail nor PAB1 is required in yeast for discrimination of nonsense-codon-containing mRNA from normal by NMD.

The RNA exosome is one of the main 3′ to 5′ exoribonucleases in eukaryotic cells. Although it is responsible for degradation or processing of a wide variety of substrate RNAs, it is very specific and distinguishes between substrate and non-substrate RNAs as well as between substrates that need to be 3′ processed and those that need to be completely degraded. This specificity does not appear to be determined by the exosome itself but rather by about a dozen other proteins. Four of these exosome cofactors have enzymatic activity, namely, the nuclear RNA-dependent ATPase Mtr4, its cytoplasmic paralog Ski2 and the nuclear non-canonical poly(A) polymerases, Trf4 and Trf5. Mtr4 and either Trf4 or Trf5 assemble into a TRAMP complex. However, how these enzymes assemble into a TRAMP complex and the functional consequences of TRAMP complex assembly remain unknown. Here, we identify an important interaction site between Mtr4 and Trf5, and show that disrupting the Mtr4/Trf interaction disrupts specific TRAMP and exosome functions, including snoRNA processing.

Translation is an important mechanism to monitor the quality of messenger RNAs (mRNAs), as exemplified by the translation-dependent recognition and degradation of transcripts harboring premature termination codons (PTCs) by the nonsense-mediated mRNA decay (NMD) pathway. We demonstrate in yeast that mRNAs lacking all termination codons are as labile as nonsense transcripts. Decay of “nonstop” transcripts in yeast requires translation but is mechanistically distinguished from NMD and the major mRNA turnover pathway that requires deadenylation, decapping, and 5′-to-3′ exonucleolytic decay. These data suggest that nonstop decay is initiated when the ribosome reaches the 3′ terminus of the message. We demonstrate multiple physiologic sources of nonstop transcripts and conservation of their accelerated decay in mammalian cells. This process regulates the stability and expression of mRNAs that fail to signal translational termination.

Post-transcriptional control provides bacterial pathogens a method by which they can rapidly adapt to environmental change. Dual exo- and endonucleolytic activities of RNase J enzymes contribute to Gram-positive RNA processing and decay. First discovered in Bacillus subtilis, RNase J1 plays a key role in mRNA maturation and degradation, while the function of the paralogue RNase J2 is largely unknown. Previously, we discovered that deletion of the Enterococcus faecalis rnjB gene significantly attenuates expression of a major virulence factor involved in enterococcal pathogenesis, the Ebp pili. In this work, we demonstrate that E. faecalis rnjB encodes an active RNase J2, and that the ribonuclease activity of RNase J2 is required for regulation of Ebp pili. To further investigate how rnjB affects E. faecalis gene expression on a global scale, we compared transcriptomes of the E. faecalis strain OG1RF with its isogenic rnjB deletion mutant (ΔrnjB). In addition to Ebp pili regulation, previously demonstrated to have a profound effect on the ability of E. faecalis to form biofilm or establish infection, we identified that rnjB regulates the expression of several other genes involved in bacterial virulence and fitness, including gls24 (a virulence factor important in stress response). We further demonstrated that the E. faecalis RNase J2 deletion mutant is more sensitive to bile salt and greatly attenuated in in vivo organ infection as determined by an IV-sublethal challenge infection mouse model, indicating that E. faecalis RNase J2 plays an important role in E. faecalis virulence.

The exosome is a protein complex consisting of a variety of 3'-to-5' exonucleases that functions both in 3'-to-5' trimming of rRNA precursors and in 3'-to-5' degradation of mRNA. To determine additional exosome functions, we examined the processing of a variety of RNAs, including tRNAs, small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), RNase P, RNase MRP, and SRP RNAs, and 5S rRNAs in mutants defective in either the core components of the exosome or in other proteins required for exosome function. These experiments led to three important conclusions. First, exosome mutants accumulate 3'-extended forms of the U4 snRNA and a wide variety of snoRNAs, including snoRNAs that are independently transcribed or intron derived. This finding suggests that the exosome functions in the 3' end processing of these species. Second, in exosome mutants, transcripts for U4 snRNA and independently transcribed snoRNAs accumulate as 3'-extended polyadenylated species, suggesting that the exosome is required to process these 3'-extended transcripts. Third, processing of 5.8S rRNA, snRNA, and snoRNA by the exosome is affected by mutations of the nuclear proteins Rrp6p and Mtr4p, whereas mRNA degradation by the exosome required Ski2p and was not affected by mutations in RRP6 or MTR4. This finding suggests that the cytoplasmic and nuclear forms of the exosome represent two functionally different complexes involved in distinct 3'-to-5' processing and degradation reactions.

