Optimization of Codon Translation Rates via tRNA Modifications Maintains Proteome Integrity

SummaryProteins begin to fold as they emerge from translating ribosomes. The kinetics of ribosome transit along a given mRNA can influence nascent chain folding, but the extent to which individual codon translation rates impact proteome integrity remains unknown. Here, we show that slower decoding of discrete codons elicits widespread protein aggregation in vivo. Using ribosome profiling, we find that loss of anticodon wobble uridine (U34) modifications in a subset of tRNAs leads to ribosome pausing at their cognate codons in S. cerevisiae and C. elegans. Cells lacking U34 modifications exhibit gene expression hallmarks of proteotoxic stress, accumulate aggregates of endogenous proteins, and are severely compromised in clearing stress-induced protein aggregates. Overexpression of hypomodified tRNAs alleviates ribosome pausing, concomitantly restoring protein homeostasis. Our findings demonstrate that modified U34 is an evolutionarily conserved accelerator of decoding and reveal an unanticipated role for tRNA modifications in maintaining proteome integrity

The Gcn4 transcription factor reduces protein synthesis capacity and extends yeast lifespan

In Saccharomyces cerevisiae, deletion of large ribosomal subunit protein-encoding genes increases the replicative lifespan in a Gcn4-dependent manner. However, how Gcn4, a key transcriptional activator of amino acid biosynthesis genes, increases lifespan, is unknown. Here we show that Gcn4 acts as a repressor of protein synthesis. By analyzing the messenger RNA and protein abundance, ribosome occupancy and protein synthesis rate in various yeast strains, we demonstrate that Gcn4 is sufficient to reduce protein synthesis and increase yeast lifespan. Chromatin immunoprecipitation reveals Gcn4 binding not only at genes that are activated, but also at genes, some encoding ribosomal proteins, that are repressed upon Gcn4 overexpression. The promoters of repressed genes contain Rap1 binding motifs. Our data suggest that Gcn4 is a central regulator of protein synthesis under multiple perturbations, including ribosomal protein gene deletions, calorie restriction, and rapamycin treatment, and provide an explanation for its role in longevity and stress response

Arterivirus Nsp1 Modulates the Accumulation of Minus-Strand Templates to Control the Relative Abundance of Viral mRNAs

The gene expression of plus-strand RNA viruses with a polycistronic genome depends on translation and replication of the genomic mRNA, as well as synthesis of subgenomic (sg) mRNAs. Arteriviruses and coronaviruses, distantly related members of the nidovirus order, employ a unique mechanism of discontinuous minus-strand RNA synthesis to generate subgenome-length templates for the synthesis of a nested set of sg mRNAs. Non-structural protein 1 (nsp1) of the arterivirus equine arteritis virus (EAV), a multifunctional regulator of viral RNA synthesis and virion biogenesis, was previously implicated in controlling the balance between genome replication and sg mRNA synthesis. Here, we employed reverse and forward genetics to gain insight into the multiple regulatory roles of nsp1. Our analysis revealed that the relative abundance of viral mRNAs is tightly controlled by an intricate network of interactions involving all nsp1 subdomains. Distinct nsp1 mutations affected the quantitative balance among viral mRNA species, and our data implicate nsp1 in controlling the accumulation of full-length and subgenome-length minus-strand templates for viral mRNA synthesis. The moderate differential changes in viral mRNA abundance of nsp1 mutants resulted in similarly altered viral protein levels, but progeny virus yields were greatly reduced. Pseudorevertant analysis provided compelling genetic evidence that balanced EAV mRNA accumulation is critical for efficient virus production. This first report on protein-mediated, mRNA-specific control of nidovirus RNA synthesis reveals the existence of an integral control mechanism to fine-tune replication, sg mRNA synthesis, and virus production, and establishes a major role for nsp1 in coordinating the arterivirus replicative cycle

Arterivirus Subgenomic mRNA Synthesis and Virion Biogenesis Depend on the Multifunctional nsp1 Autoprotease▿

