Identifying eIF4E-binding protein translationally-controlled transcripts reveals links to mRNAs bound by specific PUF proteins.

eIF4E-binding proteins (4E-BPs) regulate translation of mRNAs in eukaryotes. However the extent to which specific mRNA targets are regulated by 4E-BPs remains unknown. We performed translational profiling by microarray analysis of polysome and monosome associated mRNAs in wild-type and mutant cells to identify mRNAs in yeast regulated by the 4E-BPs Caf20p and Eap1p; the first-global comparison of 4E-BP target mRNAs. We find that yeast 4E-BPs modulate the translation of >1000 genes. Most target mRNAs differ between the 4E-BPs revealing mRNA specificity for translational control by each 4E-BP. This is supported by observations that eap1Δ and caf20Δ cells have different nitrogen source utilization defects, implying different mRNA targets. To account for the mRNA specificity shown by each 4E-BP, we found correlations between our data sets and previously determined targets of yeast mRNA-binding proteins. We used affinity chromatography experiments to uncover specific RNA-stabilized complexes formed between Caf20p and Puf4p/Puf5p and between Eap1p and Puf1p/Puf2p. Thus the combined action of each 4E-BP with specific 3'-UTR-binding proteins mediates mRNA-specific translational control in yeast, showing that this form of translational control is more widely employed than previously thought.

Upstream sequence elements direct post-transcriptional regulation of gene expression under stress conditions in yeast.

BACKGROUND: The control of gene expression in eukaryotic cells occurs both transcriptionally and post-transcriptionally. Although many genes are now known to be regulated at the translational level, in general, the mechanisms are poorly understood. We have previously presented polysomal gradient and array-based evidence that translational control is widespread in a significant number of genes when yeast cells are exposed to a range of stresses. Here we have re-examined these gene sets, considering the role of UTR sequences in the translational responses of these genes using recent large-scale datasets which define 5' and 3' transcriptional ends for many yeast genes. In particular, we highlight the potential role of 5' UTRs and upstream open reading frames (uORFs). RESULTS: We show a highly significant enrichment in specific GO functional classes for genes that are translationally up- and down-regulated under given stresses (e.g. carbohydrate metabolism is up-regulated under amino acid starvation). Cross-referencing these data with the stress response data we show that translationally upregulated genes have longer 5' UTRs, consistent with their role in translational regulation. In the first genome-wide study of uORFs in a set of mapped 5' UTRs, we show that uORFs are rare, being statistically under-represented in UTR sequences. However, they have distinct compositional biases consistent with their putative role in translational control and are more common in genes which are apparently translationally up-regulated. CONCLUSION: These results demonstrate a central regulatory role for UTR sequences, and 5' UTRs in particular, highlighting the significant role of uORFs in post-transcriptional control in yeast. Yeast uORFs are more highly conserved than has been suggested, lending further weight to their significance as functional elements involved in gene regulation. It also suggests a more complex and novel mechanism of control, whereby uORFs permit genes to escape from a more general attenuation of translation under conditions of stress. However, since uORFs are relatively rare (only ~13% of yeast genes have them) there remain many unanswered questions as to how UTR elements can direct translational control of many hundreds of genes under stress.

ABF1-binding sites promote efficient global genome nucleotide excision repair.

Global genome nucleotide excision repair (GG-NER) removes DNA damage from nontranscribing DNA. In Saccharomyces cerevisiae, the RAD7 and RAD16 genes are specifically required for GG-NER. We have reported that autonomously replicating sequence-binding factor 1 (ABF1) protein forms a stable complex with Rad7 and Rad16 proteins. ABF1 functions in transcription, replication, gene silencing, and NER in yeast. Here we show that binding of ABF1 to its DNA recognition sequence found at multiple genomic locations promotes efficient GG-NER in yeast. Mutation of the I silencer ABF1-binding site at the HMLalpha locus caused loss of ABF1 binding, which resulted in a domain of reduced GG-NER efficiency on one side of the ABF1-binding site. During GG-NER, nucleosome positioning at this site was not altered, and this correlated with an inability of the GG-NER complex to reposition nucleosomes in vitro.We discuss how the GG-NER complex might facilitate GG-NER while preventing unregulated gene transcription during this process.

Global translational responses to oxidative stress impact upon multiple levels of protein synthesis.

