Insertion of the Biogenesis Factor Rei1 Probes the Ribosomal Tunnel during 60S Maturation

Abstract

Eukaryotic ribosome biogenesis depends on several hundred assembly factors to produce functional 40S and 60S ribosomal subunits. The final phase of 60S subunit biogenesis is cytoplasmic maturation, which includes the proofreading of functional centers of the 60S subunit and the release of several ribosome biogenesis factors. We report the cryo-electron microscopy (cryo-EM) structure of the yeast 60S subunit in complex with the biogenesis factors Rei1, Arx1, and Alb1 at 3.4 Å resolution. In addition to the network of interactions formed by Alb1, the structure reveals a mechanism for ensuring the integrity of the ribosomal polypeptide exit tunnel. Arx1 probes the entire set of inner-ring proteins surrounding the tunnel exit, and the C terminus of Rei1 is deeply inserted into the ribosomal tunnel, where it forms specific contacts along almost its entire length. We provide genetic and biochemical evidence that failure to insert the C terminus of Rei1 precludes subsequent steps of 60S maturation.

A Puzzle of Life: Crafting Ribosomal Subunits

“…Subsequently, Arx1 is dissociated by Jjj1 and Rei1 [103]. Interestingly, as shown by high-resolution cryo-EM, Rei1 and the closely related Reh1 probe the exit tunnel in a manner similar to Nog1 [25,28,30]. During P-stalk assembly, recruitment of uL10 is coupled with the release of Yvh1, which has previously displaced Mrt4 from pre-60S particles [103,104].…”

“…A few of these preribosomal particles were visualized at relatively low resolution by EM [21][22][23][24]. Recent progress in cryo-EM has enabled a quantum jump by permitting the visualization of preribosomal particles at atomic or near-atomic resolution [25][26][27][28][29][30][31][32][33]. These studies not only provided a detailed view of the overall architecture of Glossary Biogenesis factors: the efficient and accurate assembly of ribosomal subunits is promoted by more than 200 proteins that are transiently associated with preribosomal particles; these proteins are collectively referred to as biogenesis factors, or alternatively as transacting or assembly factors.…”

Mechanistic insight into eukaryotic 60S ribosomal subunit biogenesis by cryo-electron microscopy

“…A central cavity in the fold of Arx1, which corresponds to the catalytic pocket of homologous MAP enzymes, has been suggested to serve as a binding site for FG-repeat nucleoporins of the NPC, thereby mediating nuclear export ). However, the structures of the natively purified Arx1 particle Leidig et al 2014), as well as the higher-resolution structures of in vitro reconstituted complexes of the yeast 60S subunit and Arx1 (Greber et al 2012(Greber et al , 2016, showed that this pocket faces the ribosomal subunit surface near the polypeptide tunnel exit, and that access to it is restricted by several Arx1-specific extensions to the conserved MAPlike core fold that are involved in interactions with the 60S subunit ( Fig. 7A; Greber et al 2016).…”

“…However, the structures of the natively purified Arx1 particle Leidig et al 2014), as well as the higher-resolution structures of in vitro reconstituted complexes of the yeast 60S subunit and Arx1 (Greber et al 2012(Greber et al , 2016, showed that this pocket faces the ribosomal subunit surface near the polypeptide tunnel exit, and that access to it is restricted by several Arx1-specific extensions to the conserved MAPlike core fold that are involved in interactions with the 60S subunit ( Fig. 7A; Greber et al 2016). Even though these observations do not exclude that highly flexible FG-repeats of nucleoporins can access the Arx1 cavity, it is possible that structural elements of Arx1 other than the putative nucleoporin-binding pocket are involved in mediating nuclear export.…”

“…The structure of the 60S-Arx1-Alb1-Rei1 complex (Fig. 3C) allowed the building of atomic coordinate models for Arx1 and Rei1 and demonstrated the insertion of Rei1 into the ribosomal polypeptide exit tunnel (Greber et al 2016), while reconstructions of complexes of the anti-association factor eIF6 (Tif6 in yeast) and its release factors SBDS and EFL1 (Sdo1 and Efl1/Ria1 in yeast, respectively) on the 60S subunit ( Fig. 3D) provided important insights into the functional activation of the 60S particle during the final steps of cytoplasmic maturation (Weis et al 2015).…”