Many groups of plus-stranded RNA viruses produce additional, subgenomic mRNAs to regulate the expression of part of their genome. Arteriviruses and coronaviruses (order Nidovirales) are unique among plus-stranded RNA viruses for using a mechanism of discontinuous RNA synthesis to produce a nested set of 5′- and 3′-coterminal subgenomic mRNAs, which serve to express the viral structural protein genes. The discontinuous step presumably occurs during minus-strand synthesis and joins noncontiguous sequences copied from the 3′- and 5′-proximal domains of the genomic template. Nidovirus genome amplification (“replication”) and subgenomic mRNA synthesis (“transcription”) are driven by 13 to 16 nonstructural proteins (nsp's), generated by autocatalytic processing of two large “replicase” polyproteins. Previously, using a replicon system, the N-terminal nsp1 replicase subunit of the arterivirus equine arteritis virus (EAV) was found to be dispensable for replication but crucial for transcription. Using reverse genetics, we have now addressed the role of nsp1 against the background of the complete EAV life cycle. Mutagenesis revealed that nsp1 is in fact a multifunctional regulatory protein. Its papain-like autoprotease domain releases nsp1 from the replicase polyproteins, a cleavage essential for viral RNA synthesis. Several mutations in the putative N-terminal zinc finger domain of nsp1 selectively abolished transcription, while replication was either not affected or even increased. Other nsp1 mutations did not significantly affect either replication or transcription but still dramatically reduced the production of infectious progeny. Thus, nsp1 is involved in at least three consecutive key processes in the EAV life cycle: replicase polyprotein processing, transcription, and virion biogenesis

Relative specific infectivities of virus particles from nsp1 mutants with imbalanced mRNA profiles.

a<p>Virus titers were determined by plaque assays of culture supernatants harvested at 24 h post-transfection and normalized to the pfu/ml value of the wt control, which was set at 1.</p>b<p>EAV genomic RNA levels were quantified in virion preparations obtained from culture supernatants harvested at 24 h post-transfection by reverse transcription and quantitative PCR. Values for mutant virions were obtained by comparing their threshold cycle (Ct) against the qPCR standard curve, and were normalized relative to the genomic RNA level in the wt control, which was set at 1.</p>c<p>Relative specific infectivity values were calculated by dividing the relative infectivity (mutant∶wt pfu ratio) by the relative genomic RNA content for each construct.</p>d<p>The data shown are derived from two independent experiments.</p

Minus-strand RNA accumulation is also modulated by mutations in nsp1.

<p>(A–D) Analysis and quantification of EAV minus-strand accumulation by a two-cycle RNase protection assay. (A) Schematic representation of the nested set of viral minus-strand RNA [(−)RNA] species produced in EAV-infected cells. The anti-leader sequence is depicted in light green. The in vitro-transcribed plus-strand probes used for detection of (−)RNA1 (pRNA1), (−)RNA6 (pRNA6) and (−) RNA7 (pRNA7) are shown. pRNA6 and pRNA7 target the leader-body junction sequences of (−)RNA6 and (−)RNA7, respectively. Note that hybridization with pRNA1 results in the protection of a single fragment, while the probes for (−)RNAs 6 and 7 each protect three fragments – one derived from the full-length sg minus strand, and two fragments derived in part from partial hybridization of these probes to larger viral (−)RNAs in which the target sequences are noncontiguous (exemplified for pRNA6). For simplicity, non-EAV sequences present near the termini of the three probes were omitted from the scheme. (B) Viral (−)RNA accumulation was analyzed at 11 h post-transfection for the ZCH, A1 and A4 mutants, and a wt control. Protected fragments were resolved on denaturing 5% polyacrylamide/8M urea gels and visualized by phosphorimaging. The constructs analyzed are labeled above the lanes (M, mock-transfected cells; (−), no-RNase control that shows a band corresponding to 0.2 fmol of the full-length probe). Sizes (nt) of RNA markers have been indicated on the left. The single 327-nt protected fragment resulting from hybridization with the positive-sense probe for RNA1(−) is indicated. The probes for subgenome-length minus strands protected fragments derived from the full-length (−)RNA6 and (−)RNA7 (327 nt and 319 nt, respectively; denoted with LB), as well as from the (−)RNA6 and (−)RNA7 body sequences (188 nt and 180 nt, respectively; denoted with B) and the anti-leader sequence (139 nt; denoted with L). The presence of two bands in the size range of the anti-leader fragment has been described previously <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000772#ppat.1000772-denBoon3" target="_blank">[55]</a>. (C) The relative levels of minus-strand accumulation were quantified by phosphorimaging. For (−)RNAs 6 and 7, only the bands resulting from protection of full-length sg minus strands (denoted with LB in panel [B]) were quantified. The values correspond to the means from three independent transfections that were normalized to the level of accumulation of each minus-strand RNA in the wt control, which was set at 1. Intracellular RNA from the same transfection samples for which plus-strand accumulation was quantified (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000772#ppat-1000772-g004" target="_blank">Fig. 4B</a>) was used. Genomic minus-strand RNA levels are represented as dark blue bars. Error bars denote standard deviation. (D) The ratio of plus-strand to minus-strand accumulation for RNAs 1, 6 and 7 was calculated using the mean relative values obtained in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000772#ppat-1000772-g004" target="_blank">Fig. 4B</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000772#ppat-1000772-g005" target="_blank">Fig. 5C</a>.</p

Analysis of viral protein accumulation and virus production by selected nsp1 mutants.

