Role of NSD/NGD: identification of targets and factors involved in these processes in Saccharomyces cerevisiae – NGD-NSD

To decrypt the cytoplasmic surveillance mechanisms presented above, our project is divided into two tasks:
The first one is to identify and characterize the different molecular factors involved in these regulatory pathways. For this we use genetics, biochemistry and molecular biology approaches. We selected and identified several mutants involved in the degradation pathways of aberrant proteins. We are currently characterizing biochemically and functionally complexes involved in these pathways to define the specific role of the different proteins.
The second task is to identify, at a genomic scale, genes regulated by the NGD/NSD pathways. For this we have implemented a combination of innovative approaches. We have developed approaches for studying gene expression across the cell. We combine transcriptomic approaches (to study the transcription of all RNA in a cell), translatomics approaches (to determine the amount and position of translating ribosomes onto mRNA ). These approaches are associated with proteomic approaches to analyse the protein content of the cell. These three approaches cover all steps of gene expression and should allow us to identify, using bioinformatics analysis, ribosomes blocking sites and target genes of these degradation pathways. The use of mutants affecting NGD / NSD pathways will allow us to better define their physiological importance.

We have characterized a novel complex involved in the degradation of peptides derived from the translation of aberrant mRNAs. This characterization was achieved through a combination of genetic and biochemical approaches and clarified the steps leading to the degradation of peptides still associated with ribosomes. These results are important because they not only allow to precisely identify the actors involved in the degradation of peptides derived from the NSD, but also because they allow us to specify the steps between pausing the ribosome and degradation of newly synthesized peptide.

The perspectives opened by a better understanding of the coupling between translation and degradation of mRNA / proteins are mainly fundamental. Indeed, these pathways may be a new step in gene regulation, yet their importance in regulating gene expression is currently poorly known.
It is too early to talk about societal or environmental impact.

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4. Defenouillère Q, Yao Y, Mouaikel J, Namane A, Galopier A, Decourty L, Doyen A, Malabat C, Saveanu C, Jacquier A, Fromont-Racine M. Cdc48-associated complex bound to 60S particles is required for the clearance of aberrant translation products. Proc Natl Acad Sci U S A. 2013 Mar 26;110(13):5046-51.

In eukaryotic cells, mRNA turnover is highly regulated and plays important roles in gene expression or in antiviral responses. Moreover the integrity of mRNAs is strictly controlled by different quality control mechanisms to prevent the expression of deleterious proteins. Among these pathways, nonstop mRNA decay (NSD) and no-go mRNA (NGD) decay are the most recently identified quality control mechanisms in Saccharomyces cerevisiae. Both these pathways depend on the translational status of the mRNA: NSD targets either an mRNA lacking a stop codon or an mRNA with a ribosome translating downstream the stop codon, NGD targets mRNA with a ribosome stalled during the translation elongation cycle. Both NSD and NGD promote rapid degradation of the mRNA, dissociation of ribosome and degradation of the no-go peptide (NGP) and the nonstop peptide (NSP).
Mechanistically few factors involved in these two pathways have been identified so far. Recently, it has been proposed that the complex Dom34p/Hbs1p, required for NGD would act as a "termination like" complex independently of the codon present in the ribosomal A-site. Indeed biochemical evidences suggest that this complex promotes translation termination and ribosome dissociation when ribosome is stalled. However the involvement of other factors in NGD remains unknown. The mRNA is degraded by the exosome and also probably decapped to prevent new round of translation. In the NSD, it would be Ski7p that would mimic eRF3 within the empty A-site and recruit the Ski-exosome complex. The behaviour of the NSP/NGP synthetised until the translational arrest remains very elusive. Up to now only one protein, Ltn1p, an E3 ubiquitin ligase, has been involved to the degradation of these peptides, but evidences exist demonstrating that others genes are also involved in this process.
Our project aims to identify cellular genes regulated by NGD/NSD pathways, to analyse to what extent these pathways are globally involved in the normal process of gene expression and to characterize new factors involved in the degradation of aberrant proteins (NGP/NSP). To reach this objective we will develop a combination of state-of-the-art approaches (transcriptomics, translatomics and proteomics) to analyse the accumulation of RNA, Ribosome and proteins in yeast strains defective for NGD/NSD (Teams1, 2, 4). These approaches will be associated with a robust bioinformatics analysis to identify signals promoting NGD/NSD (Team 3). These signals will be experimentally validated and will be used to search genes carrying such signals in the yeast genome (Teams 1 and 3). In parallel a functional and molecular characterization of the genes identified by these global approaches will be performed (Teams 1 and 2). This study will be completed by a characterization of factors involved in NGP/NSP degradation, starting from new genes already identified by one team of the consortium (Team 2).
These data will contribute to a better understanding of these highly conserved mRNA quality control mechanisms in eukaryotes and to better apprehend their physiological importance in diverse conditions. They will provide new information about factors involved in the control of aberrant protein degradation, which is also an important quality control preventing the appearance of diseases linked to misfolded proteins or peptides (like alpha-synuclein in Parkinson's disease).