Loss of a Conserved tRNA Anticodon Modification Perturbs Cellular Signaling

Transfer RNA (tRNA) modifications enhance the efficiency, specificity and fidelity of translation in all organisms. The anticodon modification mcm[superscript 5]s[superscript 2]U[superscript 34] is required for normal growth and stress resistance in yeast; mutants lacking this modification have numerous phenotypes. Mutations in the homologous human genes are linked to neurological disease. The yeast phenotypes can be ameliorated by overexpression of specific tRNAs, suggesting that the modifications are necessary for efficient translation of specific codons. We determined the in vivo ribosome distributions at single codon resolution in yeast strains lacking mcm[superscript 5]s[superscript 2]U. We found accumulations at AAA, CAA, and GAA codons, suggesting that translation is slow when these codons are in the ribosomal A site, but these changes appeared too small to affect protein output. Instead, we observed activation of the GCN4-mediated stress response by a non-canonical pathway. Thus, loss of mcm[superscript 5]s[superscript 2]U causes global effects on gene expression due to perturbation of cellular signaling.National Institutes of Health (U.S.) (Grant GM081399

Translation initiation factor eIF4G1 preferentially binds yeast transcript leaders containing conserved oligo-uridine motifs

Translational control of gene expression plays essential roles in cellular stress responses and organismal development by enabling rapid, selective, and localized control of protein production. Translational regulation depends on context-dependent differences in the protein output of mRNAs, but the key mRNA features that distinguish efficiently translated mRNAs are largely unknown. Here, we comprehensively determined the RNA-binding preferences of the eukaryotic initiation factor 4G (eIF4G) to assess whether this core translation initiation factor has intrinsic sequence preferences that may contribute to preferential translation of specific mRNAs. We identified a simple RNA sequence motif-oligo-uridine-that mediates high-affinity binding to eIF4G in vitro. Oligo(U) motifs occur naturally in the transcript leader (TL) of hundreds of yeast genes, and mRNAs with unstructured oligo(U) motifs were enriched in immunoprecipitations against eIF4G. Ribosome profiling following depletion of eIF4G in vivo showed preferentially reduced translation of mRNAs with long TLs, including those that contain oligo(U). Finally, TL oligo(U) elements are enriched in genes with regulatory roles and are conserved between yeast species, consistent with an important cellular function. Taken together, our results demonstrate RNA sequence preferences for a general initiation factor, which cells potentially exploit for translational control of specific mRNAs. Keywords: RNA binding; eIF4G; ribosome footprint profiling; transcript leaders; translation initiationNational Institutes of Health (U.S.) (Grant GM094303)National Institutes of Health (U.S.) (Grant GM081399)National Institutes of Health (U.S.) (Grant T32GM007287

Protein kinase A regulates gene-specific translational adaptation in differentiating yeast

Cellular differentiation is driven by coordinately regulated changes in gene expression. Recent discoveries suggest that translation contributes as much as transcription to regulating protein abundance, but the role of translational regulation in cellular differentiation is largely unexplored. Here we investigate translational reprogramming in yeast during cellular adaptation to the absence of glucose, a stimulus that induces invasive filamentous differentiation. Using ribosome footprint profiling and RNA sequencing to assay gene-specific translation activity genome-wide, we show that prolonged glucose withdrawal is accompanied by gene-specific changes in translational efficiency that significantly affect expression of the majority of genes. Notably, transcripts from a small minority (<5%) of genes make up the majority of translating mRNA in both rapidly dividing and starved differentiating cells, and the identities of these highly translated messages are almost nonoverlapping between conditions. Furthermore, these two groups of messages are subject to condition-dependent translational privilege. Thus the “housekeeping” process of translation does not stay constant during cellular differentiation but is highly adapted to different growth conditions. By comparing glucose starvation to growth-attenuating stresses that do not induce invasive filamentation, we distinguish a glucose-specific translational response mediated through signaling by protein kinase A (PKA). Together, these findings reveal a high degree of growth-state specialization of the translatome and identify PKA as an important regulator of gene-specific translation activity.National Institutes of Health (U.S.) (R01 GM094303

EDF1 coordinates cellular responses to ribosome collisions

Translation of aberrant mRNAs induces ribosomal collisions, thereby triggering pathways for mRNA and nascent peptide degradation and ribosomal rescue. Here we use sucrose gradient fractionation combined with quantitative proteomics to systematically identify proteins associated with collided ribosomes. This approach identified Endothelial differentiation-related factor 1 (EDF1) as a novel protein recruited to collided ribosomes during translational distress. Cryo-electron microscopic analyses of EDF1 and its yeast homolog Mbf1 revealed a conserved 40S ribosomal subunit binding site at the mRNA entry channel near the collision interface. EDF1 recruits the translational repressors GIGYF2 and EIF4E2 to collided ribosomes to initiate a negative-feedback loop that prevents new ribosomes from translating defective mRNAs. Further, EDF1 regulates an immediate-early transcriptional response to ribosomal collisions. Our results uncover mechanisms through which EDF1 coordinates multiple responses of the ribosome-mediated quality control pathway and provide novel insights into the intersection of ribosome-mediated quality control with global transcriptional regulation

The Myxobacterial Antibiotic Myxovalargin: Biosynthesis, Structural Revision, Total Synthesis, and Molecular Characterization of Ribosomal Inhibition