Nuclease I enzymes are responsible for the degradation of RNA and single-stranded DNA during several plant growth and developmental processes, including senescence. However, in the case of senescence the corresponding genes have not been reported. We describe the identification and characterization of BFN1 of Arabidopsis, and demonstrate that it is a senescence-associated nuclease I gene. BFN1 nuclease shows high similarity to the sequence of a barley nuclease induced during germination and a zinnia (Zinnia elegans) nuclease induced during xylogenesis. In transgenic plants overexpressing the BFN1 cDNA, a nuclease activity of about 38 kD was detected on both RNase and DNase activity gels. Levels of BFN1 mRNA were extremely low or undetectable in roots, leaves, and stems. In contrast, relatively highBFN1 mRNA levels were detected in flowers and during leaf and stem senescence. BFN1 nuclease activity was also induced during leaf and stem senescence. The strong response of theBFN1 gene to senescence indicated that it would be an excellent tool with which to study the mechanisms of senescence induction, as well as the role of the BFN1 enzyme in senescence using reverse genetic approaches in Arabidopsis.

In plants, as in other eukaryotes, most synonymous codons of the genetic code are not used with equal frequency, but instead some codons are preferred, whereas others are rare. Circumstantial evidence led to the suggestion that rare codons have a negative influence on mRNA stability. To address this question experimentally, rare codons encoded by a Bacillus thuringiensis (B.t.) toxin gene (cryIA(c)) or a synthetic sequence were introduced into a phytohemagglutinin (PHA) reporter gene. In neither case was the mRNA stability appreciably diminished in stably transformed tobacco cell cultures nor was the accumulation of mRNA in transgenic plants affected. Thus rare codons do not appear to be sufficient to cause rapid degradation of the PHA mRNA and potentially other mRNAs in plants.

Premature termination of translation has often been associated with decreased mRNA accumulation in plants, but the affected step in gene expression has not been identified. To investigate this problem, the expression of wild-type and mutant alleles of the bean phytohemagglutinin (PHA) gene has been examined in tobacco cells and transgenic plants. Measurement of mRNA decay rates in stably transformed cell lines demonstrated that premature nonsense codons markedly destabilized the mRNA. This decreased stability was also reflected by decreased accumulation of transcripts containing premature nonsense codons in transgenic plants. The positional dependence of the nonsense codon effect was evaluated by introducing premature nonsense codons at different distances from the PHA AUG start codon. Transcripts with nonsense codons about 20, 40 or 60% of the way through the normal PHA coding region yielded highly unstable mRNAs, whereas a transcript with a nonsense codon at 80% was as stable as wild-type. The ability to recognize and rapidly degrade certain transcripts with early nonsense codons could provide plant cells with a means to minimize the production of wasteful and possible deleterious truncated proteins.

The RNA exosome is responsible for a wide variety of RNA processing and degradation reactions. The activity and specificity of the RNA exosome is thought to be controlled by a number of cofactors. Mtr4 is an essential RNA-dependent adenosine triphosphatase that is required for all of the nuclear functions of the RNA exosome. The crystal structure of Mtr4 uncovered a domain that is conserved in the RNA exosome cofactors Mtr4 and Ski2 but not in other helicases, suggesting it has an important role related to exosome activation. Rrp6 provides the nuclear exosome with one of its three nuclease activities, and previous findings suggested that the arch domain is specifically required for Rrp6 functions. Here, we report that the genetic interactions between the arch domain of Mtr4 and Rrp6 cannot be explained by the arch domain solely acting in Rrp6-dependent processing reactions. Specifically, we show that the arch domain is not required for all Rrp6 functions, and that the arch domain also functions independently of Rrp6. Finally, we show that the arch domain of Ski2, the cytoplasmic counterpart of Mtr4, is required for Ski2's function, thereby confirming that the arch domains of these cofactors function independently of Rrp6.

