|Director : Alain Jacquier (email@example.com)|
We study various aspects of RNA metabolism in the yeast S. cerevisiaecerevisiae (maturation, transport, and degradation). 1) We take advantage of our own two-hybrid database to identify new factors involved in RNA maturation processes (ribosomal RNA and messager RNA). We select in the database previously uncharacterized proteins, which are now embedded within a specific network of interactions. We first use different generic approaches, including genetic screens and biochemical affinity purification) to characterize these candidates. More specific functional assays are then used in order to more precisely define the functions of these proteins. 2) We are characterizing pre-ribosomal complexes involved in the maturation of the 60S particle.
Some candidates of unknown function and embedded within a network of interactions specific for a given metabolic pathway are selected for functional analyses. In order to biochemically confirm that the selected candidates indeed belong to a given network of physical interactions, we use a double-affinity biochemical purification technique. The proteins from the purified complexes are identified by mass-spectrometry, in collaboration with the "Plateau Technique Protéomique" at the Institut Pasteur (A. Namane). In some cases, we apply genetic screens, such as synthetic-lethality screens, to provide information of a functional nature. These are complementary to the physical information provided by the two-hybrid and purification methodologies. The combination of the three methods points to the step in which the candidate is involved. Finally, classical functional assays, specific for the identified pathway, are performed.
- Components of the small mitochondrial ribosomal sub-unit
The Ykl155c protein was initially linked, by two-hybrid, to a number of other proteins, including splicing and transport factors, making it an attractive candidate for functional analyses. In fact, the Ykl155c associated complex turned out to correspond to the small mitochondrial ribosomal sub-unit. This let us to identify 12 new proteins of this ribosomal particle (Saveanu et al. (2001) J. Biol. Chem., 276, 15861-15867). We confirmed that these new factors were indeed located into the mitochondria and that they were required for its function. This work illustrates the limits of the two-hybrid approach since, in this case, the technique selected non-specific interactions. While this study gave rise to interesting results, we do not pursue it because it lies too far away from the general interests of the laboratory.
- Yel015wp and mRNA degradation
The two-hybrid network built around the eight Lsm proteins revealed a strong link between Yel015wp and the mRNA degradation pathway (Fromont-Racine et al. (2000) Yeast, 17, 95-110). We are now exploiting the results of colethality screens, tandem affinity purification and DNA micro-arrays analyses in order to define the function of Yel015wp.
Role of a protein associated with the nucleopore complex
We selected the Yar002w protein for functional studies because it had multiple connections with proteins involved in transport, suggesting that it was itself implicated in the nucleo-cytoplasmic transport pathway. Part of the two-hybrid links was confirmed by the biochemical approach which identified the factors most stably associated with this protein. This protein has since been identified as the nucleoporin Nup60 (Rout et al. J Cell Biol, 148, 635-651). Two-hybrid links, together with genetic links that we identified, suggest a role for this protein in RNA export. Yet, the results, as a whole, indicate that this protein must play a more pleiotropic function, at the crossing of different transport pathways, by interacting with proteins of the nucleoplasm.
- Nup60 and the nucleo-cytoplasmic architecture
This work has been performed in collaboration with the group of Ulf Nehrbass at the "Institut Pasteur". Starting from the nucleoplasmic nucleoporin Nup60p we have identified structural constituents of silent domains in yeast. By structure/function analyses we demonstrate that silent domains play an active role in transcriptional repression. Our data support a model in which domains of immobilized transcriptional regulators allow chromatin to determine transcriptional states by relative positioning (Feuerbach et al., Nature Cell Biology in press).
- Nucleo-cytoplasmic architecture and splicing
Vincent Gali, in the group of Ulf Nehrbass, has found a genetic link between Mlp1p and prp18p, a splicing factor. We have demonstrated that the depletion of Mlp1p affects the retention of intron containing premRNA without affecting splicing. This work is pursued in collaboration with Olivier Gadal and Ulf Nehrbass (manuscript in preparation).
Characterisation of nuclear preribosomal complexes
Nog2p (Ynr053c), the Saccharomyces cerevisiae homologue of the human Ngp-1 autoantigen is located in the nucleus/nucleolus. We found Nog2p to be physically associated within large complexes containing, beside a subset of ribosomal proteins, at least two additional factors, Nog1p and Rlp24p. In the absence of Nog2p, the pre-60S ribosomal complexes, containing rRNA precursors, left the nucleolus, but were retained in the nucleoplasm. We also observed a defect in 5.8S and 25S maturation with increased levels of both 27SBS and 7SS precursors. Nog1p and Nog2p are conserved putative GTP-binding proteins. The precise role of these proteins remains to be elucidated.
|Publications of the unit on Pasteur's references database|
|Office staff||Researchers||Scientific trainees||Other personnel|
LABOUISE, Odile, firstname.lastname@example.org
FROMONT-RACINE, Micheline, CNRS CR1, email@example.com
LEGRAIN, Pierre, CNRS DR1, firstname.lastname@example.org, on leave
SAVEANU Cosmin, Postdoc, email@example.com
BREARD, Gwenaël, PhD student, firstname.lastname@example.org
CLERTE, Caroline, Postdoc, email@example.com
COST, Grégory, Postdoc
HANTRAYE, Florence, Engineer, firstname.lastname@example.org
DECOURTY, Florence, Technician, email@example.com