Dynamic Remodeling Events Drive the Removal of the ITS2 Spacer Sequence During Assembly of 60S Ribosomal Subunits in S. cerevisiae

Ribosome assembly in eukaryotes require ~200 trans-acting proteins that drive the concerted processes of pre-rRNA processing and modification, RNA folding and rprotein binding. Previous work has identified the steps of ribosome assembly in which most trans-acting ‘assembly factors’ and r-proteins function. Yet, we are only beginning to understand the mechanistic details of remodeling events facilitated by assembly factors and r-proteins during ribosome assembly. The objective of my work is to understand mechanistic details underlying the removal of spacer sequences ITS1 and ITS2 in pre-ribosomal RNA (pre-rRNA) during assembly of 60S ribosomal subunits in S. cerevisiae. The steps I focused on are (1) the exonucleolytic processing of the ITS1 spacer in 27SA3 pre-rRNA, and (2) the removal of the ITS2 spacer sequence in 27SB pre-rRNA and 7S pre-rRNAs. In the first part of my work, I explored the functions of the evolutionarily conserved assembly factor Erb1 in 60S subunit assembly. Previous research from our lab demonstrated that depletion of Erb1 blocks the removal of ITS1 spacer sequence and affects the association of the interdependent A3 factors with pre-ribosomes. This results in destabilization of the pre-ribosomes resulting in their turnover. Since depletion of Erb1 disrupts its multiple contacts in pre-ribosomes, we hypothesized that more careful mutagenesis perturbing specific intermolecular interactions of Erb1 could reveal additional functions, if any, for Erb1 in 60S ribosomal subunit assembly. I constructed internal deletion mutations targeting the evolutionarily conserved N-terminal half of Erb1 and explored the effects of these mutations on ribosome assembly using molecular and proteomic approaches. My studies revealed a new role for Erb1 in the removal of the ITS2 spacer sequence, in addition to its initial role in processing the ITS1 spacer sequence. I demonstrated that the folding of 5.8S rRNA and stable association of rRNA domain V binding assembly factors are affected in erb1 mutants, thus blocking ITS2 removal. I also demonstrate a role for ES26/ES20 helical structures in domain III of 25S rRNA for 60S ribosomal subunit assembly. Based on these observations, I predict a model in which remodeling events triggered by the removal of Erb1 together with its interacting partner Ytm1 facilitate rearrangements in domain III of 25S rRNA in preribosomes necessary to initiate the removal of the ITS2 spacer sequence. In the second part of my thesis research, I investigated the roles of r-proteins L21 and L28 in processing of the ITS2 spacer sequence. Based on comparative analysis of structures of pre-ribosomes and ribosomes, I hypothesized that these r-proteins facilitate RNA folding in between domains II /V of 25S rRNA. Biochemical analysis of the composition of pre-ribosomes revealed specific changes in the association of assembly factors binding to the domain II/V interface of 25S rRNA, adjacent to the peptidyl transferase center, revealing that the organization of this interface is crucial to processing of the ITS2 spacer in 7S pre-rRNA. In addition to understanding specific mechanisms driving 60S ribosomal subunit assembly, my studies demonstrate coordination between the organization of the active centers in the 60S subunit (peptidyl transferase activity and tRNA binding sites in domain V and the polypeptide exit tunnel in domain III of 25S rRNA) and removal of the ITS2 spacer sequence. Thus, the ITS2 spacer might have evolved in eukaryotes as a mechanism to ensure accurate assembly of the translational apparatus.