Talk on Saturday 08:54-09:06am submitted by Beril Kumcuoglu
Eukaryote-specific extensions of ribosomal proteins are involved in 60S ribosomal subunit assembly
Beril Kumcuoglu (Carnegie Mellon University), Jelena Jakovljevic (Carnegie Mellon University), John L. Woolford (Carnegie Mellon University)
Recently, our lab demonstrated that the domains of eukaryotic 60S ribosomal subunits are formed in a hierarchical manner. Importantly, the function of ribosomal proteins (r-proteins) in pre-rRNA processing correlates with their location in the mature 60S subunit. Depletion phenotypes reveal the earliest defect in the assembly pathway, after which pre-ribosomes are turned over. Therefore, to understand the functions of r-proteins in more detail, we need to construct more specific mutations.
One of our goals is to distinguish the functions of flexible eukaryote-specific extensions of r-proteins from these of their globular domains. Interestingly, eukaryotic elements are clustered at the solvent-exposed side of the 60S subunit while the peptidyl-transferase center is devoid of these additions. We suggest that these eukaryotic elements have a role in stabilization of pre-ribosomes by providing additional protein-protein and protein-rRNA interactions. To test this hypothesis, we systematically truncated and introduced missense mutations to the eukaryotic extensions of four r-proteins (L7, L8, L25, L35). Our data indicate that the extensions of these four proteins are essential for viability. Truncation mutations of L25 and L35 resulted in the same pre-rRNA processing defects as depletions of each protein. In contrast, a partial truncation mutation of L8, along with a missense mutation of L7 resulted in different pre-rRNA processing defects than their depletions. These results led us to propose a model where the N-terminal extension of L8 is involved in a later step of ribosome assembly than its globular domain, potentially rearranging the 5’ end of 25S and the 3’ end of 5.8S rRNAs in domain V.
Our findings indicate that the flexible eukaryotic extensions of ribosomal proteins are involved in 60S subunit maturation. Additionally, our approach provides insights into the evolution of protein-protein and protein-rRNA interactions in ribosomes from bacteria to eukaryotes.
Gamalinda, M., Ohmayer, U., Jakovljevic, J., Kumcuoglu, B., Woolford, J., Mbom, B., Lin, L., et al. (2014). A hierarchical model for assembly of eukaryotic 60S ribosomal subunit domains. Genes & development, 28(2), 198–210. doi:10.1101/gad.228825.113
Keywords: pre-rRNA processing, ribosome assembly, ribosomal proteins