The unifying theme of the laboratory is the study of eukaryotic RNA cellular metabolism in a model organism, the yeast S. cerevisiae. Several intertwined topics have evolved in our laboratory from this general theme. We described molecular mechanisms that are essential for ribosome formation, discovered a novel class of cellular RNAs (CUTs), identified mRNA degradation factors and developed a genome-scale genetic screen.

Genetic interaction networks.

Genome-wide genetic interaction experiments analyze the effects on growth of combining several mutations in a single cell. Such results should lead to a better understanding of the principles underlying genetic interactions, answer fundamental biological questions about how cellular pathways are organized and should provide the basis for a better understanding of diseases resulting from multiple allelic interactions.

In order to obtain insights into gene function on a genomic scale, we measured the growth impact of combining mutations in S. cerevisiaecells. To obtain quantitative and sensitive measurements of these genetic interactions we developed a novel method, called GIM for Genetic Interactions Mapping, and used it in a pilot experiment covering 140,000 tested combinations of non-essential gene deletions (Decourty et al., PNAS2008). Surprisingly, a large amount of functional information could be extracted from many of the weak measured effects that generate genetic interaction signature highly specific for most biological pathways. Moreover, we could find functional links between a large number of genes unrelated to the mutations used for screening. In addition, a lower number of strong effects, classically described as synthetic lethality, allowed the identification of novel mRNA degradation factors (Milligan et al., Mol Cell Biol2008). Using variants of essential genes and a broad selection of mutants affecting various cellular pathways we are now in the process of obtaining results for a larger fraction of the yeast genetic “interactome”. We recently identified several new factors involved in the degradation of proteins synthesized from aberrant mRNAs, a pathway which was very poorly characterized in eukaryotes, opening a novel line of research in the laboratory.

Ribosome biogenesis

Ribosomes are abundant protein factories and their formation is an essential process, highly conserved in eukaryotes. While ribosome formation starts into the nucleolus, it continues into the nucleoplasm and ends into the cytoplasm. This pathway requires the ribosomal RNA (rRNA), transcribed under the form of precursors, ribosomal proteins and a large number of pre-ribosomal factors that have to be imported into the nucleus. In contrast to the ribosomal proteins, which associate early to the particles and remain tightly bound to the mature subunits, the pre-ribosomal factors bind only transiently to the assembly intermediates and must dissociate to allow formation of the final, functional ribosomal particles.

During the past few years, we identified and characterised many of these pre-60S factors and contributed to the establishment of general ribosome assembly mechanisms. We have recently focused our studies on the late steps of pre-60S maturation. We are now investigating the export of pre-60S particles and recycling of the shuttling pre-ribosomal factors after their release from the particles (Demoinet et al., RNA2007; Lebreton et al., Nucleic Acids Res2008; Yao et al., RNA2010).

CUTs - Cryptic Unstable Transcripts.

We previously described a novel class of ubiquitous transcripts that are very efficiently degraded by the combined action of a poly-adenylation complex (TRAMP) and of the nuclear exosome (La Cava et al., Cell 2005; Wyers et al., Cell 2005). We generated a detailed genomic map of these transcripts, collectively known as CUTs - for Cryptic Unstable Transcripts. The transcription of CUTs predominantly arises from nucleosome-free regions, most of which correspond to promoter regions of bona fide genes. Some of the CUTs start upstream from messenger RNAs and overlap their 5' end. Our study of genes involved in the nucleotide biosynthetic pathway and glycolysis, as well as recent results from the literature, indicate that such concurrent transcription is potentially associated with regulatory mechanisms. Our data reveal numerous new CUTs with such a potential regulatory role. However, most of the identified CUTs corresponded to transcripts divergent from the promoter regions of genes, indicating that they represent by-products of divergent transcription occurring at many and possibly most promoters. Eukaryotic promoter regions are thus intrinsically bidirectional, a fundamental property that escaped previous analyses because in most cases divergent transcription generates short-lived unstable transcripts present at very low steady-state levels (Neil et al., Nature 2009; Jacquier, Nat Rev Genet 2009). We now study how this phenomenon can be involved in novel specific or global gene regulation mechanisms.

Keywords: RNA metabolism, RNA degradation, RNA quality control, ribosome biogenesis, genetic screens, Saccharomyces cerevisiae

Yao, YH, Demoinet, E, Saveanu, C, Lenormand, P, Jacquier, A, Fromont-Racine, M.2010. Ecm1 is a new pre-ribosomal factor involved in pre-60S particle export. RNA16:1007-1017

Neil, H., Malabat, C., d'Aubenton-Carafa, Y., Xu, Z., Steinmetz, L.M. and A. Jacquier2009. Widespread bidirectional promoters are the major source of cryptic transcripts in yeast. Nature457:1038-1042

Decourty, L., Saveanu, C., Zemam, K., Hantraye, F., Frachon, E., Rousselle, J-C., Fromont-Racine M., and A. Jacquier 2008. Linking functionally related genes by sensitive and quantitative characterization of genetic interaction profiles PNAS105:5821-5826

Lebreton, A., Rousselle, J.C., Lenormand, P., Namane, A., Jacquier, A., Fromont-Racine, M. and Saveanu, C. 2008. 60S ribosomal subunit assembly dynamics defined by semi-quantitative mass spectrometry of purified complexes. Nucleic Acids Res36:4988-4999.