Structural basis of the unfolded protein response.

The unfolded protein response (UPR) is a network of intracellular signaling pathways that maintain the protein-folding capacity of the endoplasmic reticulum (ER) in eukaryotic cells. Dedicated molecular sensors embedded in the ER membrane detect incompletely folded or unfolded proteins in the ER lumen and activate a transcriptional program that increases the abundance of the ER according to need. In metazoans the UPR additionally regulates translation and thus relieves unfolded protein load by globally reducing protein synthesis. If homeostasis in the ER cannot be reestablished, the metazoan UPR switches from the prosurvival to the apoptotic mode. The UPR involves a complex, coordinated action of many genes that is controlled by one ER-embedded sensor, Ire1, in yeasts, and three sensors, Ire1, PERK, and ATF6, in higher eukaryotes, including human. We discuss the emerging molecular understanding of the UPR and focus on the structural biology of Ire1 and PERK, the two recently crystallized UPR sensors.

Human cDC1s display constitutive activation of the UPR sensor IRE1.

The intracellular mechanisms safeguarding DC function are of biomedical interest in several immune-related diseases. Type 1 conventional DCs (cDC1s) are prominent targets of immunotherapy typified by constitutive activation of the unfolded protein response (UPR) sensor IRE1. Through its RNase domain, IRE1 regulates key processes in cDC1s including survival, ER architecture and function. However, most evidence linking IRE1 RNase with cDC1 biology emerges from mouse studies and it is currently unknown whether human cDC1s also activate the enzyme to preserve cellular homeostasis. In this work, we report that human cDC1s constitutively activate IRE1 RNase in steady state, which is evidenced by marked expression of IRE1, XBP1s, and target genes, and low levels of mRNA substrates of the IRE1 RNase domain. On a functional level, pharmacological inhibition of the IRE1 RNase domain curtailed IL-12 and TNF production by cDC1s upon stimulation with TLR agonists. Altogether, this work demonstrates that activation of the IRE1/XBP1s axis is a conserved feature of cDC1s across species and suggests that the UPR sensor may also play a relevant role in the biology of the human lineage.

Quasi-essentiality of RNase Y in Bacillus subtilis is caused by its critical role in the control of mRNA homeostasis.

RNA turnover is essential in all domains of life. The endonuclease RNase Y (rny) is one of the key components involved in RNA metabolism of the model organism Bacillus subtilis. Essentiality of RNase Y has been a matter of discussion, since deletion of the rny gene is possible, but leads to severe phenotypic effects. In this work, we demonstrate that the rny mutant strain rapidly evolves suppressor mutations to at least partially alleviate these defects. All suppressor mutants had acquired a duplication of an about 60 kb long genomic region encompassing genes for all three core subunits of the RNA polymerase-α, ß, ß'. When the duplication of the RNA polymerase genes was prevented by relocation of the rpoA gene in the B. subtilis genome, all suppressor mutants carried distinct single point mutations in evolutionary conserved regions of genes coding either for the ß or ß' subunits of the RNA polymerase that were not tolerated by wild type bacteria. In vitro transcription assays with the mutated polymerase variants showed a severe decrease in transcription efficiency. Altogether, our results suggest a tight cooperation between RNase Y and the RNA polymerase to establish an optimal RNA homeostasis in B. subtilis cells.

MazF toxin causes alterations in Staphylococcus aureus transcriptome, translatome and proteome that underlie bacterial dormancy.

Antibiotic resistance is a serious problem which may be caused by bacterial dormancy. It has been suggested that bacterial toxin-antitoxin systems induce dormancy. We analyzed the genome-wide role of Staphylococcus aureus endoribonuclease toxin MazF using RNA-Seq, Ribo-Seq and quantitative proteomics. We characterized changes in transcriptome, translatome and proteome caused by MazF, and proposed that MazF decreases translation directly by cleaving mRNAs, and indirectly, by decreasing translation factors and by promoting ribosome hibernation. Important pathways affected during the early stage of MazF induction were identified: MazF increases cell wall thickness and decreases cell division; MazF activates SsrA-system which rescues stalled ribosomes, appearing as a result of MazF mRNA cleavage. These pathways may be promising targets for new antibacterial drugs that prevent bacteria dormancy. Finally, we described the overall impact of MazF on S. aureus cell physiology, and propose one of the mechanisms by which MazF might regulate cellular changes leading to dormancy.

