Global analysis of PTC-containing RNA regulation by the NMD pathway and the EJC-like in intron-rich and intron-poor yeasts – YeastIntrons

Our strategy is using the strengths of the molecular genetics applied to these yeasts to build different mutant strains and also the capacity of the last generation of sequencing technologies. Using very deep sequencing, we are studding the transcriptome structure of both yeasts under different growth conditions. We are also studying how the mutations we are making in these yeasts alter this trancriptome structure and more specifically intron retention

We have re-annotated the C. neoformans var. grubii genome and identified more than 4000 introns We changed the annotation for more than 40% of the genes and identified more than 1000 5’ or 3’UTR introns. We have also identified the polyadenylation site(s) for more than 80% of the genes.
We have performed a first round of re-annotation of the Y. lipolytica genome increasing the introns number by 20%. For the first time, 3’ and 5’ UTR have been identified in this yeast.
We have constructed EJC and NMD mutant strains in C. neoformans and Y. lipolytica. The analysis of the consequences of such mutations on introns retention remains the main theme of research for this project.

The data on the transcriptome structure we producing will be of course very important for the accomplishments of the present project objectives but they will also represent great information for the analysis of the RNA metabolism in general. Our date will certainly be one of the most complete ever produced in any organism.

We have published a paper on the alternative splicing in Y . lipolytica this year and another one is describing the role of introns on gene expression in C. neoformans is under revision. We also participated in a number of scientific meetings during which we have presented our data and discussed their perpectives.
Thon, Goebels, Gonzalez-Hilarion, Rolland, Moyrand, Beilhartz, Janbon, Introns regulate gene expression in Cryptococcus neoformans in a Pab2p dependent pathway (submitted)
Kabran, Rossignol, Gaillardin, Nicaud, Neuvéglise. 2012. Alternative Splicing Regulates Targeting of Malate Dehydrogenase in Yarrowia lipolytica. DNA Research doi:10.1093/dnares/dss007
Brunel, Proux, Coppée, Neuvéglise. Transcriptome analysis and expression regulation by the NMD pathway in the yeast Yarrowia lipolytica. San Feliu, Spain, Octobre 2011 (poster)
Thom, Rolland, Moyrand, Janbon G. Regulation of gene expression by intron in C. neoformans Keystone Symposia on Molecular and Cellular Biology. Fungal Pahtogens: From Basic Biology to Drug Discovery. Santa Fe, USA January 2012 USA (oral presentation)
Janbon G. Annual meeting of the Korean Society for Microbiology and Biotechnology GyeongJu, South Korea June 2011.Role of the introns in regulating gene expression in C. neoformans. USA (oral presentation)
Goebels, Gonzalez-Hilarion, Moyrand, Janbon Analysis of the role of introns on gene expression in Cryptococcus neoformans. October 2012 The complex life of mRNA EMBO EMBL symposium Heidelberg Germany. (poster)
Gonzalez-Hilarion, Mogensen, Janbon EJC and NMD mutant in Cryptococcus neoformans. June 2012. 17th annual meeting of the RNA Society, Ann Harbor, USA (poster)
Neuvéglise. Yeast genome evolution: comparison of divergent clades. ICY, Madison 26-30 août 2012, USA (oral presentation)

Alternative splicing of pre-mRNA molecules is a source of protein diversity but also a mean for gene expression regulation. Thus, one highly common possibility of generating alternative isoforms is due to “intron retention”. In this case, one or more introns remain unspliced resulting often in an mRNA molecule containing a premature termination codon (PTC). These PTC are then supposed to trigger mRNA degradation by the non sense mediated mRNA decay (NMD) pathway. There is an obvious discrepancy between the intuitive prediction of a wide regulation of gene expression by alternative splicing and NMD, and the very poor transcriptomic consequences of the deletion or knock down of the major player of this pathway. In this project, we are going to use two very different yeasts (Cryptococcus neoformans and Yarrowia lipolytica) that combine, a least in part, the complexity of the RNA metabolism of metazoans and the easiness of manipulation of the yeast S. cerevisiae to investigate this fascinating question. Actually, despite numerous studies in metazoans, the extent, the complexity, the regulation and the roles of alternative splicing in fungi has been very little studied. Here, we will use the latest ultrahigh-throughput RNA sequencing (RNAseq) approaches and a new bioinformatic “intron pipeline” tool we are going to set up, for cataloguing and quantifying alternative splicing in these yeasts. We will also be interested in the analysis of the influence of EJC-like and NMD mutations on the transcriptome so as in the potential existence of a parallel pathway regulating PCT-containing RNA degradation.