Impact of Nonsense-Mediated mRNA Decay on the Global Expression Profile of Budding Yeast

Nonsense-mediated mRNA decay (NMD) is a eukaryotic mechanism of RNA surveillance that selectively eliminates aberrant transcripts coding for potentially deleterious proteins. NMD also functions in the normal repertoire of gene expression. In Saccharomyces cerevisiae, hundreds of endogenous RNA Polymerase II transcripts achieve steady-state levels that depend on NMD. For some, the decay rate is directly influenced by NMD (direct targets). For others, abundance is NMD-sensitive but without any effect on the decay rate (indirect targets). To distinguish between direct and indirect targets, total RNA from wild-type (Nmd(+)) and mutant (Nmd(−)) strains was probed with high-density arrays across a 1-h time window following transcription inhibition. Statistical models were developed to describe the kinetics of RNA decay. 45% ± 5% of RNAs targeted by NMD were predicted to be direct targets with altered decay rates in Nmd(−) strains. Parallel experiments using conventional methods were conducted to empirically test predictions from the global experiment. The results show that the global assay reliably distinguished direct versus indirect targets. Different types of targets were investigated, including transcripts containing adjacent, disabled open reading frames, upstream open reading frames, and those prone to out-of-frame initiation of translation. Known targeting mechanisms fail to account for all of the direct targets of NMD, suggesting that additional targeting mechanisms remain to be elucidated. 30% of the protein-coding targets of NMD fell into two broadly defined functional themes: those affecting chromosome structure and behavior and those affecting cell surface dynamics. Overall, the results provide a preview for how expression profiles in multi-cellular eukaryotes might be impacted by NMD. Furthermore, the methods for analyzing decay rates on a global scale offer a blueprint for new ways to study mRNA decay pathways in any organism where cultured cell lines are available

Multiple Means to the Same End: The Genetic Basis of Acquired Stress Resistance in Yeast

In nature, stressful environments often occur in combination or close succession, and thus the ability to prepare for impending stress likely provides a significant fitness advantage. Organisms exposed to a mild dose of stress can become tolerant to what would otherwise be a lethal dose of subsequent stress; however, the mechanism of this acquired stress tolerance is poorly understood. To explore this, we exposed the yeast gene-deletion libraries, which interrogate all essential and non-essential genes, to successive stress treatments and identified genes necessary for acquiring subsequent stress resistance. Cells were exposed to one of three different mild stress pretreatments (salt, DTT, or heat shock) and then challenged with a severe dose of hydrogen peroxide (H2O2). Surprisingly, there was little overlap in the genes required for acquisition of H2O2 tolerance after different mild-stress pretreatments, revealing distinct mechanisms of surviving H2O2 in each case. Integrative network analysis of these results with respect to protein–protein interactions, synthetic–genetic interactions, and functional annotations identified many processes not previously linked to H2O2 tolerance. We tested and present several models that explain the lack of overlap in genes required for H2O2 tolerance after each of the three pretreatments. Together, this work shows that acquired tolerance to the same severe stress occurs by different mechanisms depending on prior cellular experiences, underscoring the context-dependent nature of stress tolerance

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Ebs1p, a Negative Regulator of Gene Expression Controlled by the Upf Proteins in the Yeast Saccharomyces cerevisiae

Mutations in EBS1 were identified in Saccharomyces cerevisiae that cosuppress missense, frameshift, and nonsense mutations. Evidence from studies of loss of function and overexpression of EBS1 suggests that Ebs1p affects gene expression by inhibiting translation and that a loss of EBS1 function causes suppression by increasing the rate of translation. Changes in EBS1 expression levels alter the expression of wild-type genes, but, in general, no changes in mRNA abundance were associated with a loss of function or overexpression of EBS1. Translation of a lacZ reporter was increased in strains carrying an ebs1-Δ mutant gene, whereas translation was decreased when EBS1 was overexpressed. The cap binding protein eIF-4E copurifies with Ebs1p in the absence of RNA, suggesting that the two proteins interact in vivo. Although physical and genetic interactions were detected between Ebs1p and Dcp1p, copurification was RNase sensitive, and changes in the expression of Ebs1p had little to no effect on decapping of the MFA2 transcript. The combined results suggest that Ebs1p inhibits translation, most likely through effects on eIF-4E rather than on decapping. Finally, EBS1 transcript levels are under the control of nonsense-mediated mRNA decay (NMD), providing the first example of an NMD-sensitive transcript whose protein product influences a step in gene expression required for NMD

Nutritional control of epigenetic processes in yeast and human cells.

