Deciphering the function of proteins involved in ribosome biogenesis – BIORIB

In eukaryotes, the ribosome cannot assemble spontaneously from isolated components. Ribosome biogenesis takes place in the nucleolus, a specialized nuclear compartment, and requires the coordinated action of over 200 non ribosomal proteins. The function of these non ribosomal factors is generally indispensable for cell viability but remains poorly understood. Assembly of the ribosomal proteins onto the rRNA is highly coordinated with rRNA processing and takes place during rRNA transcription. The proteins assemble on the nascent rRNA forming huge ribonucleoprotein complexes containing both ribosomal and non-ribosomal proteins. One of the best characterized complexes is a 2 MDa RNP called the SSU processome or 90S pre-ribosome which comprises the U3 snoRNP and more than 40 proteins. This large RNP can be visualised as terminal knobs on transcribed rRNA still attached to the rDNA chromatin. Understanding ribosome biogenesis is challenging for many reasons. - The maturation of ribosomes is a multistep dynamic process in which the protein complexes are remodeled and recruited following specific maturation events. The complexes formed are therefore transient and are difficult to detect and validate experimentally. - A minority of proteins of the ribosome biogenesis regulon have a demonstrated functions (helicases, nucleases, RNA modification enzymes,…). Sequence analysis by bioinformatics often does not provide any clue on the function of these proteins. - Most of these proteins are essential, making in vivo deletion studies non conclusive. The aim of the project is to obtain functional information on the proteins of the ribosome biogenesis regulon by determining their three dimensional structure. Our experience from structural genomics shows that the three dimensional structure sometimes provides an unambiguous functional assignment. In the most difficult cases, useful insight on the function of the protein can still be extracted. Co-crystallisation with co-factors or activity assays can then be used to confirm the predicted function. The interactome of ribosome biogenesis proteins is extremely complex and dynamic, but it has been demonstrated that complexes containing two or three proteins often act as small independent molecular modules inside the larger ribonucleoprotein complex. The project will focus on the validation and on the structure determination of protein complexes, using recombinant proteins. Solving the structure of protein complexes will undoubtedly provide a wealth of information on not only the function of the proteins but also on the organization of the larger complexes such as the SSU processome. The different techniques envisaged for the projects are : - Bioinfomatics analysis and literature datamining for the choice of protein targets - Cloning of genes and bacterial expression tests for isolated proteins - Crystallogenesis of purified proteins - Reconstitution and validation of complexes from purified proteins and crystallogenesis - Purification and validation of complexes by co-expression of proteins - Conception of mutants for in vitro and in vivo functional assays The deregulation of ribosome synthesis has drastic consequences on the cell. Several genetics diseases, such as X-linked dyskeratosis congenital, are the consequence of mutations in proteins of the the ribosome biogenesis regulon. Cancer is also often associated with deregulation of protein biosynthesis apparatus. Due to their essential nature, these proteins are therefore emerging as new potential antimicrobial or tumour inhibitory drug targets. Our results will shed light on the mechanism of deregulation on one hand and provide structures for rational drug design on the other.