Stringency of start codon selection modulates autoregulation of translation initiation factor eIF5

An AUG in an optimal nucleotide context is the preferred translation initiation site in eukaryotic cells. Interactions among translation initiation factors, including eIF1 and eIF5, govern start codon selection. Experiments described here showed that high intracellular eIF5 levels reduced the stringency of start codon selection in human cells. In contrast, high intracellular eIF1 levels increased stringency. High levels of eIF5 induced translation of inhibitory upstream open reading frames (uORFs) in eIF5 mRNA that initiate with AUG codons in conserved poor contexts. This resulted in reduced translation from the downstream eIF5 start codon, indicating that eIF5 autoregulates its own synthesis. As with eIF1, which is also autoregulated through translation initiation, features contributing to eIF5 autoregulation show deep evolutionary conservation. The results obtained provide the basis for a model in which auto- and cross-regulation of eIF5 and eIF1 translation establish a regulatory feedback loop that would stabilize the stringency of start codon selection

Insertion of Horizontally Transferred Genes within Conserved Syntenic Regions of Yeast Genomes

Horizontal gene transfer has been occasionally mentioned in eukaryotic genomes, but such events appear much less numerous than in prokaryotes, where they play important functional and evolutionary roles. In yeasts, few independent cases have been described, some of which corresponding to major metabolic functions, but no systematic screening of horizontally transferred genes has been attempted so far. Taking advantage of the synteny conservation among five newly sequenced and annotated genomes of Saccharomycetaceae, we carried out a systematic search for HGT candidates amidst genes present in only one species within conserved synteny blocks. Out of 255 species-specific genes, we discovered 11 candidates for HGT, based on their similarity with bacterial proteins and on reconstructed phylogenies. This corresponds to a minimum of six transfer events because some horizontally acquired genes appear to rapidly duplicate in yeast genomes (e.g. YwqG genes in Kluyveromyces thermotolerans and serine recombinase genes of the IS607 family in Saccharomyces kluyveri). We show that the resulting copies are submitted to a strong functional selective pressure. The mechanisms of DNA transfer and integration are discussed, in relation with the generally small size of HGT candidates. Our results on a limited set of species expand by 50% the number of previously published HGT cases in hemiascomycetous yeasts, suggesting that this type of event is more frequent than usually thought. Our restrictive method does not exclude the possibility that additional HGT events exist. Actually, ancestral events common to several yeast species must have been overlooked, and the absence of homologs in present databases leaves open the question of the origin of the 244 remaining species-specific genes inserted within conserved synteny blocks

A proteogenomic update to Yersinia: enhancing genome annotation

<p>Abstract</p> <p>Background</p> <p>Modern biomedical research depends on a complete and accurate proteome. With the widespread adoption of new sequencing technologies, genome sequences are generated at a near exponential rate, diminishing the time and effort that can be invested in genome annotation. The resulting gene set contains numerous errors in even the most basic form of annotation: the primary structure of the proteins.</p> <p>Results</p> <p>The application of experimental proteomics data to genome annotation, called proteogenomics, can quickly and efficiently discover misannotations, yielding a more accurate and complete genome annotation. We present a comprehensive proteogenomic analysis of the plague bacterium, <it>Yersinia pestis KIM</it>. We discover non-annotated genes, correct protein boundaries, remove spuriously annotated ORFs, and make major advances towards accurate identification of signal peptides. Finally, we apply our data to 21 other <it>Yersinia </it>genomes, correcting and enhancing their annotations.</p> <p>Conclusions</p> <p>In total, 141 gene models were altered and have been updated in RefSeq and Genbank, which can be accessed seamlessly through any NCBI tool (e.g. blast) or downloaded directly. Along with the improved gene models we discover new, more accurate means of identifying signal peptides in proteomics data.</p

Pichia sorbitophila, an Interspecies Yeast Hybrid, Reveals Early Steps of Genome Resolution After Polyploidization

