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

ABSTRACT: 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.

SUBMITTER: Mekouar M

PROVIDER: S-EPMC2911113 | biostudies-literature | 2010

REPOSITORIES: biostudies-literature

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

Mekouar Meryem M Blanc-Lenfle Isabelle I Ozanne Christophe C Da Silva Corinne C Cruaud Corinne C Wincker Patrick P Gaillardin Claude C Neuvéglise Cécile C

Genome biology 20100623 6

<h4>Background</h4>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.<h4>Results</h4>Experimental investigation of Y. lip ...[more]

PMID: 20573210

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Project description:Eukaryotic polycistronic transcription units are rare and only a few examples are known, mostly being the outcome of serendipitous discovery. We claim that nonsense-mediated mRNA decay (NMD) immune structure is a common characteristic of polycistronic transcripts, and that this immunity is an emergent property derived from all functional CDSs. The human RefSeq transcriptome was computationally screened for transcripts capable of eliciting NMD, and which contain an additional ORF(s) potentially capable of rescuing the transcript from NMD. Transcripts were further analyzed implementing domain-based strategies in order to estimate the potential of the candidate ORF to encode a functional protein. Consequently, we predict the existence of forty nine novel polycistronic transcripts. Experimental verification was carried out utilizing two different types of analyses. First, five Gene Expression Omnibus (GEO) datasets from published NMD-inhibition studies were used, aiming to explore whether a given mRNA is indeed insensitive to NMD. All known bicistronic transcripts and eleven out of the twelve predicted genes that were analyzed, displayed NMD insensitivity using various NMD inhibitors. For three genes, a mixed expression pattern was observed presenting both NMD sensitivity and insensitivity in different cell types. Second, we used published global translation initiation sequencing data from HEK293 cells to verify the existence of translation initiation sites in our predicted polycistronic genes. In five of our genes, the predicted rescuing uORFs are indeed identified as translation initiation sites, and in two additional genes, one of two predicted rescuing uORF is verified. These results validate our computational analysis and reinforce the possibility that NMD-immune architecture is a parameter by which polycistronic genes can be identified. Moreover, we present evidence for NMD-mediated regulation controlling the production of one or more proteins encoded in the polycistronic transcript.

Dataset Information

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

Publications

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

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