The small DUF1127 protein CcaF1 from Rhodobacter sphaeroides is an RNA-binding protein involved in sRNA maturation and RNA turnover.

Many different protein domains are conserved among numerous species, but their function remains obscure. Proteins with DUF1127 domains number >17 000 in current databases, but a biological function has not yet been assigned to any of them. They are mostly found in alpha- and gammaproteobacteria, some of them plant and animal pathogens, symbionts or species used in industrial applications. Bioinformatic analyses revealed similarity of the DUF1127 domain of bacterial proteins to the RNA binding domain of eukaryotic Smaug proteins that are involved in RNA turnover and have a role in development from Drosophila to mammals. This study demonstrates that the 71 amino acid DUF1127 protein CcaF1 from the alphaproteobacterium Rhodobacter sphaeroides participates in maturation of the CcsR sRNAs that are processed from the 3' UTR of the ccaF mRNA and have a role in the oxidative stress defense. CcaF1 binds to many cellular RNAs of different type, several mRNAs with a function in cysteine / methionine / sulfur metabolism. It affects the stability of the CcsR RNAs and other non-coding RNAs and mRNAs. Thus, the widely distributed DUF1127 domain can mediate RNA-binding, affect stability of its binding partners and consequently modulate the bacterial transcriptome, thereby influencing different physiological processes.

Dimeric structure of pseudokinase RNase L bound to 2-5A reveals a basis for interferon-induced antiviral activity.

RNase L is an ankyrin repeat domain-containing dual endoribonuclease-pseudokinase that is activated by unusual 2,'5'-oligoadenylate (2-5A) second messengers and which impedes viral infections in higher vertebrates. Despite its importance in interferon-regulated antiviral innate immunity, relatively little is known about its precise mechanism of action. Here we present a functional characterization of 2.5 Å and 3.25 Å X-ray crystal and small-angle X-ray scattering structures of RNase L bound to a natural 2-5A activator with and without ADP or the nonhydrolysable ATP mimetic AMP-PNP. These studies reveal how recognition of 2-5A through interactions with the ankyrin repeat domain and the pseudokinase domain, together with nucleotide binding, imposes a rigid intertwined dimer configuration that is essential for RNase catalytic and antiviral functions. The involvement of the pseudokinase domain of RNase L in 2-5A sensing, nucleotide binding, dimerization, and ribonuclease functions highlights the evolutionary adaptability of the eukaryotic protein kinase fold.

Determinants of heterochromatic siRNA biogenesis and function.

Endogenous small interfering RNAs (siRNAs) and other classes of small RNA provide the specificity signals for silencing of transposons and repeated DNA elements at the posttranscriptional and transcriptional levels. However, the determinants that define an siRNA-producing region or control the silencing function of siRNAs are poorly understood. Here we show that convergent antisense transcription and availability of the Dicer ribonuclease are the key determinants for primary siRNA generation. Surprisingly, Dicer makes dual contributions to heterochromatin formation, promoting histone H3 lysine 9 methylation independently of its catalytic activity, in addition to its well-known role in catalyzing siRNA generation. Furthermore, sequences in the 3' UTR of an mRNA-coding gene inhibit the ability of siRNAs to promote heterochromatin formation, providing another layer of control that prevents the silencing of protein-coding RNAs. Our results reveal distinct mechanisms that limit siRNA generation to centromeric DNA repeats and prevent spurious siRNA-mediated silencing at euchromatic loci.

IRE1A Stimulates Hepatocyte-Derived Extracellular Vesicles That Promote Inflammation in Mice With Steatohepatitis.

