Function of yeast eRF1 methyltransferase in translation termination and mRNA decay – RF-MTase

Methylation is one of the major post-translational modifications present in prokaryotic and eukaryotic cells. Protein N-methylation in yeast is often involved in regulation, such as in the case of histones, where it is clearly linked to gene repression through heterochromatin fomation. This process is also essential to translation, since methylation of ribosomal proteins seems important for ribosome biogenesis. Class I release factors (RF) are also methylated both in E.coli and S.cerevisiae. It is important to note that besides a common function, the only conserved part is a small tripeptide motif (GGQ) which is in both cases methylated on its glutamine residue. This tripeptide enters in the peptidyl-transferase center of the ribosome, triggering peptidyl-tRNA hydrolysis. In absence of the ribosome, eRF1 forms a complex with eRF3, the class II RF whose function is less clear than that of its bacterial counterpart, involved in RF1 and RF2 recycling. In E.coli, PrmC, which methylates RF1 and RF2, was the first characterized protein Gln methyltransferase. In S. cerevisiae, eRF1 methylation is catalyzed by a methyltransferase comprising two subunits: Ydr140w and Ynr046w, and is dependent on the presence of eRF3 and GTP. Deletion of Ydr140w gene leads to a severe growth defect. My objectives are to clarify Ydr140w function, studying its potential role in several other processes related to translation. Towards this goal I plan to study: (i) the mechanism of eRF1 methylation (ii) the role of eRF1 methylation in termination efficiency (iii) the role of eRF1 methylation in the nonsense-mediated mRNA decay (NMD) process - 1.Mechanism of eRF1 methylation: Ydr140w methylates eRF1 but requires a partner, Ynr046w, which belongs to a large family of totally uncharacterized proteins. A structural approach, in collaboration with Herman van Tilbeurgh's crystallography group (Université Paris Sud), will be an important part of elucidating the mechanism of methylation and Ynr046w function. A functional approach will be pursued in the archaeae Halobacterium salinarum, where there exist homologs of Ynr140w and Ynr046w, but no known equivalent of RF3. 2. Role of eRF1 methylation in termination efficiency: Preliminary experiments show that the absence of eRF1 methylation has a negative effect on in vivo termination in S. cerevisiae. However, this has to be investigated in further detail to determine the exact function of methylation. New experiments are necessary to determine if methylation has the same effect independently on the nature of the stop codon. This work will be done in collaboration with the group of J-P. Rousset (Université Paris Sud), who developed experimental procedures for the qualitative and quantitative study of termination activity. We will also examine by immunoprecipitation experiments whether eRF1 methylation might be involved in post-termination steps such as the interaction between RFs and the poly(A) binding protein. 3. Role of eRF1 methylation in the NMD process: The nonsense-mediated mRNA decay pathway in eukaryotic cells is a mechanism allowing rapid degradation of mRNA having an in frame premature stop codon. The processes of NMD and translation termination are intimately connected. In addition of the physical interaction between the NMD factors and translation termination factors, mutations in genes encoding the NMD factors (Upfp) not only lead to the stabilization of nonsense-containing mRNAs, but also result in a nonsense suppression phenotype. I plan to study the possibility of a link between eRF1 methylation and the stability of nonsense mRNA. Two kinds of experiments will be done: (i) mRNA stability measurements on ydr140w mutant, upf mutants and double mutants upf / ydr140w, (ii) immunoprecipitation experiments to study the effect of methylation on eRF3 / Upf factor interactions. The possibility of a physical interaction between the methylation complex Ydr140w.Ynr046w and NMD factors will also be studied carefull...