Global inhibition of protein synthesis is a common response to stress conditions. We have analyzed the regulation of protein synthesis in response to oxidative stress induced by exposure to H(2)O(2) in the yeast Saccharomyces cerevisiae. Our data show that H(2)O(2) causes an inhibition of translation initiation dependent on the Gcn2 protein kinase, which phosphorylates the alpha-subunit of eukaryotic initiation factor-2. Additionally, our data indicate that translation is regulated in a Gcn2-independent manner because protein synthesis was still inhibited in response to H(2)O(2) in a gcn2 mutant. Polysome analysis indicated that H(2)O(2) causes a slower rate of ribosomal runoff, consistent with an inhibitory effect on translation elongation or termination. Furthermore, analysis of ribosomal transit times indicated that oxidative stress increases the average mRNA transit time, confirming a post-initiation inhibition of translation. Using microarray analysis of polysome- and monosome-associated mRNA pools, we demonstrate that certain mRNAs, including mRNAs encoding stress protective molecules, increase in association with ribosomes following H(2)O(2) stress. For some candidate mRNAs, we show that a low concentration of H(2)O(2) results in increased protein production. In contrast, a high concentration of H(2)O(2) promotes polyribosome association but does not necessarily lead to increased protein production. We suggest that these mRNAs may represent an mRNA store that could become rapidly activated following relief of the stress condition. In summary, oxidative stress elicits complex translational reprogramming that is fundamental for adaptation to the stress.

Global gene expression profiling reveals widespread yet distinctive translational responses to different eukaryotic translation initiation factor 2B-targeting stress pathways.

Global inhibition of protein synthesis is a hallmark of many cellular stress conditions. Even though specific mRNAs defy this (e.g., yeast GCN4 and mammalian ATF4), the extent and variation of such resistance remain uncertain. In this study, we have identified yeast mRNAs that are translationally maintained following either amino acid depletion or fusel alcohol addition. Both stresses inhibit eukaryotic translation initiation factor 2B, but via different mechanisms. Using microarray analysis of polysome and monosome mRNA pools, we demonstrate that these stress conditions elicit widespread yet distinct translational reprogramming, identifying a fundamental role for translational control in the adaptation to environmental stress. These studies also highlight the complex interplay that exists between different stages in the gene expression pathway to allow specific preordained programs of proteome remodeling. For example, many ribosome biogenesis genes are coregulated at the transcriptional and translational levels following amino acid starvation. The transcriptional regulation of these genes has recently been connected to the regulation of cellular proliferation, and on the basis of our results, the translational control of these mRNAs should be factored into this equation.

Differential activation of M26-containing meiotic recombination hot spots in Schizosaccharomyces pombe.

Certain genomic loci, termed hot spots, are predisposed to undergo genetic recombination during meiosis at higher levels relative to the rest of the genome. The factors that specify hot-spot potential are not well understood. The M26 hot spot of Schizosaccharomyces pombe is dependent on certain trans activators and a specific nucleotide sequence, which can function as a hot spot in a position- and orientation-independent fashion within ade6. In this report we demonstrate that a linear element (LE) component, Rec10, has a function that is required for activation of some, but not all, M26-containing hot spots and from this we propose that, with respect to hot-spot activity, there are three classes of M26-containing sequences. We demonstrate that the localized sequence context in which the M26 heptamer is embedded is a major factor governing whether or not this Rec10 function is required for full hot-spot activation. Furthermore, we show that the rec10-144 mutant, which is defective in full activation of ade6-M26, but proficient for activation of other M26-containing hot spots, is also defective in the formation of LEs, suggesting an intimate link between higher-order chromatin structure and local influences on hot-spot activation.

The unique centromeric chromatin structure of Schizosaccharomyces pombe is maintained during meiosis.

In meiosis I sister centromeres are unified in their polarity on the spindle, and this unique behavior is known to require the function of meiosis-specific factors that set some intrinsic property of the centromeres. The fission yeast, Schizosaccharomyces pombe, possesses complex centromeres consisting of repetitive DNA elements, making it an excellent model in which to study the behavior of complex centromeres. In mitosis, during which sister centromeres mediate chromosome segregation by establishing bipolar chromosome attachments to the spindle, the central core of the S. pombe centromere chromatin has a unique irregular nucleosome pattern. Deletion of repeats flanking this core structure have no effect on mitotic chromosome segregation, but have profound effects during meiosis. While this demonstrates that the outer repeats are critical for normal meiotic sister centromere behavior, exactly how they function and how monopolarity is established remains unclear. In this study we provide the first analysis of the chromatin structure of a complex centromere during meiosis. We show that the nature and extent of the unique central core chromatin structure is maintained with no measurable expansion. This demonstrates that monopolarity of sister centromeres, and subsequent reversion to bipolarity, does not involve a global change to the centromeric chromatin structure.