BACKGROUND & AIMS: Endoplasmic reticulum to nucleus signaling 1 (ERN1, also called IRE1A) is a sensor of the unfolded protein response that is activated in the livers of patients with nonalcoholic steatohepatitis (NASH). Hepatocytes release ceramide-enriched inflammatory extracellular vesicles (EVs) after activation of IRE1A. We studied the effects of inhibiting IRE1A on release of inflammatory EVs in mice with diet-induced steatohepatitis. METHODS: C57BL/6J mice and mice with hepatocyte-specific disruption of Ire1a (IRE1αΔhep) were fed a diet high in fat, fructose, and cholesterol to induce development of steatohepatitis or a standard chow diet (controls). Some mice were given intraperitoneal injections of the IRE1A inhibitor 4µ8C. Mouse liver and primary hepatocytes were transduced with adenovirus or adeno-associated virus that expressed IRE1A. Livers were collected from mice and analyzed by quantitative polymerase chain reaction and chromatin immunoprecipitation assays; plasma samples were analyzed by enzyme-linked immunosorbent assay. EVs were derived from hepatocytes and injected intravenously into mice. Plasma EVs were characterized by nanoparticle-tracking analysis, electron microscopy, immunoblots, and nanoscale flow cytometry; we used a membrane-tagged reporter mouse to detect hepatocyte-derived EVs. Plasma and liver tissues from patients with NASH and without NASH (controls) were analyzed for EV concentration and by RNAscope and gene expression analyses. RESULTS: Disruption of Ire1a in hepatocytes or inhibition of IRE1A reduced the release of EVs and liver injury, inflammation, and accumulation of macrophages in mice on the diet high in fat, fructose, and cholesterol. Activation of IRE1A, in the livers of mice, stimulated release of hepatocyte-derived EVs, and also from cultured primary hepatocytes. Mice given intravenous injections of IRE1A-stimulated, hepatocyte-derived EVs accumulated monocyte-derived macrophages in the liver. IRE1A-stimulated EVs were enriched in ceramides. Chromatin immunoprecipitation showed that IRE1A activated X-box binding protein 1 (XBP1) to increase transcription of serine palmitoyltransferase genes, which encode the rate-limiting enzyme for ceramide biosynthesis. Administration of a pharmacologic inhibitor of serine palmitoyltransferase to mice reduced the release of EVs. Levels of XBP1 and serine palmitoyltransferase were increased in liver tissues, and numbers of EVs were increased in plasma, from patients with NASH compared with control samples and correlated with the histologic features of inflammation. CONCLUSIONS: In mouse hepatocytes, activated IRE1A promotes transcription of serine palmitoyltransferase genes via XBP1, resulting in ceramide biosynthesis and release of EVs. The EVs recruit monocyte-derived macrophages to the liver, resulting in inflammation and injury in mice with diet-induced steatohepatitis. Levels of XBP1, serine palmitoyltransferase, and EVs are all increased in liver tissues from patients with NASH. Strategies to block this pathway might be developed to reduce liver inflammation in patients with NASH.

RNase L Amplifies Interferon Signaling by Inducing Protein Kinase R-Mediated Antiviral Stress Granules.

Virus infection leads to activation of the interferon (IFN)-induced endoribonuclease RNase L, which results in degradation of viral and cellular RNAs. Both cellular and viral RNA cleavage products of RNase L bind pattern recognition receptors (PRRs), like retinoic acid-inducible I (Rig-I) and melanoma differentiation-associated protein 5 (MDA5), to further amplify IFN production and antiviral response. Although much is known about the mechanics of ligand binding and PRR activation, how cells coordinate RNA sensing with signaling response and interferon production remains unclear. We show that RNA cleavage products of RNase L activity induce the formation of antiviral stress granules (avSGs) by regulating activation of double-stranded RNA (dsRNA)-dependent protein kinase R (PKR) and recruit the antiviral proteins Rig-I, PKR, OAS, and RNase L to avSGs. Biochemical analysis of purified avSGs showed interaction of a key stress granule protein, G3BP1, with only PKR and Rig-I and not with OAS or RNase L. AvSG assembly during RNase L activation is required for IRF3-mediated IFN production, but not IFN signaling or proinflammatory cytokine induction. Consequently, cells lacking avSG formation or RNase L signaling produced less IFN and showed higher susceptibility during Sendai virus infection, demonstrating the importance of avSGs in RNase L-mediated host defense. We propose a role during viral infection for RNase L-cleaved RNAs in inducing avSGs containing antiviral proteins to provide a platform for efficient interaction of RNA ligands with pattern recognition receptors to enhance IFN production to mount an effective antiviral response.IMPORTANCE Double-stranded RNAs produced during viral infections serve as pathogen-associated molecular patterns (PAMPs) and bind pattern recognition receptors to stimulate IFN production. RNase L is an IFN-regulated endoribonuclease that is activated in virus-infected cells and cleaves single-stranded viral and cellular RNAs. The RNase L-cleaved dsRNAs signal to Rig-like helicases to amplify IFN production. This study identifies a novel role of antiviral stress granules induced by RNase L as an antiviral signaling hub to coordinate the RNA ligands with cognate receptors to mount an effective host response during viral infections.

