Pre-ribosomal particles in yeast

Many of the basic mechanisms of eukaryotic ribosomal formation are gleaned from genetic and biochemical experimentation in Saccharomyces cerevisiae . Recent advances in proteomics have allowed the characterization of nascent ribosome assembly intermediates.; A combination of genetic, biochemical, and proteomic approaches was utilized to characterize the role of the essential evolutionarily conserved nucleolar protein Rrp1p in 60S ribosomal subunit biogenesis. The formation of 27SB _S, 25.5S and 7S pre-rRNAs is inhibited while the relative amounts of 27SA₂, 27SA₃, 27SB_L pre-rRNAs increase upon inactivation or depletion of RRP1. Furthermore, inactivation of Rrp1p may disrupt the association between ribosome assembly factors and a subset of 66S pre-ribosomes. Consistent with this role in ribosome assembly, affinity chromatography with Rrp1p enables the enrichment of 27SA, 27SB, 25.5S, and 7S pre-rRNAs as well as numerous 60S assembly factors. Rrp1p fulfills a functional role in production of the mature 60S ribosomal subunit and is an integral component of 66S pre-ribosomes.; Two additional essential nucleolar 60S assembly factors were characterized utilizing proteomic approaches. Affinity chromatography with Nip7p enriches for 66S pre-ribosomes and super-stoichiometric amounts of the putative methyltransferase Nop2p. This reveals a possible role for a Nip7p-Nop2p heterodimer in 60S ribosomal assembly and rRNA methylation.; Epitope-tagged Nop4p enriches for 35S, 27S, 25.5S, and 7S pre-rRNAs in addition to 90S and 60S specific assembly intermediates. Thus, these associations reflect a role for Nop4p in both the early stages of 90S particle formation as well as the later stages of 66S pre-ribosomal particle assembly. Taken together, these data demonstrate the utility of orthogonal approaches in resolving the complexity and dynamics of eukaryotic ribosomal subunit assembly.