The Somatic Reproductive Tissues of C. elegans Promote Longevity through Steroid Hormone Signaling

Removal of the germ cells of C. elegans extends lifespan in part because signals from the somatic reproductive tissues activate the nuclear hormone receptor DAF-12

Coordinated Destruction of Cellular Messages in Translation Complexes by the Gammaherpesvirus Host Shutoff Factor and the Mammalian Exonuclease Xrn1

Several viruses encode factors that promote host mRNA degradation to silence gene expression. It is unclear, however, whether cellular mRNA turnover pathways are engaged to assist in this process. In Kaposi's sarcoma-associated herpesvirus this phenotype is enacted by the host shutoff factor SOX. Here we show that SOX-induced mRNA turnover is a two-step process, in which mRNAs are first cleaved internally by SOX itself then degraded by the cellular exonuclease Xrn1. SOX therefore bypasses the regulatory steps of deadenylation and decapping normally required for Xrn1 activation. SOX is likely recruited to translating mRNAs, as it cosediments with translation initiation complexes and depletes polysomes. Cleaved mRNA intermediates accumulate in the 40S fraction, indicating that recognition occurs at an early stage of translation. This is the first example of a viral protein commandeering cellular mRNA turnover pathways to destroy host mRNAs, and suggests that Xrn1 is poised to deplete messages undergoing translation in mammalian cells

Canagliflozin and renal outcomes in type 2 diabetes and nephropathy

BACKGROUND Type 2 diabetes mellitus is the leading cause of kidney failure worldwide, but few effective long-term treatments are available. In cardiovascular trials of inhibitors of sodium–glucose cotransporter 2 (SGLT2), exploratory results have suggested that such drugs may improve renal outcomes in patients with type 2 diabetes. METHODS In this double-blind, randomized trial, we assigned patients with type 2 diabetes and albuminuric chronic kidney disease to receive canagliflozin, an oral SGLT2 inhibitor, at a dose of 100 mg daily or placebo. All the patients had an estimated glomerular filtration rate (GFR) of 30 to <90 ml per minute per 1.73 m2 of body-surface area and albuminuria (ratio of albumin [mg] to creatinine [g], >300 to 5000) and were treated with renin–angiotensin system blockade. The primary outcome was a composite of end-stage kidney disease (dialysis, transplantation, or a sustained estimated GFR of <15 ml per minute per 1.73 m2), a doubling of the serum creatinine level, or death from renal or cardiovascular causes. Prespecified secondary outcomes were tested hierarchically. RESULTS The trial was stopped early after a planned interim analysis on the recommendation of the data and safety monitoring committee. At that time, 4401 patients had undergone randomization, with a median follow-up of 2.62 years. The relative risk of the primary outcome was 30% lower in the canagliflozin group than in the placebo group, with event rates of 43.2 and 61.2 per 1000 patient-years, respectively (hazard ratio, 0.70; 95% confidence interval [CI], 0.59 to 0.82; P=0.00001). The relative risk of the renal-specific composite of end-stage kidney disease, a doubling of the creatinine level, or death from renal causes was lower by 34% (hazard ratio, 0.66; 95% CI, 0.53 to 0.81; P<0.001), and the relative risk of end-stage kidney disease was lower by 32% (hazard ratio, 0.68; 95% CI, 0.54 to 0.86; P=0.002). The canagliflozin group also had a lower risk of cardiovascular death, myocardial infarction, or stroke (hazard ratio, 0.80; 95% CI, 0.67 to 0.95; P=0.01) and hospitalization for heart failure (hazard ratio, 0.61; 95% CI, 0.47 to 0.80; P<0.001). There were no significant differences in rates of amputation or fracture. CONCLUSIONS In patients with type 2 diabetes and kidney disease, the risk of kidney failure and cardiovascular events was lower in the canagliflozin group than in the placebo group at a median follow-up of 2.62 years

Genes acting downstream of sensory neurons to influence longevity, dauer formation and pathogen responses in C. elegans.