Flavivirus internalization is regulated by a size-dependent endocytic pathway.

Flaviviruses enter host cells through the process of clathrin-mediated endocytosis, and the spectrum of host factors required for this process are incompletely understood. Here we found that lymphocyte antigen 6 locus E (LY6E) promotes the internalization of multiple flaviviruses, including West Nile virus, Zika virus, and dengue virus. Perhaps surprisingly, LY6E is dispensable for the internalization of the endogenous cargo transferrin, which is also dependent on clathrin-mediated endocytosis for uptake. Since viruses are substantially larger than transferrin, we reasoned that LY6E may be required for uptake of larger cargoes and tested this using transferrin-coated beads of similar size as flaviviruses. LY6E was indeed required for the internalization of transferrin-coated beads, suggesting that LY6E is selectively required for large cargo. Cell biological studies found that LY6E forms tubules upon viral infection and bead internalization, and we found that tubule formation was dependent on RNASEK, which is also required for flavivirus internalization, but not transferrin uptake. Indeed, we found that RNASEK is also required for the internalization of transferrin-coated beads, suggesting it functions upstream of LY6E. These LY6E tubules resembled microtubules, and we found that microtubule assembly was required for their formation and flavivirus uptake. Since microtubule end-binding proteins link microtubules to downstream activities, we screened the three end-binding proteins and found that EB3 promotes virus uptake and LY6E tubularization. Taken together, these results highlight a specialized pathway required for the uptake of large clathrin-dependent endocytosis cargoes, including flaviviruses.

The bacterial endoribonuclease RNase E can cleave RNA in the absence of the RNA chaperone Hfq.

RNase E is a component of the RNA degradosome complex and plays a key role in RNA degradation and maturation in Escherichia coli RNase E-mediated target RNA degradation typically involves the RNA chaperone Hfq and requires small guide RNAs (sRNAs) acting as a seed by binding to short (7-12-bp) complementary regions in target RNA sequences. Here, using recombinantly expressed and purified proteins, site-directed mutagenesis, and RNA cleavage and protein cross-linking assays, we investigated Hfq-independent RNA decay by RNase E. Exploring its RNA substrate preferences in the absence of Hfq, we observed that RNase E preferentially cleaves AU-rich sites of single-stranded regions of RNA substrates that are annealed to an sRNA that contains a monophosphate at its 5'-end. We further found that the quaternary structure of RNase E is also important for complete, Hfq-independent cleavage at sites both proximal and distal to the sRNA-binding site within target RNAs containing monophosphorylated 5'-ends. Of note, genetic RNase E variants with unstable quaternary structure exhibited decreased catalytic activity. In summary, our results show that RNase E can degrade its target RNAs in the absence of the RNA chaperone Hfq. We conclude that RNase E-mediated, Hfq-independent RNA decay in E. coli requires a cognate sRNA sequence for annealing to the target RNA, a 5'-monophosphate at the RNA 5'-end, and a stable RNase E quaternary structure.

RNase L Antiviral Activity Is Not a Critical Component of the Oas1b-Mediated Flavivirus Resistance Phenotype.

