Molecular Basis for Ribosomal Protein Protection from Cellular Degradation

Abstract

Ribosomes are large macromolecular machineries composed of both protein and RNA constituents with a species-dependent molecular mass of at least ~3.3 MDa for the fully assembled eukaryotic 80S ribosome. Their catalytic activity is dependent on ribosomal RNA; therefore, ribosomes are bona fide ribozymes, and as such they mediate the final step of gene expression from DNA to RNA to protein by peptide bond formation between amino acids. Importantly, spatial separation of ribosome function and biogenesis into distinct cellular compartments allows for intricate regulatory mechanisms and rigorous quality control. Ribosome biogenesis occurs predominantly in the nucleolus and nucleus of the cell with final cytoplasmic maturation and quality control steps. Briefly, nucleolar ribosomal RNA together with ~200 trans-acting assembly factors co-transcriptionally forms the 40S and 60S pre-ribosomal subunits into which ~80 ribosomal proteins are incorporated in a hierarchical fashion. Recent studies, including this thesis, have identified a novel class of dedicated ribosome assembly chaperones, in addition to the ~200 trans-acting ribosome assembly factors, which facilitate ribosomal protein shuttling. Ribosomal proteins are generated in the cytoplasm, and with only few exceptions they all have to enter the nucleus for incorporation into the pre-ribosomal subunits. Assembly chaperones can bind and protect unassembled ribosomal proteins either co-translationally or following nuclear import and shuttle them in a timely fashion to their destination sites at the maturing pre-ribosomal subunits. The first chapter of this thesis describes the identification and characterization of a dedicated assembly chaperone for the large ribosomal subunit protein RpL4, termed Acl4. Interestingly, Acl4 and likely also other dedicated assembly chaperones not only interact with ribosomal proteins to avoid aggregation and to shield them from unfavorable interactions, but also protect their client proteins from cellular degradation by the ubiquitin-proteasome machinery. Ribosomes are built by assembling equimolar amounts of ribosomal proteins, which generates a challenge for the cell to ensure stoichiometric quantities of ribosomal proteins. Recent studies have demonstrated that stoichiometric levels of ribosomal proteins are established by cellular degradation of excess protein via ubiquitination of unassembled components. The second chapter of this thesis describes a conserved degradation pathway, which is dependent on the E3 ubiquitin ligase Tom1 to mark unprotected and unassembled ribosomal proteins and target them for degradation. Moreover, it is demonstrated in the third chapter for the first time how an assembly chaperone protects its client ribosomal protein from ubiquitination and proteasome-mediated degradation. High resolution structures of the Acl4•RpL4 complex as well as RpL4 in complex with the nuclear transport factor Kap104 visualize the molecular interactions of those proteins and uncover the molecular mechanism of protecting conserved Tom1-target sites within RpL4. Together, the reported results identify and characterize both a novel degradation pathway as well as a protection mechanism for ribosomal proteins and advance the understanding of the intricate regulation of ribosome biogenesis.