Structural And Functional Analysis of Yeast Cleavage and Polyadenylation Factors – SAFAPOLYA

We plan to study in details a protein complex that present multiple essential features. It can specifically interact with mRNA, it interacts physically with the RNA polymerase that is responsible for its synthesis, it binds ATP, and finally, it makes multiple contacts with the other multi-subunits factor of the polyadenylation machinery. Another layer of complexity is reached through the discovery that two of the four subunits dimerize within the complex. To reach a comprehensive understanding of the necessity for such a molecular scaffolding, we combine scientific and technological strategies with the aim to reach a low-resolution structure of the complex by means of cryo-electron microscopy, within which the high-resolution data obtained by both nuclear magnetic resonance and X-ray crystallography take place. Simultaneously, a functional study by means of molecular genetics, conducted in vivo and tested in vitro, of the factor subunits is developed, using information already collected or newly obtained through the structural approach, and conversely, when mutants already available will be analyzed with structural methods which will help understand the defects of these mutants, which will allow us to deduce the function of the «normal« proteins. These architectural studies of the poyladenylation complex are supplemented by the powerful and accurate tools of mass spectrometry.

We made large progress in the structural and functional aspects of one of the sub-units of the complex, involved in both polyadenylation and transcription termination. Two new domains of this protein have been revealed. The atomic structure of the first one has been solved (article in press) and its direct implication in termination of transcription has been revealed, too. The second domain analyzed showed a role in the polyadenylation reaction itself; determination of its structure is underway.
Concerning the multimeric organization of the complex, two parallel approaches have been used, using MS with native recombinant factors, or after employing stabilizing agents such as cross-linkers. Both techniques allow us to believe that the complex is mainly found as two different multimeric factors. The putative function of these alternative assemblies is currently analyzed genetically and in vitro.

The new findings obtained during this early step of the project primarily lead to publications in international peer-reviewed journals, and contribute to the broad scientific community through participation in seminars, workshops and conferences. Direct applications, other than those that may of interest for the scientific community, for instance for the direct benefit of the society or the environment, is difficult to appreciate at this stage of the project.
With regards to new research programs, plenty of questions arose from our data. On the technical side, we have to improve stabilization methods, in particular to help improve EM studies. On the scientific side, the different forms of the complexes analyzed raised new questions, in particular in view of alternative polyadenylation

Last year, one of the partner published the structure of a mutant subunit of the factor. This report expands our knowledge of the factor by bringing explaination of the mutant phenotype at the atomic level.

Xu, X., Perebaskine, N., Minvielle-Sebastia, L., Fribourg, S., Mackereth, C.D. (2015). «Chemical shift assignments of a new folded domain from yeast Pcf11«. Biomol. NMR Assign.
Based on project common to three of the partners, the atomic structure of a newly identified domain of Pcf11 has been recently published. The function of this domain has also been extensively studied in yeast and will lead to another common publication soon.

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.