We are interested in RNA metabolism and our results cover a large array of different cellular processes involving RNA, like the identification of novel transcripts, the assembly and the transport of ribonucleoprotein particles or the mechanisms of mRNA degradation in the cytoplasm. We use the yeast Saccharomyces cerevisiae as a model organism and try to develop novel genomic-scale tools for this unicellular eukaryote. We spend our research time between different projects on several main topics:

Dynamics of assembly and export of the large ribosomal subunit in S. cerevisiae.

We identified novel factors involved in late 60S ribosomal subunit assembly steps, in the cytoplasm of yeast cells, like the conserved ATPase Drg1 (Pertschy et al., Mol Cell Biol 2007 - in collaboration with Helmut Bergler’s laboratory, Graz University, Austria). We also identified a network of functional and physical interactions between Kap121, Rei1, Arx1, Alb1 and Tif6 involved in the recycling of pre-ribosomal proteins that shuttle between the nucleus and the cytoplasm and required for proper 60S ribosomal subunit maturation (Lebreton et al., J Cell Biol 2006). The Jjj1 Hsp40 chaperone, a physical and genetical partner of Rei1, is also involved in the dissociation/recycling of the pre-60S shuttling factors. However, while the functions of Jjj1 and Rei1 partially overlap, Jjj1 is preferentially required for the release of Arx1 and Alb1 shuttling factors from the cytoplasmic pre-60S particles while Rei1 is preferentially involved in their recycling (Demoinet et al., RNA 2007). In the earlier steps of 60S biogenesis, we recently described the role of the nucleolar pre-60S WD40 repeats protein Mak11 in maintaining the levels of the ribosomal-like protein Rlp24 and its association with nascent ribosomal particles (Saveanu et al., Mol Cell Biol 2007). For the global description of the pathway, we used quantitative mass-spectrometry coupled with affinity purification of pre-ribosomal complexes from various mutant strains (Lebreton et al., Nucl Acids Res 2008).

Figure ribosome biogenesis

A summary of sequential association to and dissociation from the nuclear and cytoplasmic pre-60S particles. The pre-60S factors studied in our group are shown as color shapes. Many others preribosomal factors (more than 80) are involved in the large ribosomal subunit biogenesis. They are symbolized by grey shapes with a white contour. This figure illustrates the existence of several classes of preribosomal factors. Some of them such as Drg1, Rei1 and Jjj1 are strictly cytoplasmic; others, such as Mak11 and Nsa2, are strictly nuclear and recycled within the nucleus; and finally others, such as Rlp24, Nog1, Arx1/Alb1 are shuttling factors. Their release from the preribosomal particles requires cytoplasmic pre-60S factors and their recycling involves karyopherins.

We currently work on new mass-spectrometry based tools that give a dynamic view of the maturation process in vivo and have identified new 60S associated factors that could link translation and ribosome biosynthesis.

Mechanisms of mRNA degradation.

One of the major steps in mRNA degradation is the decapping reaction, usually triggered by a reduction in the size of the poly(A) tail. Sometimes, decapping can occur without deadenylation. We identified a mechanism of deadenylation-independent mRNA decapping and degradation that involves the protein Edc3 - a factor physically and genetically linked to the decapping machinery. The auto-regulation mechanism involved in the fine-tuning of the RPS28B mRNA levels depends on Edc3 (Badis et al., Mol Cell 2004). We analyzed other candidate proteins that are involved either in general decapping or in the regulation of specific transcripts levels. The general method for quantitative tests of the effect of combining mutations on a large scale, allow the identification of novel functional links in RNA synthesis and decay (Decourty et al., PNAS 2008).

RPS28 degradation

CUTs - Cryptic Unstable Transcripts.

Browse our genome-wide yeast CUTs abundance and localization results (Neil et al., Nature 2009)

CUTs - Cryptic Unstable Transcripts.

A few years ago, in collaboration with three other laboratories, we described the existence of a novel class of ubiquitous transcripts that are normally 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). These transcripts, collectively known as CUTs - for Cryptic Unstable Transcripts, are very abundant in mutant strains that lack the degradation or poly-adenylation activities. The study of CUTs gives hints to understand cellular processes as diverse as transcription initiation and termination (Gudipati et al., EMBO J 2012), nucleo-cytoplasmic transport and translation. We have exhaustively mapped the position and abundance of yeast CUTs (Neil et al., Nature 2009). The association of CUTs with gene promoters strongly suggests that eucaryotic promoters are intrinsically bidirectional (for a review, see Jacquier A, Nat Rev Genet 2009).

Genome-wide genetic screens by GIM (Genetic Interactions Mapping).

To explore the effects of combining mutations and thus, be able to better understand complex functional interactions in a cell, we developed a novel genetic screening method that combines the exhaustivity of the yeast systematic gene deletion collection, with a novel haploid specific selection marker and detection of the effects of combining gene mutations on growth by the use of microarrays (Decourty et al., PNAS 2008) [full text]. Microarray based methods for genome-wide testing of resistance to toxic chemicals or antibiotics can also give indications about the target of a drug or how the cells adapt to a specific stress (Peyroche, Saveanu et al., PLOS One 2012, Zeidler et al., J Antimicrob Chemoter 2013).

Genetic interactions

As a side effect of doing large scale screens, we are developing tools for data analysis and visualization. These tools use combinations of database, Python, R and JavaScript programs.

Prototype for data exploration (screenshot)

Screenshot of the web interface developed by Christophe Malabat for genetic interactions screens results exploration (also a custom made LIMS).

For further details, please download the annual scientific report for 2013. Our laboratory belongs to the “Genomes and Genetics” department.

2014-02-20 10:20:00