In mice, resistance to central nervous system (CNS) disease induced by members of the genus Flavivirus is conferred by an allele of the 2'-5' oligoadenylate synthetase 1b gene that encodes the inactive full-length protein (Oas1b-FL). The susceptibility allele encodes a C-terminally truncated protein (Oas1b-tr). We show that the efficiency of neuron infection in the brains of resistant and susceptible mice is similar after an intracranial inoculation of two flaviviruses, but amplification of viral proteins and double-stranded RNA (dsRNA) is inhibited in infected neurons in resistant mouse brains at later times. Active OAS proteins detect cytoplasmic dsRNA and synthesize short 2'-5'-linked oligoadenylates (2'-5'A) that interact with the latent endonuclease RNase L, causing it to dimerize and cleave single-stranded RNAs. To evaluate the contribution of RNase L to the resistance phenotype in vivo, we created a line of resistant RNase L-/- mice. Evidence of RNase L activation in infected RNase L+/+ mice was indicated by higher levels of viral RNA in the brains of infected RNase L-/- mice. Activation of type I interferon (IFN) signaling was detected in both resistant and susceptible brains, but Oas1a and Oas1b mRNA levels were lower in RNase L+/+ mice of both types, suggesting that activated RNase L also has a proflaviviral effect. Inhibition of virus replication was robust in resistant RNase L-/- mice, indicating that activated RNase L is not a critical factor in mediating this phenotype.IMPORTANCE The mouse genome encodes a family of Oas proteins that synthesize 2'-5'A in response to dsRNA. 2'-5'A activates the endonuclease RNase L to cleave single-stranded viral and cellular RNAs. The inactive, full-length Oas1b protein confers flavivirus-specific disease resistance. Although similar numbers of neurons were infected in resistant and susceptible brains after an intracranial virus infection, viral components amplified only in susceptible brains at later times. A line of resistant RNase L-/- mice was used to evaluate the contribution of RNase L to the resistance phenotype in vivo Activation of RNase L antiviral activity by flavivirus infection was indicated by increased viral RNA levels in the brains of RNase L-/- mice. Oas1a and Oas1b mRNA levels were higher in infected RNase L-/- mice, indicating that activated RNase L also have a proflaviviral affect. However, the resistance phenotype was equally robust in RNase L-/- and RNase L+/+ mice.

A novel mechanism of RNase L inhibition: Theiler's virus L* protein prevents 2-5A from binding to RNase L.

The OAS/RNase L pathway is one of the best-characterized effector pathways of the IFN antiviral response. It inhibits the replication of many viruses and ultimately promotes apoptosis of infected cells, contributing to the control of virus spread. However, viruses have evolved a range of escape strategies that act against different steps in the pathway. Here we unraveled a novel escape strategy involving Theiler's murine encephalomyelitis virus (TMEV) L* protein. Previously we found that L* was the first viral protein binding directly RNase L. Our current data show that L* binds the ankyrin repeats R1 and R2 of RNase L and inhibits 2'-5' oligoadenylates (2-5A) binding to RNase L. Thereby, L* prevents dimerization and oligomerization of RNase L in response to 2-5A. Using chimeric mouse hepatitis virus (MHV) expressing TMEV L*, we showed that L* efficiently inhibits RNase L in vivo. Interestingly, those data show that L* can functionally substitute for the MHV-encoded phosphodiesterase ns2, which acts upstream of L* in the OAS/RNase L pathway, by degrading 2-5A.

Ferroptosis mediated by the interaction between Mfn2 and IREα promotes arsenic-induced nonalcoholic steatohepatitis.