Polyploidization is an important process in the evolution of eukaryotic genomes, but ensuing molecular mechanisms remain to be clarified. Autopolyploidization or whole-genome duplication events frequently are resolved in resulting lineages by the loss of single genes from most duplicated pairs, causing transient gene dosage imbalance and accelerating speciation through meiotic infertility. Allopolyploidization or formation of interspecies hybrids raises the problem of genetic incompatibility (Bateson-Dobzhansky-Muller effect) and may be resolved by the accumulation of mutational changes in resulting lineages. In this article, we show that an osmotolerant yeast species, Pichia sorbitophila, recently isolated in a concentrated sorbitol solution in industry, illustrates this last situation. Its genome is a mosaic of homologous and homeologous chromosomes, or parts thereof, that corresponds to a recently formed hybrid in the process of evolution. The respective parental contributions to this genome were characterized using existing variations in GC content. The genomic changes that occurred during the short period since hybrid formation were identified (e.g., loss of heterozygosity, unilateral loss of rDNA, reciprocal exchange) and distinguished from those undergone by the two parental genomes after separation from their common ancestor (i.e., NUMT (NUclear sequences of MiTochondrial origin) insertions, gene acquisitions, gene location movements, reciprocal translocation). We found that the physiological characteristics of this new yeast species are determined by specific but unequal contributions of its two parents, one of which could be identified as very closely related to an extant Pichia farinosa strain

Ribosome formation from subunits studied by stopped-flow and Rayleigh light scattering

Light scattering and standard stopped-flow techniques were used to monitor rapid association of ribosomal subunits during initiation of eubacterial protein synthesis. The effects of the initiation factors IF1, IF2, IF3 and buffer conditions on subunit association were studied along with the role of GTP in this process. The part of light scattering theory that is essential for kinetic measurements is high-lighted in the main text and a more general treatment of Rayleigh scattering from macromolecules is given in an appendix

Tye7 regulates yeast Ty1 retrotransposon sense and antisense transcription in response to adenylic nucleotides stress

Transposable elements play a fundamental role in genome evolution. It is proposed that their mobility, activated under stress, induces mutations that could confer advantages to the host organism. Transcription of the Ty1 LTR-retrotransposon of Saccharomyces cerevisiae is activated in response to a severe deficiency in adenylic nucleotides. Here, we show that Ty2 and Ty3 are also stimulated under these stress conditions, revealing the simultaneous activation of three active Ty retrotransposon families. We demonstrate that Ty1 activation in response to adenylic nucleotide depletion requires the DNA-binding transcription factor Tye7. Ty1 is transcribed in both sense and antisense directions. We identify three Tye7 potential binding sites in the region of Ty1 DNA sequence where antisense transcription starts. We show that Tye7 binds to Ty1 DNA and regulates Ty1 antisense transcription. Altogether, our data suggest that, in response to adenylic nucleotide reduction, TYE7 is induced and activates Ty1 mRNA transcription, possibly by controlling Ty1 antisense transcription. We also provide the first evidence that Ty1 antisense transcription can be regulated by environmental stress conditions, pointing to a new level of control of Ty1 activity by stress, as Ty1 antisense RNAs play an important role in regulating Ty1 mobility at both the transcriptional and post-transcriptional stages

Nuclear Mitochondrial DNA Activates Replication in Saccharomyces cerevisiae

The nuclear genome of eukaryotes is colonized by DNA fragments of mitochondrial origin, called NUMTs. These insertions have been associated with a variety of germ-line diseases in humans. The significance of this uptake of potentially dangerous sequences into the nuclear genome is unclear. Here we provide functional evidence that sequences of mitochondrial origin promote nuclear DNA replication in Saccharomyces cerevisiae. We show that NUMTs are rich in key autonomously replicating sequence (ARS) consensus motifs, whose mutation results in the reduction or loss of DNA replication activity. Furthermore, 2D-gel analysis of the mrc1 mutant exposed to hydroxyurea shows that several NUMTs function as late chromosomal origins. We also show that NUMTs located close to or within ARS provide key sequence elements for replication. Thus NUMTs can act as independent origins, when inserted in an appropriate genomic context or affect the efficiency of pre-existing origins. These findings show that migratory mitochondrial DNAs can impact on the replication of the nuclear region they are inserted in