The vitamin folate is required for methionine homeostasis in all organisms. In addition to its role in protein synthesis, methionine is the precursor to S-adenosyl-methionine (SAM), which is used in myriad cellular methylation reactions, including all histone methylation reactions. Here, we demonstrate that folate and methionine deficiency led to reduced methylation of lysine 4 of histone H3 (H3K4) in Saccharomyces cerevisiae. The effect of nutritional deficiency on H3K79 methylation was less pronounced, but was exacerbated in S. cerevisiae carrying a hypomorphic allele of Dot1, the enzyme responsible for H3K79 methylation. This result suggested a hierarchy of epigenetic modifications in terms of their susceptibility to nutritional limitations. Folate deficiency caused changes in gene transcription that mirrored the effect of complete loss of H3K4 methylation. Histone methylation was also found to respond to nutritional deficiency in the fission yeast Schizosaccharomyces pombe and in human cells in culture

Brand-Specific Toxicity of Tire Tread Particles Helps Identify the Determinants of Toxicity

Thewidespread occurrence of tire tread particles (TPs) has arousedincreasing concerns over their impacts. However, how they affect thesoil fauna remains poorly understood. Here, based on systematicallyassessing the toxicity of TPs on soil model speciesEnchytraeus crypticusat environmentally relevantconcentrations through both soil and food exposure routes, we reportedthat TPs affected gut microbiota, intestinal histopathology, and metabolitesof the worms both through particulate- and leachate-induced effects,while TP leachates exerted stronger effects. The dominant role ofTP leachates in TP toxicity was further explained by the findingsthat worms did not ingest TPs with a particle size of over 150 & mu;mand actively avoided consuming TP particles. Moreover, by comparingthe effects of different brands of TPs as well as new and aged TPs,we demonstrated that it was mainly TP leachates that resulted in theubiquity of the disturbance in the worm's gut microbiota amongdifferent brands of TPs. Notably, the large variations in leachatecompositions among different brands of TPs provided us a unique opportunityto identify the determinants of TP toxicity. These results providenovel insights into the toxicity of TPs to soil fauna and a referencefor toxicity reduction of tires. HowTPs affect soil fauna remains poorly understood. Wedemonstrate the dominant role leachate-induced effects play in TPtoxicity to soil fauna due to the limited ingestion of TPs, and brand-specifictoxicity helps identify determinants of TP toxicity

Targeting through uORFs

<div><p>(A) Two uORF start codons are present in the <i>FZF1</i> sequence. The uORF stop codon is located upstream of the start codon of the coding ORF in the −1 reading frame. The position of a sequence resembling a downstream element reported to be required for NMD [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020203#pgen-0020203-b078" target="_blank">78</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020203#pgen-0020203-b079" target="_blank">79</a>] is shown. uORF start codons were changed to AGG sense codons. DSE, downstream element.</p><p>(B) Steady-state RNA levels for <i>FZF1, fzf1-Δ1, fzf1-Δ2,</i> and <i>fzf1-Δ1,-Δ2</i> determined by Northern blotting.</p><p>(C) Comparison of <i>FZF1</i> and <i>fzf1-Δ1,-Δ2</i> RNA half-lives in Nmd⁺ and Nmd⁻ strains. Half-lives, FCRs, and <i>p</i>-values were calculated for <i>n</i> = 5.</p><p>(D) Potential uORFs with end points between −100 and +100 nucleotides of predicted direct targets as a function of start position and total length. Left: upper and lower numbers refer to uORFs that end downstream and upstream of the coding ORF start codon, respectively.</p></div

Nutritional control of epigenetic processes in yeast and human cells.

The vitamin folate is required for methionine homeostasis in all organisms. In addition to its role in protein synthesis, methionine is the precursor to S-adenosyl-methionine (SAM), which is used in myriad cellular methylation reactions, including all histone methylation reactions. Here, we demonstrate that folate and methionine deficiency led to reduced methylation of lysine 4 of histone H3 (H3K4) in Saccharomyces cerevisiae. The effect of nutritional deficiency on H3K79 methylation was less pronounced, but was exacerbated in S. cerevisiae carrying a hypomorphic allele of Dot1, the enzyme responsible for H3K79 methylation. This result suggested a hierarchy of epigenetic modifications in terms of their susceptibility to nutritional limitations. Folate deficiency caused changes in gene transcription that mirrored the effect of complete loss of H3K4 methylation. Histone methylation was also found to respond to nutritional deficiency in the fission yeast Schizosaccharomyces pombe and in human cells in culture

Targeting through Out-of-Frame Initiation of Translation

<div><p>(A) A_UGCAI(r) scores were calculated to evaluate the influence of context on the efficiency of translation initiation. The bit score indicates relative sequence conservation at a given nucleotide position and the height of nucleotide symbols indicates the frequency of nucleotide use. Standard numbering (parentheses) differs from Web logo numbering. The bar chart shows the distribution of A_UGCAI(r) scores for protein-coding transcripts that satisfy criteria for targeting by leaky scanning. The dot plot shows the distribution of candidate transcripts where the A_UGCAI(r) score of the initiator AUG is plotted against the score for the downstream out-of-frame AUG. Red dots correspond to transcripts considered to be likely candidates for leaky scanning based solely on the presence of U or C at the −3 position.</p><p>(B) Sequence changes in <i>RDR1</i> DNA and comparison of <i>RDR1</i> and <i>rdr1-AUG</i> transcript half-lives in Nmd⁺ and Nmd⁻ strains.</p><p>(C) Sequence changes in <i>ASF2</i> DNA and comparison of <i>ASF2</i> and <i>asf2-AUG</i> transcript half-lives in Nmd⁺ and Nmd⁻ strains.</p></div