Exposure to arsenic is a risk factor for nonalcoholic steatohepatitis (NASH). Ferroptosis is a form of regulated cell death defined by the accumulation of lipid peroxidation. In the current study, we observed the occurrence of ferroptosis in arsenic-induced NASH by assessing ferroptosis related hallmarks. In vitro, we found that ferrostatin-1 effectively attenuated the executing of ferroptosis and NASH. Simultaneously, the expression of ACSL4 (acyl-CoA synthetase long-chain family member 4) was upregulated in rat's liver and L-02 cells exposed to arsenic. While, suppression of ACSL4 with rosiglitazone or ACSL4 siRNA remarkably alleviated arsenic-induced NASH and ferroptosis through diminishing 5-hydroxyeicosatetraenoic acid (5-HETE) content. Additionally, Mitofusin 2 (Mfn2), a physical tether between endoplasmic reticulum and mitochondria, has rarely been explored in the ferroptosis. Using Mfn2 siRNA or inositol-requiring enzyme 1 alpha (IRE1α) inhibitor, we found NASH and ferroptosis were obviously mitigated through reducing 5-HETE content. Importantly, Co-IP assay indicated that Mfn2 could interact with IRE1α and promoted the production of 5-HETE, ultimately led to ferroptosis and NASH. Collectively, our data showed that ferroptosis is involved in arsenic-induced NASH. These data provide insightful viewpoints into the mechanism of arsenic-induced NASH.

Epigenetic Regulation of Skeletal Tissue Integrity and Osteoporosis Development.

Bone turnover is sophisticatedly balanced by a dynamic coupling of bone formation and resorption at various rates. The orchestration of this continuous remodeling of the skeleton further affects other skeletal tissues through organ crosstalk. Chronic excessive bone resorption compromises bone mass and its porous microstructure as well as proper biomechanics. This accelerates the development of osteoporotic disorders, a leading cause of skeletal degeneration-associated disability and premature death. Bone-forming cells play important roles in maintaining bone deposit and osteoclastic resorption. A poor organelle machinery, such as mitochondrial dysfunction, endoplasmic reticulum stress, and defective autophagy, etc., dysregulates growth factor secretion, mineralization matrix production, or osteoclast-regulatory capacity in osteoblastic cells. A plethora of epigenetic pathways regulate bone formation, skeletal integrity, and the development of osteoporosis. MicroRNAs inhibit protein translation by binding the 3'-untranslated region of mRNAs or promote translation through post-transcriptional pathways. DNA methylation and post-translational modification of histones alter the chromatin structure, hindering histone enrichment in promoter regions. MicroRNA-processing enzymes and DNA as well as histone modification enzymes catalyze these modifying reactions. Gain and loss of these epigenetic modifiers in bone-forming cells affect their epigenetic landscapes, influencing bone homeostasis, microarchitectural integrity, and osteoporotic changes. This article conveys productive insights into biological roles of DNA methylation, microRNA, and histone modification and highlights their interactions during skeletal development and bone loss under physiological and pathological conditions.

[SLFN14 inhibits LINE-1 transposition activity].

Long interspersed nuclear element-1 (LINE-1) is the only active autonomous transposon in the human genome. Its transposition frequently induces host genome instability, leading to a variety of genetic diseases, including cancers. The host factors play important roles in inhibiting LINE-1 retrotransposition. As an important component of the immune system, the host factor SLFN14 has antiviral activity. Our laboratory shows that SLFN14 possesses potent inhibitory activity against LINE-1 retrotransposition. To explore the potential mechanism of SLFN14 inhibition, we analyzed its effects on transcription, translation, reverse transcription and insertion in the LINE-1 replication cycle. We confirmed that SLFN14 could suppress the LINE-1 mRNA level by affecting its transcription and degradation, thereby diminishing the protein and cDNA levels of LINE-1, which eventually block the LINE-1 retrotransposition. Further, by mapping the active domains of SLFN14, we found its inhibitory activity on LINE-1 being closely related to its endoribonuclease and ribosome binding domains. These results demonstrate the mechanism of SLFN14 in regulating LINE-1 replication, which further provide new insights for improving the regulation network of host factors for controlling genomic instability caused by LINE-1 replication.

The serine/threonine-protein kinase/endoribonuclease IRE1α protects the heart against pressure overload-induced heart failure.

