The "Unité de Génétique des Interactions Macromoléculaires" studies various aspects of RNA metabolism in the yeast S. cerevisiae. We take advantage of our own two-hybrid database to identify new factors involved in these processes. We select in the database previously uncharacterized proteins, which are now embedded within a specific network of interactions. We first use different generic approaches to characterize these candidates. Then, more specific functional assays are used in order to more precisely define the function of these proteins.
Example of a functional network issued from the two-hybrid database: involvement of Lsm proteins in mRNA degradation
The two-hybrid network built around the eight Lsm proteins revealed that they are closely linked between themselves, suggesting the existence of an Lsm complex. At this time, data from the literature suggested that two Lsm proteins were involved in splicing. Our results showed that the Lsm proteins were indeed connected to splicing factors specific for a given step of the spliceosome assembly pathway. More surprisingly, we also found that the Lsm proteins are also connected to the cytoplasmic mRNA degradation pathway by the way of the proteins Dcp1, Dcp2, Xrn1 and Mrt1 (Fromont-Racine et al. (2000) Yeast, 17, 95-110). This study suggested that Lsm complexes could be involved in two different pathways, one located in the nucleus, the other in the cytoplasm. This hypothesis has now been demonstrated by several independent studies.
- Biochemical purification of complexes
In order to biochemically confirm that the selected candidates indeed belonged 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).
At the present time, we have analyzed six biochemical complexes, from which more than 40 proteins were identified. The functional analysis of these complexes is ongoing. For example, we have characterized 12 novel proteins from the small mitochondrial ribosomal sub-unit (see below). Likewise, we now characterize new complexes involved in rRNA maturation (see below).
Synthetic-lethality genetic screens
Synthetic-lethality screens provide information of a functional nature. They are thus very complementary to the physical information provided by the two-hybrid methodology. The general principal of the approach is to look for mutation inducing a growth phenotype only when combined with another defined mutation. The characterization of a phenotype expressed only when both mutations are present simultaneously reveals functional redundancies that often mask the function of the genes. In order to simplify and standardize key steps of the procedure, we bring some modifications to the methodology. Proteins of unknown function, physically linked to the splicing pathway or to an mRNA degradation pathway are being analyzed.
Specific functional analyses
For some local networks defined within the two-hybrid database, the functional analysis is performed in the framework of specific collaborations. We describe below functional analyses directly performed in the laboratory.
Components of the small mitochondrial ribosomal sub-unit
The Ykl155c protein is linked by two-hybrid to a number of other proteins, including both 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, it is not pursued by us because too far away from the general centers of interest of the laboratory.
Roles 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 y the biochemical approach which identified the most stable interactions 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. This work is performed in collaboration with the group of Ulf Nehrbass at the "Institut Pasteur".
Characterization of a nuclear pre-ribosomal complex
The protein Ynr053c, that we now call Nog2 (Nucleolar-G-Protein 2), is nuclear/nucleolar. It is the yeast homologue of the human Ngp-1 protein. The affinity purification of its associated complex revealed that it is associated to a nuclear pre-60S particle. In addition to ribosomal proteins, this complex contains two new factors, Nog1 and Rlp24. We have shown that Nog2 is essential for the last steps of the 60S ribosomal sub-unit biogenesis. Nog2, as Nog1, possess a potential GTP binding domain and could be involved in the coordination of large ribosomal sub-unit maturation steps and export.