A New Role for Translation Initiation Factor 2 in Maintaining Genome Integrity

Escherichia coli translation initiation factor 2 (IF2) performs the unexpected function of promoting transition from recombination to replication during bacteriophage Mu transposition in vitro, leading to initiation by replication restart proteins. This function has suggested a role of IF2 in engaging cellular restart mechanisms and regulating the maintenance of genome integrity. To examine the potential effect of IF2 on restart mechanisms, we characterized its influence on cellular recovery following DNA damage by methyl methanesulfonate (MMS) and UV damage. Mutations that prevent expression of full-length IF2-1 or truncated IF2-2 and IF2-3 isoforms affected cellular growth or recovery following DNA damage differently, influencing different restart mechanisms. A deletion mutant (del1) expressing only IF2-2/3 was severely sensitive to growth in the presence of DNA-damaging agent MMS. Proficient as wild type in repairing DNA lesions and promoting replication restart upon removal of MMS, this mutant was nevertheless unable to sustain cell growth in the presence of MMS; however, growth in MMS could be partly restored by disruption of sulA, which encodes a cell division inhibitor induced during replication fork arrest. Moreover, such characteristics of del1 MMS sensitivity were shared by restart mutant priA300, which encodes a helicase-deficient restart protein. Epistasis analysis indicated that del1 in combination with priA300 had no further effects on cellular recovery from MMS and UV treatment; however, the del2/3 mutation, which allows expression of only IF2-1, synergistically increased UV sensitivity in combination with priA300. The results indicate that full-length IF2, in a function distinct from truncated forms, influences the engagement or activity of restart functions dependent on PriA helicase, allowing cellular growth when a DNA–damaging agent is present

Amplification of a Zygosaccharomyces bailii DNA Segment in Wine Yeast Genomes by Extrachromosomal Circular DNA Formation

We recently described the presence of large chromosomal segments resulting from independent horizontal gene transfer (HGT) events in the genome of Saccharomyces cerevisiae strains, mostly of wine origin. We report here evidence for the amplification of one of these segments, a 17 kb DNA segment from Zygosaccharomyces bailii, in the genome of S. cerevisiae strains. The copy number, organization and location of this region differ considerably between strains, indicating that the insertions are independent and that they are post-HGT events. We identified eight different forms in 28 S. cerevisiae strains, mostly of wine origin, with up to four different copies in a single strain. The organization of these forms and the identification of an autonomously replicating sequence functional in S. cerevisiae, strongly suggest that an extrachromosomal circular DNA (eccDNA) molecule serves as an intermediate in the amplification of the Z. bailii region in yeast genomes. We found little or no sequence similarity at the breakpoint regions, suggesting that the insertions may be mediated by nonhomologous recombination. The diversity between these regions in S. cerevisiae represents roughly one third the divergence among the genomes of wine strains, which confirms the recent origin of this event, posterior to the start of wine strain expansion. This is the first report of a circle-based mechanism for the expansion of a DNA segment, mediated by nonhomologous recombination, in natural yeast populations

Trypanosoma cruzi Gene Expression in Response to Gamma Radiation

Trypanosoma cruzi is an organism highly resistant to ionizing radiation. Following a dose of 500 Gy of gamma radiation, the fragmented genomic DNA is gradually reconstructed and the pattern of chromosomal bands is restored in less than 48 hours. Cell growth arrests after irradiation but, while DNA is completely fragmented, RNA maintains its integrity. In this work we compared the transcriptional profiles of irradiated and non-irradiated epimastigotes at different time points after irradiation using microarray. In total, 273 genes were differentially expressed; from these, 160 were up-regulated and 113 down-regulated. We found that genes with predicted functions are the most prevalent in the down-regulated gene category. Translation and protein metabolic processes, as well as generation of precursor of metabolites and energy pathways were affected. In contrast, the up-regulated category was mainly composed of obsolete sequences (which included some genes of the kinetoplast DNA), genes coding for hypothetical proteins, and Retrotransposon Hot Spot genes. Finally, the tyrosyl-DNA phosphodiesterase 1, a gene involved in double-strand DNA break repair process, was up-regulated. Our study demonstrated the peculiar response to ionizing radiation, raising questions about how this organism changes its gene expression to manage such a harmful stress