The assembly factor Erb1 functions in multiple remodeling events during 60S ribosomal subunit assembly inS. cerevisiae

Abstract

A major gap in our understanding of ribosome assembly is knowing the precise function of each of the ∼200 assembly factors. The steps in subunit assembly in which these factors participate have been examined for the most part by depleting each protein from cells. Depletion of the assembly factor Erb1 prevents stable assembly of seven other interdependent assembly factors with pre-60S subunits, resulting in turnover of early preribosomes, before the ITS1 spacer can be removed from 27SA3 pre-rRNA. To investigate more specific functions of Erb1, we constructed eight internal deletions of 40–60 amino acid residues each, spanning the amino-terminal half of Erb1. The erb1Δ161–200 and erb1Δ201–245 deletion mutations block a later step than depletion of Erb1, namely cleavage of the C2 site that initiates removal of the ITS2 spacer. Two other remodeling events fail to occur in these erb1 mutants: association of twelve different assembly factors with domain V of 25S rRNA, including the neighborhood surrounding the peptidyl transferase center, and stable association of ribosomal proteins with rRNA surrounding the polypeptide exit tunnel. This suggests that successful initiation of construction of these functional centers is a checkpoint for committing to spacer removal.

Modular assembly of the nucleolar large subunit processome

“…3B, C). This location is in agreement with previous cross-linking data and explains why deletions within this region prevent incorporation into pre-60S particles (10,11). Nop16 interconnects RNA elements of the 5.8S rRNA and regions of domain I, and additionally contains a bipartite binding site by interacting with ribosomal proteins Rpl8 and Rpl13 (Fig.…”

Modular assembly of the nucleolar pre-60S ribosomal subunit

“…3b, c). This location is consistent with previous cross-linking data and explains why deletions in this region prevent the incorporation of Erb1 into pre-60S particles 10,11 . Nop16 interconnects RNA elements of the 5.8S rRNA and regions of domain I. Additionally, Nop16 interacts with both Rpl8 and Rpl13 (Fig.…”

Visualizing the Assembly Pathway of Nucleolar Pre-60S Ribosomes

“…A central assembly factor already present on the earliest pre-60S particles is Erb1, for which a large number of biochemical, structural, and genetic data have been obtained in previous studies ( Konikkat et al., 2017 , Miles et al., 2005 , Thoms et al., 2016 , Wegrecki et al., 2015 ). It is composed of an N-terminal region containing the nuclear localization signal, a long N-terminal stretch, and a C-terminal β-propeller domain ( Konikkat et al., 2017 , Pestov et al., 2001 , Thoms et al., 2016 , Wegrecki et al., 2015 ). Whereas part of its N terminus is already visible in states A–C, most of Erb1 (amino acids 125 to 807 of 807) can only be seen in the states D and E particles ( Figure 3 A).…”