Heart failure is associated with induction of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). The serine/threonine protein kinase/endoribonuclease IRE1α is a key protein in ER stress signal transduction. IRE1α activity can induce both protective UPR and apoptotic downstream signaling events, but the specific role for IRE1α activity in the heart is unknown. A major aim of this study was to characterize the specific contribution of IRE1α in cardiac physiology and pathogenesis. We used both cultured myocytes and a transgenic mouse line with inducible and cardiomyocyte-specific IRE1α overexpression as experimental models to achieve targeted IRE1α activation. IRE1α expression induced a potent but transient ER stress response in cardiomyocytes and did not cause significant effects in the intact heart under normal physiological conditions. Furthermore, the IRE1α-activated transgenic heart responding to pressure overload exhibited preserved function and reduced fibrotic area, associated with increased adaptive UPR signaling and with blunted inflammatory and pathological gene expression. Therefore, we conclude that IRE1α induces transient ER stress signaling and confers a protective effect against pressure overload-induced pathological remodeling in the heart. To our knowledge, this report provides first direct evidence of a specific and protective role for IRE1α in the heart and reveals an interaction between ER stress signaling and inflammatory regulation in the pathologically stressed heart.

The non-stop decay mRNA surveillance pathway is required for oxidative stress tolerance.

Reactive oxygen species (ROS) are toxic by-products of normal aerobic metabolism. ROS can damage mRNAs and the translational apparatus resulting in translational defects and aberrant protein production. Three mRNA quality control systems monitor mRNAs for translational errors: nonsense-mediated decay, non-stop decay (NSD) and no-go decay (NGD) pathways. Here, we show that factors required for the recognition of NSD substrates and components of the SKI complex are required for oxidant tolerance. We found an overlapping requirement for Ski7, which bridges the interaction between the SKI complex and the exosome, and NGD components (Dom34/Hbs1) which have been shown to function in both NSD and NGD. We show that ski7 dom34 and ski7 hbs1 mutants are sensitive to hydrogen peroxide stress and accumulate an NSD substrate. We further show that NSD substrates are generated during ROS exposure as a result of aggregation of the Sup35 translation termination factor, which increases stop codon read-through allowing ribosomes to translate into the 3΄-end of mRNAs. Overexpression of Sup35 decreases stop codon read-through and rescues oxidant tolerance consistent with this model. Our data reveal an unanticipated requirement for the NSD pathway during oxidative stress conditions which prevents the production of aberrant proteins from NSD mRNAs.

Downstream element determines RNase Y cleavage of the saePQRS operon in Staphylococcus aureus.

In gram-positive bacteria, RNase J1, RNase J2 and RNase Y are thought to be major contributors to mRNA degradation and maturation. In Staphylococcus aureus, RNase Y activity is restricted to regulating the mRNA decay of only certain transcripts. Here the saePQRS operon was used as a model to analyze RNase Y specificity in living cells. A RNase Y cleavage site is located in an intergenic region between saeP and saeQ. This cleavage resulted in rapid degradation of the upstream fragment and stabilization of the downstream fragment. Thereby, the expression ratio of the different components of the operon was shifted towards saeRS, emphasizing the regulatory role of RNase Y activity. To assess cleavage specificity different regions surrounding the sae CS were cloned upstream of truncated gfp, and processing was analyzed in vivo using probes up- and downstream of CS. RNase Y cleavage was not determined by the cleavage site sequence. Instead a 24-bp double-stranded recognition structure was identified that was required to initiate cleavage 6 nt upstream. The results indicate that RNase Y activity is determined by secondary structure recognition determinants, which guide cleavage from a distance.

RNase E action at a distance: degradation of target mRNAs mediated by an Hfq-binding small RNA in bacteria.

A major class of bacterial small RNAs (sRNAs), along with RNA-binding protein Hfq and endoribonuclease RNase E, acts on target mRNAs through base-pairing, leading to translational repression and rapid degradation of the mRNAs. In this issue of Genes & Development, Prévost and colleagues (pp. 385-396) demonstrate by using the well-characterized sRNA RyhB that RNase E cleavage at sites distal from the pairing region triggers degradation of target mRNAs. The study has provided an important insight into the initial events of sRNA-induced degradation of target mRNAs.