Poster abstracts
Poster number 14 submitted by Stephanie Biedka
Three major remodeling events within assembling ribosomes are required for irreversible removal of a pre-rRNA spacer sequence
Stephanie Biedka (Department of Biological Sciences, Carnegie Mellon University), Jelena Jakovljevic (Department of Biological Sciences, Carnegie Mellon University), Shan Wu (School of Life Sciences, Tsinghua University), Ning Gao (School of Life Sciences, Tsinghua University), John Woolford (Department of Biological Sciences, Carnegie Mellon University)
Abstract:
Three interdependent processes drive ribosome biosynthesis in vivo: pre-rRNA folding and processing and binding of ribosomal proteins to the pre-rRNA. In yeast, ribosome assembly is facilitated by the actions of ~200 protein assembly factors. Assembly is hierarchical and progresses through establishment and restructuring of RNP interaction networks. A major goal at this time is to establish what these networks are, how they are remodeled, and how their reconstruction drives assembly forward in an efficient and accurate manner. We have focused on the events required for one particular remodeling event- irreversible removal of the ITS2 spacer RNA from the pre-60S subunit. Our approach has been to isolate mutants that cannot carry out this pre-rRNA processing event and assay effects of these mutations on pre-ribosome protein composition. Critical to our analysis was our recent determination of near atomic resolution cryo-EM structures of three consecutive late nuclear pre-60S subunit assembly intermediates. From these structures we were able to identify RNP networks affected in these mutants. Based on their mutant pre-rRNA processing phenotype, ~30 ribosomal proteins and assembly factors are known to be required for cleavage at the C2 site to initiate removal of ITS2. Analysis of these mutants revealed three major remodeling events that are necessary for C2 cleavage. (1) A group of assembly factors bound proximal to ITS2 must be released. (2) Several assembly factors that bind to the nascent peptidyl transferase center must associate with the pre-60S subunit. (3) Seven ribosomal proteins surrounding the exit of the polypeptide exit tunnel must become stably associated with the pre-60S subunit. We believe that these three events reveal the existence of a complicated checkpoint that prevents pre-60S subunits with improperly structured functional centers from undergoing the irreversible step of ITS2 removal.
Keywords: ribosome assembly, pre-ribosome remodeling, pre-rRNA processing