Bud23 promotes the final disassembly of the small subunit Processome in Saccharomyces cerevisiae

Abstract

The first metastable assembly intermediate of the eukaryotic ribosomal small subunit (SSU) is the SSU Processome, a large complex of RNA and protein factors that is thought to represent an early checkpoint in the assembly pathway. Transition of the SSU Processome towards continued maturation requires the removal of the U3 snoRNA and biogenesis factors as well as ribosomal RNA processing. While the factors that drive these events are largely known, how they do so is not. The methyltransferase Bud23 has a role during this transition, but its function, beyond the nonessential methylation of ribosomal RNA, is not characterized. Here, we have carried out a comprehensive genetic screen to understand Bud23 function. We identified 67 unique extragenic bud23Δ-suppressing mutations that mapped to genes encoding the SSU Processome factors DHR1, IMP4, UTP2 (NOP14), BMS1 and the SSU protein RPS28A. These factors form a physical interaction network that links the binding site of Bud23 to the U3 snoRNA and many of the amino acid substitutions weaken protein-protein and protein-RNA interactions. Importantly, this network links Bud23 to the essential GTPase Bms1, which acts late in the disassembly pathway, and the RNA helicase Dhr1, which catalyzes U3 snoRNA removal. Moreover, particles isolated from cells lacking Bud23 accumulated late SSU Processome factors and ribosomal RNA processing defects. We propose a model in which Bud23 dissociates factors surrounding its binding site to promote SSU Processome progression.

Release of the ribosome biogenesis factor Bud23 from small subunit precursors in yeast

“…Indeed, ectopic expression of RPS2 partially restored growth in the bud23∆ cells (Figure 2A). Our previous analyses of extragenic suppressors of bud23∆ showed that these suppressors partially alleviate the 40S biogenesis defect of bud23∆ cells (Sardana et al 2013;Black et al 2020). To assess whether the partial suppression of bud23∆ due to ectopic RPS2 also improved 40S production, we analyzed ribosome subunit levels by separating extracts on sucrose density gradients.…”

“…Bud23 is a conserved methyltransferase that modifies G1575 of 18S rRNA in yeast (G1539 in humans) during 40S biogenesis (White et al 2008;Figaro et al 2012;Létoquart et al 2014;Zorbas et al 2015). We previously proposed that Bud23 binds to a partially disassembled Processome where it coordinates the activities of the GTPase Bms1 and the RNA helicase Dhr1 to ensure the productive folding of the central pseudoknot (CPK) as the nascent particle transitions into the pre-40S (Black et al 2020;Black and Johnson 2021). Consistent with the role of Bud23 in CPK formation, we found here that the overexpression of Rps2, which binds to the CPK, partially bypassed the defects caused by bud23∆ (Figures 2A & 2B).…”

“…The cells were then poured over ice, collected by centrifugation, frozen in liquid nitrogen, and stored at -80°C. Sucrose density gradients were performed as previously described (Black et al 2020), except the Lysis Buffer consisted of 50mM Tris-HCl pH 7.6 (25°C), 100mM KCl, 5mM MgCl2, 150µg/ml CHX, 5mM βME, 1 mM PMSF and benzamidine, and 1µM leupeptin and pepstatin.…”

“…CPK formation is a crucial step in 40S biogenesis and proper folding of the CPK appears to be a highly coordinated event requiring the timely release of the U3 snoRNA from the partially disassembled Processome. U3 release is catalyzed by the helicase Dhr1 (Sardana et al 2015), and we recently proposed that the binding of Bud23 to the partially disassembled Processome, the Dis-C complex, coordinates the enzymatic activities of Dhr1 and the GTPase Bms1 to promote CPK folding (Figure 1) (Black et al 2020;Black and Johnson 2021). Rps2 (uS5) then binds to the CPK in the subsequent pre-40S intermediates (Ameismeier et al 2018).…”

“…Black et al 2020) AJY2665 MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 ENP1-TAP::HIS3MX6(Ghaemmaghami et al 2003) AJY2667 MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 RIO2-TAP::HIS3MX6(Ghaemmaghami et al 2003) AJY4281 MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 ENP1-6xGly-FTP::KIURA3This study.AJY4283 MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 ENP1-6xGly-FTP::KIURA3 KanMX6-MATa his3-1 leu2∆0 lys2∆0 met15∆0 ura3∆0 ENP1-6xGly-FTP::KIURA3rps21A∆::kanMX4 rps21B∆::kanMX4 + (PGAL1-RPS21A LEU2 CEN) This study. AJY4710 MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 RIO2-AID-HA_PGAL1-OsTIR1_GAL4BD-ER-VP16::CloNAT This study.…”