Resistance of bacterial pathogens against antibiotics is declared by WHO as a major global health threat. As novel antibacterial agents are urgently needed, we re-assessed the broad-spectrum myxobacterial antibiotic myxovalargin and found it to be extremely potent against Mycobacterium tuberculosis. To ensure compound supply for further development, we studied myxovalargin biosynthesis in detail enabling production via fermentation of a native producer. Feeding experiments as well as functional genomics analysis suggested a structural revision, which was eventually corroborated by the development of a concise total synthesis. The ribosome was identified as the molecular target based on resistant mutant sequencing, and a cryo-EM structure revealed that myxovalargin binds within and completely occludes the exit tunnel, consistent with a mode of action to arrest translation during a late stage of translation initiation. These studies open avenues for structure-based scaffold improvement toward development as an antibacterial agent

Pseudouridine profiling reveals regulated mRNA pseudouridylation in yeast and human cells

Post-transcriptional modification of RNA nucleosides occurs in all living organisms. Pseudouridine, the most abundant modified nucleoside in non-coding RNAs, enhances the function of transfer RNA and ribosomal RNA by stabilizing the RNA structure. Messenger RNAs were not known to contain pseudouridine, but artificial pseudouridylation dramatically affects mRNA function—it changes the genetic code by facilitating non-canonical base pairing in the ribosome decoding centre. However, without evidence of naturally occurring mRNA pseudouridylation, its physiological relevance was unclear. Here we present a comprehensive analysis of pseudouridylation in Saccharomyces cerevisiae and human RNAs using Pseudo-seq, a genome-wide, single-nucleotide-resolution method for pseudouridine identification. Pseudo-seq accurately identifies known modification sites as well as many novel sites in non-coding RNAs, and reveals hundreds of pseudouridylated sites in mRNAs. Genetic analysis allowed us to assign most of the new modification sites to one of seven conserved pseudouridine synthases, Pus1–4, 6, 7 and 9. Notably, the majority of pseudouridines in mRNA are regulated in response to environmental signals, such as nutrient deprivation in yeast and serum starvation in human cells. These results suggest a mechanism for the rapid and regulated rewiring of the genetic code through inducible mRNA modifications. Our findings reveal unanticipated roles for pseudouridylation and provide a resource for identifying the targets of pseudouridine synthases implicated in human disease.American Cancer Society (Robbie Sue Mudd Kidney Cancer Research Scholar Grant RSG-13-396-01-RMC)National Institutes of Health (U.S.) (GM094303)National Institutes of Health (U.S.) (GM081399)American Cancer Society. New England Division (Ellison Foundation Postdoctoral Fellowship)American Cancer Society (Postdoctoral Fellowship PF-13-319-01-RMC)National Institutes of Health (U.S.) (Pre-doctoral Training Grant T32GM007287

Determinants of translational efficiency in Saccharomyces cerevisiae

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2015.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.Cataloged from student-submitted PDF version of thesis.Includes bibliographical references.The goal of this thesis is to elucidate the mechanisms that govern translational efficiency (TE) - the amount of protein produced from each molecule of mRNA. While the mechanisms regulating the TE of a few specific messages are well understood, the general contribution of translational control to differences in cellular protein levels is currently unclear. Recent advances have enabled the direct measurement of protein levels and translation rates genome-wide, and studies in multiple organisms have found varying degrees of translation regulation, both at steady state, and in response to stress or developmental cues. Despite this influx of high-throughput data, the mechanisms underlying the differences in gene-specific and condition-dependent TE remain largely unknown. In this thesis, I describe the roles of two different components of the translational machinery in regulating translational efficiency. In Chapter 1, I discuss the features of mRNA coding sequences that can affect TE, thereby introducing Chapter 2, in which I investigate the role of a conserved anticodon tRNA modification in determining the rate of translation elongation and the phenotypic consequences of its loss for budding yeast. In Chapter 3, I discuss the regulation of translation initiation to introduce Chapter 4, in which I explore how the RNA binding specificity of the core translation factor, yeast eukaryotic initiation factor 4G (eIF4G), contributes to genome-wide competition between mRNAs. Finally, I will discuss future directions for this work.by Boris Zinshteyn.Ph. D

<i>GCN4</i> is induced independently of <i>GCN2</i> in MSUM strains.

<p>(<b>A</b>) Ribo-seq and RNA-seq RPKMs for the <i>GCN4</i> open reading frame. Standard deviations are indicated for strains with replicate data. (<b>B</b>) The indicated strains were transformed with a reporter containing the promoter and transcript leader of <i>GCN4</i> fused to <i>lacZ</i>. <i>LacZ</i> activity and mRNA levels were measured in log phase after overnight growth in YPD. (<b>C</b>) <i>LacZ</i> assays were performed as in panel B, with the addition of double mutant strains. P values are for t-test against WT unless otherwise indicated.</p

A single-codon occupancy metric shows that ribosome footprint accumulations at AAA, CAA, and GAA are statistically significant.

<p>(<b>A</b>) Description of the single codon occupancy metric. The occupancy for a given codon in a given site is the number of in-frame reads for that codon in that site, compared to the average in-frame read density for the parent gene. (<b>B</b>) Cumulative distributions of single-codon occupancy for select codons in <i>ncs6Δ</i> and <i>uba4Δ</i>. (<b>C</b>) Heatmap of K-S test p-values for all sense codons in all mutants. For <i>ncs6Δ</i> and <i>uba4Δ</i>, mutant and WT replicates were pooled to improve the accuracy of the metric.</p