<p>(A) Western blot analysis of EAV-specific protein accumulation. Cells transfected with wt or mutant viral genomes were harvested 11 h after transfection and equal amounts of total protein were analyzed with EAV-specific sera detecting nsp3, M and N, which are translated from RNAs 1, 6 and 7, respectively. The relative levels of each mRNA template (derived from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000772#ppat-1000772-g004" target="_blank">Fig. 4B</a>) are indicated below the gels. Beta-actin was used as a loading control. (B) Plaque phenotype and virus titers of the ZCH, A1 and A4 mutants in comparison with wt. Plaque assays were performed on BHK-21 using cell culture supernatants harvested 24 h after transfection. Virus titers represent an average of three independent experiments.</p

Relationship between viral mRNA accumulation profiles and infectious virus yield.

<p>The data obtained on the accumulation of genome and sg mRNAs were used for a quantitative assessment of nsp1 mutant phenotypes in terms of (A) changes in mRNA accumulation compared to wt and (B) the extent to which the balance between viral transcripts was disturbed (B). (A) For each mutant and pseudorevertant, a value for the accumulation of each of its 7 mRNAs at 11 h post-transfection had been assigned as compared to that of the wt control (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000772#ppat-1000772-g004" target="_blank">Fig. 4B</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000772#ppat-1000772-g007" target="_blank">7B and 7D</a>). The mean of these seven values (“mean relative mRNA accumulation”) was plotted against the corresponding progeny virus titer in culture supernatants at 24 h post-transfection (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000772#ppat-1000772-g006" target="_blank">Fig. 6B</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000772#ppat-1000772-t002" target="_blank">table 2</a>). Wild-type is depicted in orange. The engineered nsp1 mutants and nsp1 pseudorevertants are shown in blue and purple, respectively. The three pseudorevertants of mutant A4, displaying very similar phenotypes, are indicated as A4+PSR. (B) For each mRNA species, the deviation of its relative accumulation from the mean of the complete nested set of mRNAs (see panel A) was calculated. From these seven values, the mean (absolute) deviation was calculated for each mutant and plotted against virus titers as in (A). For wt, the mean deviation is 0. Engineered nsp1 mutants and their pseudorevertants are indicated as in (A). The data fit a negative exponential regression calculated using Microsoft Excel and depicted as a gray line (y = 6×10⁶+7e^−8.4306x, R² = 0.95). The inset shows an expanded view of the upper left quadrant of the graph (shaded in gray).</p

Overview of EAV nsp1 pseudorevertants described in this study.

a<p>Virus clones were isolated from plaque assays of culture supernatants harvested between 20 h and 24 h post transfection. The number of clones containing each mutation is indicated in brackets.</p>b<p>Numbers indicate the start coordinate of the codon in the EAV genome.</p>c<p>Pseudorevertants were reconstructed in the wild-type EAV background, and infectious progeny titers were determined by plaque assays of culture supernatants harvested at 24 h post-transfection. The average titers of three independent experiments are shown.</p>d<p>NA, not applicable.</p>e<p>A silent mutation changing the original alanine codon from GCG to GCC was introduced as a marker mutation upon construction of the ZCH mutant.</p

Domain organization of EAV nsp1.

<p>The partial sequence alignment shows key regions in the three subdomains previously identified in the arterivirus nsp1 region. GenBank accession numbers for the full-length arterivirus genomes used for the alignment are as follows: EAV, NC_002532; simian hemorrhagic fever virus (SHFV), NC_003092; lactate dehydrogenase-elevating virus (LDV-P and LDV-C), NC_001639 and NC_002534; PRRSV-LV, M96262.2; PRRSV-VR, AY150564. Zinc-coordinating residues are indicated in bold font; the active-site Cys and His of PCPα and PCPβ are indicated with triangles (note the loss of the active-site Cys in EAV PCPα). The positions of amino acid clusters mutated in this study are indicated with arrows. All substitutions were with Ala, with the exception of the ZCH construct, in which Cys-25 and His-27 were swapped. The positions of mutations found in pseudorevertants are indicated with open circles.</p