Comparative genomics of protoploid Saccharomycetaceae.

Our knowledge of yeast genomes remains largely dominated by the extensive studies on Saccharomyces cerevisiae and the consequences of its ancestral duplication, leaving the evolution of the entire class of hemiascomycetes only partly explored. We concentrate here on five species of Saccharomycetaceae, a large subdivision of hemiascomycetes, that we call "protoploid" because they diverged from the S. cerevisiae lineage prior to its genome duplication. We determined the complete genome sequences of three of these species: Kluyveromyces (Lachancea) thermotolerans and Saccharomyces (Lachancea) kluyveri (two members of the newly described Lachancea clade), and Zygosaccharomyces rouxii. We included in our comparisons the previously available sequences of Kluyveromyces lactis and Ashbya (Eremothecium) gossypii. Despite their broad evolutionary range and significant individual variations in each lineage, the five protoploid Saccharomycetaceae share a core repertoire of approximately 3300 protein families and a high degree of conserved synteny. Synteny blocks were used to define gene orthology and to infer ancestors. Far from representing minimal genomes without redundancy, the five protoploid yeasts contain numerous copies of paralogous genes, either dispersed or in tandem arrays, that, altogether, constitute a third of each genome. Ancient, conserved paralogs as well as novel, lineage-specific paralogs were identified.

Dicistronic tRNA-5S rRNA genes in Yarrowia lipolytica: an alternative TFIIIA-independent way for expression of 5S rRNA genes.

In eukaryotes, genes transcribed by RNA polymerase III (Pol III) carry their own internal promoters and as such, are transcribed as individual units. Indeed, a very few cases of dicistronic Pol III genes are yet known. In contrast to other hemiascomycetes, 5S rRNA genes of Yarrowia lipolytica are not embedded into the tandemly repeated rDNA units, but appear scattered throughout the genome. We report here an unprecedented genomic organization: 48 over the 108 copies of the 5S rRNA genes are located 3' of tRNA genes. We show that these peculiar tRNA-5S rRNA dicistronic genes are expressed in vitro and in vivo as Pol III transcriptional fusions without the need of the 5S rRNA gene-specific factor TFIIIA, the deletion of which displays a viable phenotype. We also report the existence of a novel putative non-coding Pol III RNA of unknown function about 70 nucleotide-long (RUF70), the 13 genes of which are devoid of internal Pol III promoters and located 3' of the 13 copies of the tDNA-Trp (CCA). All genes embedded in the various dicistronic genes, fused 5S rRNA genes, RUF70 genes and their leader tRNA genes appear to be efficiently transcribed and their products correctly processed in vivo.

Detection and analysis of alternative splicing in Yarrowia lipolytica reveal structural constraints facilitating nonsense-mediated decay of intron-retaining transcripts.

BACKGROUND: Hemiascomycetous yeasts have intron-poor genomes with very few cases of alternative splicing. Most of the reported examples result from intron retention in Saccharomyces cerevisiae and some have been shown to be functionally significant. Here we used transcriptome-wide approaches to evaluate the mechanisms underlying the generation of alternative transcripts in Yarrowia lipolytica, a yeast highly divergent from S. cerevisiae. RESULTS: Experimental investigation of Y. lipolytica gene models identified several cases of alternative splicing, mostly generated by intron retention, principally affecting the first intron of the gene. The retention of introns almost invariably creates a premature termination codon, as a direct consequence of the structure of intron boundaries. An analysis of Y. lipolytica introns revealed that introns of multiples of three nucleotides in length, particularly those without stop codons, were underrepresented. In other organisms, premature termination codon-containing transcripts are targeted for degradation by the nonsense-mediated mRNA decay (NMD) machinery. In Y. lipolytica, homologs of S. cerevisiae UPF1 and UPF2 genes were identified, but not UPF3. The inactivation of Y. lipolytica UPF1 and UPF2 resulted in the accumulation of unspliced transcripts of a test set of genes. CONCLUSIONS: Y. lipolytica is the hemiascomycete with the most intron-rich genome sequenced to date, and it has several unusual genes with large introns or alternative transcription start sites, or introns in the 5' UTR. Our results suggest Y. lipolytica intron structure is subject to significant constraints, leading to the under-representation of stop-free introns. Consequently, intron-containing transcripts are degraded by a functional NMD pathway.