Construction of the Central Protuberance and L1 Stalk during 60S Subunit Biogenesis

Abstract

Ribosome assembly is driven by numerous assembly factors, including the Rix1 complex and the AAA ATPase Rea1. These two assembly factors catalyze 60S maturation at two distinct states, triggering poorly understood large-scale structural transitions that we analyzed by cryo-electron microscopy. Two nuclear pre-60S intermediates were discovered that represent previously unknown states after Rea1-mediated removal of the Ytm1-Erb1 complex and reveal how the L1 stalk develops from a pre-mature nucleolar to a mature-like nucleoplasmic state. A later pre-60S intermediate shows how the central protuberance arises, assisted by the nearby Rix1-Rea1 machinery, which was solved in its pre-ribosomal context to molecular resolution. This revealed a Rix1 2 -Ipi3 2 tetramer anchored to the pre-60S via Ipi1, strategically positioned to monitor this decisive remodeling. These results are consistent with a general underlying principle that temporarily stabilized immature RNA domains are successively remodeled by assembly factors, thereby ensuring failsafe assembly progression.

A comprehensive landscape of 60S ribosome biogenesis factors

“…This timeline is broadly consistent with previous structural, biochemical and genetic studies (Klinge and Woolford, 2019;Kressler et al, 2010Woolford and Baserga, 2013). Nine of our baits (Ssf1, Nop16, Ytm1, Nsa1, Cic1, Rix1, Nog2, Arx1 and Lsg1) have been identified and modelled in cryo-electron microscopy (Cryo-EM) reconstructions of 60S intermediates (Kargas et al, 2019;Kater et al, 2020;Kater et al, 2017;Sanghai et al, 2018;Wu et al, 2016;Zhou et al, 2019a;Zhou et al, 2019b) and their distribution among known 60S intermediate structures is consistent with the timeline derived from our MS data (Figure 2A). Our other bait RBFs (Noc1, Rsa3 and Nop12) have not been identified in any 60S structural reconstructions to date.…”

“…Finally, the distribution of the C2 processing factors Las1/Grc3 correlates strongly with the binding of Rix1/Rea1, allowing us to narrowly define the timing of Las1 recruitment to the C2 site (Figures 2A and S5). This correlation between Las1 and the Rix1/Rea1 complex is consistent with the fact that Rix1-containing particles have distinct pools of particles with or without the ITS2 foot structure (Kater et al, 2020). Finally, we observe a close correlation between Ecm1 and the major export receptors Mex67/Mtr2, suggesting a functional link between these proteins in 60S nuclear export (Figures 2A and S5).…”

“…The release of pre-60S particles from the nucleolus into the cytoplasm coincides with the organization of the L1 stalk and the incorporation of the GTPase Nog2. Our representative nucleoplasmic pre-60S particles represent states before and after rotation of the 5S rRNA, which is marked by the release of RBFs Rpf2/Rrs1 and the recruitment of the Rix1 complex (Kater et al, 2020;Wu et al, 2016). After nuclear export, final maturation occurs in the cytoplasm, as typified by the structures of early and late cytoplasmic 60S intermediates (Kargas et al, 2019;Zhou et al, 2019b).…”

Structural insights into assembly of the ribosomal nascent polypeptide exit tunnel

“…3d ). Recent cryo-EM structures further suggest the importance of this interaction 25 . H74 is displaced ~4 Å relative to the wild-type conformation, indicating that this portion the NPET is improperly assembled (Fig.…”

AAA ATPases as therapeutic targets: Structure, functions, and small-molecule inhibitors

“…Knockdown of Mdn1 by small interfering RNA (siRNA) resulted in a severe growth defect in HeLa cells. Mdn1 is assembled by six non-equivalent AAA domains linked in a single polypeptide, which are similar to those of the microtubule-based motor protein dynein [147][148][149] (Figure 13). The ATPase activity of AAA2, AAA3, AAA4, and AAA5 and ATP-binding ability of AAA6 are likely essential for Mdn1's cellular functions [150].…”