Architecture of the Rix1–Rea1 checkpoint machinery during pre-60S-ribosome remodeling

Abstract

Ribosome synthesis is catalyzed by ∼200 assembly factors, which facilitate efficient production of mature ribosomes. Here, we determined the cryo-EM structure of a Saccharomyces cerevisiae nucleoplasmic pre-60S particle containing the dynein-related 550-kDa Rea1 AAA(+) ATPase and the Rix1 subcomplex. This particle differs from its preceding state, the early Arx1 particle, by two massive structural rearrangements: an ∼180° rotation of the 5S ribonucleoprotein complex and the central protuberance (CP) rRNA helices, and the removal of the 'foot' structure from the 3' end of the 5.8S rRNA. Progression from the Arx1 to the Rix1 particle was blocked by mutational perturbation of the Rix1-Rea1 interaction but not by a dominant-lethal Rea1 AAA(+) ATPase-ring mutant. After remodeling, the Rix1 subcomplex and Rea1 become suitably positioned to sense correct structural maturation of the CP, which allows unidirectional progression toward mature ribosomes.

A Puzzle of Life: Crafting Ribosomal Subunits

“…This phase is also marked by the association of the Rix1 subcomplex and the AAA-ATPase Rea1 [21,33,99] (Figures 2N and 5). Interestingly, the interaction between Rix1 and Rea1 may be regulated, as indicated by a recent study with the orthologous human proteins showing that their association is controlled by a dynamic SUMO conjugation/deconjugation cycle [100].…”

“…Interestingly, the interaction between Rix1 and Rea1 may be regulated, as indicated by a recent study with the orthologous human proteins showing that their association is controlled by a dynamic SUMO conjugation/deconjugation cycle [100]. The cryo-EM structure of the Rea1-containing Rix1 pre-60S particle, compared with the upstream Nog2-defined pre-60S particle [25,97], revealed several major differences [33], among them the absence of the foot structure. Most notably, recruitment of the gigantic Rix1-Rea1 machinery coincides with the release of Rpf2-Rrs1 and rotation of the 5S RNP into its mature position [33] (Figure 5).…”

“…The cryo-EM structure of the Rea1-containing Rix1 pre-60S particle, compared with the upstream Nog2-defined pre-60S particle [25,97], revealed several major differences [33], among them the absence of the foot structure. Most notably, recruitment of the gigantic Rix1-Rea1 machinery coincides with the release of Rpf2-Rrs1 and rotation of the 5S RNP into its mature position [33] (Figure 5). Once properly positioned the ATPase activity of Rea1 powers the removal of Rsa4 [21], an event that occurs The nucle(ol)ar Nog2-purified pre-60S particle contains the characteristic 'foot' structure [internal transcribed spacer 2 (ITS2) in orange] and the 5S ribonucleoprotein particle (RNP) is in its pre-mature conformation (left).…”

“…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.…”

The N-terminal extension of yeast ribosomal protein L8 is involved in two major remodeling events during late nuclear stages of 60S ribosomal subunit assembly

“…This stage includes two major remodeling events: removal of the 235 nt-long ITS2, and reorganization of the central protuberance containing 5S rRNA, and r-proteins L5 and L11 Greber et al 2012;Barrio-Garcia et al 2016). Cleavage of the C 2 site in ITS2 occurs after assembly factors Nog2, Nop53, and Rsa4 bind to pre-ribosomes, and is mediated FIGURE 6.…”