Procesos moleculares implicados en los pasos iniciales de la síntesis de los ribosomas humanos

Abstract

Ribosome synthesis is an intricate process during which the transcription, modification, folding, and processing of the rRNA precursors (pre-rRNAs) are coordinated to build mature ribosome subunits. This process is driven by more than 200 ribosome biogenesis factors (RBFs) that, together with the pre-rRNAs and ribosomal proteins, form different preribosomal complexes. These complexes migrate from the nucleolus to the cytoplasm as they mature. The composition and structure of preribosomes in human cells are ill-defined and, in the case of those formed in the most internal regions of the nucleolus, this is caused by technical limitations. The main reason for those limitations is that the highly viscous nature of the inner regions of the nucleolus precludes the extraction of the complexes. The first part of this thesis is focused on the generation of tools and the development of extraction methods to characterize early nucleolar preribosomes in human cells. The initial studies were the validation of the PSE extraction method to isolate early preribosomes and the generation of cell lines that endogenously express an early RBF fused to GFP to be used as bait for preribosome purification by affinity chromatography. Using these tools, and a combination of mass-spectrometry compositional analysis and sucrose-gradient sedimentation experiments, it was found that the composition of the preribosomal subcomplex UTP-B and the function of the UTP14A protein are conserved in humans and yeast. The second part of the thesis was devoted to the functional characterization of RRP8, a methyltransferase responsible for the m1A1322 modification in the 28S rRNA. The identification of RBFs that interact with RRP8 and the characterization of the RRP8 knockout phenotype unveiled that this protein forms a module with one 40S subunit RBF that binds to the bipartite preribosome, the initial intermediate that contains the primordial precursors of both the small and large subunits. The RRP8 module is required for the efficient formation of bipartite preribosomes. The complete loss of RRP8 is well tolerated by transformed cell lines, but not by non-transformed cells indicating that its function is essential for the viability of some cell types.