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Cut site preference allows influenza A virus PA-X to discriminate between host and viral mRNAs

Many viruses block host gene expression to take over the infected cell. This process, termed host shutoff, is thought to promote viral replication by preventing antiviral responses and redirecting cellular resources to viral processes. Several viruses from divergent families accomplish host shutoff through RNA degradation by endoribonucleases. However, viruses also need to ensure expression of their own genes. The influenza A virus endoribonuclease PA-X solves this problem by sparing viral mRNAs and some host RNAs necessary for viral replication. To understand how PA-X distinguishes between RNAs, we characterized PA-X cut sites transcriptome-wide using 5' rapid amplification of complementary DNA ends coupled to high-throughput sequencing. This analysis, along with RNA structure predictions and validation experiments using reporters, shows that PA-Xs from multiple influenza strains preferentially cleave RNAs at GCUG tetramers in hairpin loops. Importantly, GCUG tetramers are enriched in the human but not the influenza transcriptome. Moreover, optimal PA-X cut sites inserted in the influenza A virus genome are quickly selected against during viral replication in cells. This finding suggests that PA-X evolved these cleavage characteristics to preferentially target host over viral mRNAs in a manner reminiscent of cellular self versus non-self discrimination

Coordinated destruction of cellular messages in translation complexes by the gammaherpesvirus host shutoff factor and the mammalian exonuclease Xrn1.

Several viruses encode factors that promote host mRNA degradation to silence gene expression. It is unclear, however, whether cellular mRNA turnover pathways are engaged to assist in this process. In Kaposi's sarcoma-associated herpesvirus this phenotype is enacted by the host shutoff factor SOX. Here we show that SOX-induced mRNA turnover is a two-step process, in which mRNAs are first cleaved internally by SOX itself then degraded by the cellular exonuclease Xrn1. SOX therefore bypasses the regulatory steps of deadenylation and decapping normally required for Xrn1 activation. SOX is likely recruited to translating mRNAs, as it cosediments with translation initiation complexes and depletes polysomes. Cleaved mRNA intermediates accumulate in the 40S fraction, indicating that recognition occurs at an early stage of translation. This is the first example of a viral protein commandeering cellular mRNA turnover pathways to destroy host mRNAs, and suggests that Xrn1 is poised to deplete messages undergoing translation in mammalian cells

Selective Degradation of Host RNA Polymerase II Transcripts by Influenza A Virus PA-X Host Shutoff Protein

<div><p>Influenza A viruses (IAVs) inhibit host gene expression by a process known as host shutoff. Host shutoff limits host innate immune responses and may also redirect the translation apparatus to the production of viral proteins. Multiple IAV proteins regulate host shutoff, including PA-X, a ribonuclease that remains incompletely characterized. We report that PA-X selectively targets host RNA polymerase II (Pol II) transcribed mRNAs, while sparing products of Pol I and Pol III. Interestingly, we show that PA-X can also target Pol II-transcribed RNAs in the nucleus, including non-coding RNAs that are not destined to be translated, and reporter transcripts with RNA hairpin structures that block ribosome loading. Transcript degradation likely occurs in the nucleus, as PA-X is enriched in the nucleus and its nuclear localization correlates with reduction in target RNA levels. Complete degradation of host mRNAs following PA-X-mediated endonucleolytic cleavage is dependent on the host 5’->3’-exonuclease Xrn1. IAV mRNAs are structurally similar to host mRNAs, but are synthesized and modified at the 3’ end by the action of the viral RNA-dependent RNA polymerase complex. Infection of cells with wild-type IAV or a recombinant PA-X-deficient virus revealed that IAV mRNAs resist PA-X-mediated degradation during infection. At the same time, loss of PA-X resulted in changes in the synthesis of select viral mRNAs and a decrease in viral protein accumulation. Collectively, these results significantly advance our understanding of IAV host shutoff, and suggest that the PA-X causes selective degradation of host mRNAs by discriminating some aspect of Pol II-dependent RNA biogenesis in the nucleus.</p></div

PA-X accumulates and functions in the cell nucleus.

<p>(A and B) A549 cells expressing doxycycline-inducible wild-type PA-X-myc (“iPA-X wt”, line #10) or a catalytically inactive mutant (“iPA-X D108A”, line #8) were treated with doxycycline for 18 h to induce expression of PA-X. Total RNA was isolated from either nuclear or cytoplasmic fraction and RT-qPCR was performed to measure levels of the indicated endogenous RNAs. RNA levels were normalized by 18S rRNA levels and are plotted as fold change in induced (PA-X-expressing) vs. uninduced cells. In (A), target level changes in nuclear (nuc) and cytoplasmic (cyto) fractions are presented. In (B), changes in total nuclear mRNA and in unspliced pre-mRNA are compared. Error bars = s.e.m, n = 3–4. (C) Schematic diagram of the GFP fusion constructs generated in this study. (D) Amino acid sequences of the X-ORF from PR8 strain (X61) aligned to a shortened 41 amino-acid construct (X41) and the mutant X-ORF with alanine substitutions at four basic residues (X61(4A)). Amino acids that are highly conserved between IAV strains are shown in bold and the basic residues that are important for PA-X function are highlighted in blue. (E and F) Fluorescence microscopy images of 293A cells transfected with the expression constructs for the indicated GFP fusion proteins listed in (C). Two fields of view are shown for each transfection experiment. Representative cells with nuclear GFP signal accumulation are highlighted with filled arrowheads and those that have even signal distribution with open arrowheads. Nuclei were stained with Hoechst dye. (G and H) Relative Firefly (FF-luc, G) and Renilla (RE-luc, H) luciferase reporter expression in 293A cells co-transfected with these reporters and the indicated wild-type and mutant PA-X constructs. Error bars = st. dev. from four independent biological replicates. *,** = <i>p</i> value < 0.05, 0.01 (**); ns = <i>p</i> value > 0.05 (Anova single factor).</p

PA-X targets Pol II-transcribed mRNAs that are not translated.

<p>(A) HEK 293T cells were transfected with Pol II-driven GFP reporters and either PA-X or an empty vector. The hp-GFP construct contains a hairpin close to the 5’ end of the GFP mRNA that blocks its translation. Total cellular RNA was extracted and GFP mRNA levels were measured by RT-qPCR. GFP expression normalized by 18S rRNA abundance is plotted as fold change in PA-X vs. vector-transfected cells. Error bars = st. dev. (B) 293T iPA-X cells were transfected with a T7 polymerase expressing construct and an EMCV IRES-containing luciferase reporter driven by the T7 promoter, which results in T7-mediated luciferase RNA transcription. PA-X expression was induced 5 h after transfection by doxycycline addition for 18 h. Total cellular RNA was extracted and the levels of the luciferase reporter and endogenous actin mRNA were measured by RT-qPCR. RNA levels were normalized by 18S rRNA abundance and are plotted as fold change in induced (PA-X-expressing) vs. uninduced cells. (C-D) HEK 293T (C) or A549 (D) cells expressing doxycycline-inducible wild-type PA-X-myc (“iPA-X wt”), a catalytically inactive mutant (“iPA-X D108A”) or RFP (“iRFP”) were treated with doxycycline for 18 h to induce expression of PA-X. Total cellular RNA was extracted and the levels of the indicated endogenous RNAs were measured by RT-qPCR. Levels of actin mRNAs (also shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005427#ppat.1005427.g001" target="_blank">Fig 1</a>) and Pol II-driven non-coding RNAs were normalized to 18S rRNA levels and are plotted as fold change in induced (PA-X- or RFP-expressing) vs. uninduced cells. HEK293T iPA-X line #7 and A549 iPA-X wt line #10 and D108A line #8 were used for this figure. Data on additional clonal lines is presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005427#ppat.1005427.s002" target="_blank">S2B and S2C Fig</a> *,**,*** = <i>p</i> value (Student’s <i>t</i>-test) < 0.05, 0.01, 0.001 vs. 293T iRFP (C, n = 5) or A549 iPA-X D108A #8 (D, n = 3). Error bars = s.e.m.</p

Canonical processing of the 3’ end of Pol II transcripts confers sensitivity to PA-X.

<p>(A) Diagram of the GFP reporter constructs used in the figure, which bear different 3’ mRNA ends: the canonical poly(A) tail added by cellular processing machinery (poly(A)), a hammerhead ribozyme (HR), the hammerhead ribozyme proceeded by a 60-nt templated poly(A) stretch (A60-HR), or the histone stem-loop structure (hisSL). The arrows indicate where the HR self-cleaves the RNA. (B-D) HEK 293Ts were transfected with indicated Pol-II driven GFP reporters and either PA-X or empty vector. Total cellular RNA was extracted and GFP mRNA levels were measured by RT-qPCR (B) or northern blotting (C). GFP expression normalized by 18S rRNA abundance is plotted (B) or reported (C, relative levels) as fold change in PA-X-expressing vs. vector-transfected cells. (D) Levels of GFP for experiments in panel B were assessed by western blot, using tubulin as a loading control. A representative image is shown. ** = <i>p</i> value (Student’s <i>t</i>-test) < 0.01 for fold change of indicated reporter vs. fold change of poly(A) GFP. (E) HEK 293Ts were transfected with indicated Pol-II driven GFP reporters and PA-X from three different IAV strains or empty vector. Total cellular RNA was extracted and GFP or actin mRNA levels were measured by RT-qPCR. GFP expression normalized by 18S rRNA abundance is plotted as fold change in PA-X-expressing vs. vector-transfected cells. Error bars = s.e.m.</p