Yersinia polynucleotide phosphorylase (PNPase), a 3'-5' exoribonuclease, has been shown to affect growth during several stress responses. In Escherichia coli, PNPase is one of the subunits of a multiprotein complex known as the degradosome, but also has degradosome-independent functions. The carboxy-terminus of E. coli ribonuclease E (RNase E) serves as the scaffold upon which PNPase, enolase (a glycolytic enzyme), and RhlB helicase all have been shown to bind. In the yersiniae, only PNPase has thus far been shown to physically interact with RNase E. We show by bacterial two-hybrid and co-immunoprecipitation assays that RhlB and enolase also interact with RNase E. Interestingly, although PNPase is required for normal growth at cold temperatures, assembly of the yersiniae degradosome was not required. However, degradosome assembly was required for growth in the presence of reactive oxygen species. These data suggest that while the Yersinia pseudotuberculosis PNPase plays a role in the oxidative stress response through a degradosome-dependent mechanism, PNPase's role during cold stress is degradosome-independent.

Nonstop decay is the mechanism of identifying and disposing aberrant transcripts that lack in-frame stop codons. It is hypothesized that these transcripts are identified during translation when the ribosome arrives at the 3' end of the mRNA and stalls. Presumably, the ribosome stalling recruits additional cofactors, Ski7 and the exosome complex. The exosome degrades the transcript using either one of its ribonucleolytic activities, and the ribosome and the peptide are both released. Additional precautionary measures by the nonstop decay pathway may include translational repression of the nonstop transcript after translation, and proteolysis of the released peptide by the proteasome. This surveillance mechanism protects the cells from potentially harmful truncated proteins, but it may also be involved in mediating critical cellular functions of transcripts that are prone to stop codon read-through. Important advances have been made in the past decade as we learn that nonstop decay may have implications in human disease.

The mechanisms responsible for mRNA decay in mammalian cells, and how specific sequence elements accelerate decay, are unknown. Recent work indicates that ‘ARE’ instability elements recruit the exosome to promote rapid 3′-to-5′ degradation of the mRNA.

One role of messenger RNA (mRNA) degradation is to maintain the fidelity of gene expression by degrading aberrant transcripts. Recent results show that mRNAs without translation termination codons are unstable in eukaryotic cells. We used yeast mutants to demonstrate that these “nonstop” mRNAs are degraded by the exosome in a 3′-to-5′ direction. The degradation of nonstop transcripts requires the exosome-associated protein Ski7p. Ski7p is closely related to the translation elongation factor EF1A and the translation termination factor eRF3. This suggests that the recognition of nonstop mRNAs involves the binding of Ski7p to an empty aminoacyl-(RNA-binding) site (A site) on the ribosome, thereby bringing the exosome to a mRNA with a ribosome stalled near the 3′ end. This system efficiently degrades mRNAs that are prematurely polyadenylated within the coding region and prevents their expression.

The exosome consists of a core of ten essential proteins that includes the ribonuclease Rrp44p and is present in both the cytoplasm and nucleus of eukaryotic cells. The cytoplasmic exosome has been extensively characterized in the budding yeast Saccharomyces cerevisiae and some characterization of its metazoan counterpart indicates that most functional aspects are conserved. These studies have implicated the cytoplasmic exosome in the turnover of normal cellular mRNAs, as well as several mRNA surveillance pathways. For this, the exosome needs a set of four proteins that do not partake in nuclear exosome functions. These cofactors presumably direct the exosome to specific cytoplasmic RNA substrates. Here, we review cofactors and functions of the cytoplasmic exosome and provide unanswered questions on the mechanisms of cytoplasmic exosome function.

