Eukaryotic ribosome biogenesis is a crucial and very intricate process that mobilizes an impressive machinery, comprising the three RNA polymerases as well as hundreds of small RNAs and proteins. It is also a process that sequentially occurs in several distinct cellular locations, first in the nucleolus, then in the nucleoplasm and finally in the cytoplasm, where translation occurs. Ribosome production in eukaryotes is initiated by the transcription in the nucleolus by RNA pol I of a pre-rRNA precursor to 18S, 5.8S and 25S/28S rRNAs that will be chemically modified and processed by endo- and exoribonucleases to release the mature rRNAs. A precursor to 5S rRNA is transcribed separately by RNA pol III. The pre-rRNA precursor associates co-transcriptionally with ribosomal proteins, small nucleolar ribonucleoprotein particles (snoRNPs) and non-ribosomal proteins to yield a nascent pre-ribosomal particle from which the early pre-40S particle containing the 18S rRNA sequence is split mostly co-transcriptionally. The early pre-60S particle assembled onto the 5.8S and 25S sequences is released subsequently following transcription termination. These particles then follow independent maturation pathways first in the nucleus and finally in the cytoplasm.
Research during the last two decades has shown that ribosome production requires hundreds of snoRNPs and more than 200 protein co-factors, called non-ribosomal proteins, that dynamically associate with pre-ribosomal particles at various stages of their maturation and are absent from the mature cytoplasmic ribosomes. We participated actively in the discovery that box C/D and box H/ACA snoRNPs catalyze the site-specific 2’O-ribose methylation and pseudouridylation of pre-rRNAs. In contrast, while the list of non-ribosomal proteins required for ribosome biogenesis is probably close to complete in yeast, the molecular functions of most of them, several of which are enzymes, remain elusive. Our aims are to understand the precise molecular functions and modes of regulation of several key non-ribosomal factors involved in the co- and/or post-transcriptional steps of ribosome synthesis, in particular energy-consuming enzymes. To achieve this goal, we are exploring using yeast and mammalian cells the timing of their association with/dissociation from pre-ribosomal particles, dissecting their biochemical activities and post-translational modifications, identifying their targets and partners (rDNA, pre-rRNA and/or proteins) and the molecular consequences of their inactivation. We are also engaged in a collaboration with the team of P.E. Gleizes, LBME, to determine the structure of some pre-ribosomal particles using cryo-electron microscopy and the positions of our favourite proteins on these particles. In addition, in collaboration with structural biologists (S. Fribourg, Bordeaux and N. Leulliot, Paris), we are attempting to obtain the crystal structures of our favourite proteins alone and bound to their partners.
We are concentrating on the following non-ribosomal proteins: