SAFAPOLYA

Co-transcriptional pre-mRNA processing regroups essential events that contribute to both the accuracy and efficiency of gene expression. Among the different processing steps to which a pre-mRNA is subjected, polyadenylation represents a fundamental modification vital for eukaryotic cells. Pre-mRNA 3'-end processing is a modification that occurs in the nucleus of eukaryotic cells. Importantly, the polyadenylation reaction is required for the RNA polymerase to terminate its elongation. Subsequently, poly(A) tails are necessary to export the mature mRNA to the cytoplasm, for its translation into proteins, and its half-life. Protein factors required for this processing have been identified during the last 20 years. In the yeast Saccharomyces cerevisiae, a model organism of choice to study essential nuclear processing that happen in all eukaryotic cells (splicing, capping, modifications, polyadenylation...), factors have been identified by complementary genetic and biochemical investigations. They are organized in two multimeric complexes: CF I (Cleavage/polyadenylation factor I) and CPF (Cleavage and Polyadenylation Factor). Numerous subunits in yeast have found their homologues in metazoans. Other individual proteins have been identified based on their essential requirement for the specificity and the control of the reaction, such as the precise determination of the poly(A) site and the control of the length of poly(A) tails added to the mRNA. A complete in vitro system to recapitulate the pre-mRNA 3'-end processing reaction is available in yeast. A high degree of refinement to study polyadenylation in vitro has recently arisen from fast, reproducible and efficient purification procedures in yeast or the production of recombinant proteins in Escherichia coli. Yet, the function of most of the polypeptides of this large machinery (more than 20 different subunits) is still elusive. Moreover, if structural information on a few subunits is available - often of individual domains - a larger view of the factors they belong to and of their function does not exist yet. To gain more insights into the yeast polyadenylation machinery, we will employ an interdisciplinary approach, combining structural and functional techniques. We plan to use a large panel of structural and analytical biology (NMR, X-ray diffraction, SAXS, SANS, electron cryo-microscopy, mass spectrometry) and combine them with functional analyses of yeast strains. Wild-type and mutants, randomly obtained or genetically engineered based on structural information will be tested in reconstituted in vitro processing assays. Structural information from electron microscopy images, SAXS or SANS, X-ray and NMR will be combined into a single 3D model describing the overall architecture of these complexes and their interaction with the RNA target. This work should end up with the first description of the architecture of the yeast polyadenylation complex and bring new insights into the function of some of its subunits.