Defective mRNAs are degraded more rapidly than normal mRNAs in a process called mRNA surveillance. Eukaryotic cells use a variety of mechanisms to detect aberrations in mRNAs and a variety of enzymes to preferentially degrade them. Recent advances in the field of RNA surveillance have provided new information regarding how cells determine which mRNA species should be subject to destruction and novel mechanisms by which a cell tags an mRNA once such a decision has been reached. In this review, we highlight recent progress in our understanding of these processes.

The authors analyse the eukaryotic exosome structure, published in EMBO reports, in light of the known archaeal and prokaryotic exosomes, and discuss its striking flexibility and the conservation of the RNA channelling mechanism.

The essential RNA helicase, Mtr4, performs a critical role in RNA processing and degradation as an activator of the nuclear exosome. The molecular basis for this vital function is not understood and detailed analysis is significantly limited by the lack of structural data. In this study, we present the crystal structure of Mtr4. The structure reveals a new arch-like domain that is specific to Mtr4 and Ski2 (the cytosolic homologue of Mtr4). In vivo and in vitro analyses demonstrate that the Mtr4 arch domain is required for proper 5.8S rRNA processing, and suggest that the arch functions independently of canonical helicase activity. In addition, extensive conservation along the face of the putative RNA exit site highlights a potential interface with the exosome. These studies provide a molecular framework for understanding fundamental aspects of helicase function in exosome activation, and more broadly define the molecular architecture of Ski2-like helicases.

The exosome consists of a core of ten essential proteins that includes the ribonuclease Rrp44p and is present in both the cytoplasm and nucleus of eukaryotic cells. The cytoplasmic exosome has been extensively characterized in the budding yeast Saccharomyces cerevisiae and some characterization of its metazoan counterpart indicates that most functional aspects are conserved. These studies have implicated the cytoplasmic exosome in the turnover of normal cellular mRNAs, as well as several mRNA surveillance pathways. For this, the exosome needs a set of four proteins that do not partake in nuclear exosome functions. These cofactors presumably direct the exosome to specific cytoplasmic RNA substrates. Here, we review cofactors and functions of the cytoplasmic exosome and provide unanswered questions on the mechanisms of cytoplasmic exosome function.

A 3'-exoribonuclease complex, termed the exosome, has important functions in the cytoplasm, as well as in the nucleus, and is involved in 3'-processing and/or decay of many RNAs. This chapter will discuss methods to study cytoplasmic exosome function in yeast with in vivo approaches. The first section will describe mutants that are available to study the processing or decay of a specific RNA by the nuclear or cytoplasmic exosome. The second section will discuss methods to determine whether the cytoplasmic exosome is functional under a specific condition(s) with reporter mRNAs that are known substrates of this complex.

A 3'-exoribonuclease complex, termed the exosome, has important functions in the cytoplasm, as well as in the nucleus, and is involved in 3'-processing and/or decay of many RNAs. This chapter will discuss methods to study cytoplasmic exosome function in yeast with in vivo approaches. The first section will describe mutants that are available to study the processing or decay of a specific RNA by the nuclear or cytoplasmic exosome. The second section will discuss methods to determine whether the cytoplasmic exosome is functional under a specific condition(s) with reporter mRNAs that are known substrates of this complex.

The biogenesis of a number of RNA species in eukaryotic cells requires 3′ processing. To determine the enzymes responsible for these trimming events, we created yeast strains lacking specific 3′ to 5′ exonucleases. In this work, we describe the analysis of three members of the RNase D family of exonucleases (Rex1p, Rex2p and Rex3p). This work led to three important conclusions. First, each of these exonucleases is required for the processing of distinct RNAs. Specifically, Rex1p, Rex2p and Rex3p are required for 5S rRNA, U4 snRNA and MRP RNA trimming, respectively. Secondly, some 3′ exonucleases are redundant with other exonucleases. Specifically, Rex1p and Rex2p function redundantly in 5.8S rRNA maturation, Rex1p, Rex2p and Rex3p are redundant for the processing of U5 snRNA and RNase P RNA, and Rex1p and the exonuclease Rrp6p have an unknown redundant essential function. Thirdly, the demonstration that the Rex proteins can affect reactions that have been attributed previously to the exosome complex indicates that an apparently simple processing step can be surprisingly complex with multiple exonucleases working sequentially in the same pathway.