A translation-like cycle is a quality control checkpoint for maturing 40S ribosome subunits

Abstract

Assembly factors (AFs) prevent premature translation initiation on small (40S) ribosomal subunit assembly intermediates by blocking ligand binding. However, it is unclear how AFs are displaced from maturing 40S ribosomes, if or how maturing subunits are assessed for fidelity, and what prevents premature translation initiation once AFs dissociate. Here we show that maturation involves a translation-like cycle whereby the translation factor eIF5B, a GTPase, promotes joining of large (60S) subunits with pre-40S subunits to give 80S-like complexes, which are subsequently disassembled by the termination factor Rli1, an ATPase. The AFs Tsr1 and Rio2 block the mRNA channel and initiator tRNA binding site, and therefore 80S-like ribosomes lack mRNA or initiator tRNA. After Tsr1 and Rio2 dissociate from 80S-like complexes Rli1-directed displacement of 60S subunits allows for translation initiation. This cycle thus provides a functional test of 60S subunit binding and the GTPase site before ribosomes enter the translating pool.

Figure 1. Depletion of Fap7 Leads to Accumulation of 40S Assembly Factors in 80S-Like Ribosomes

10–50% sucrose gradients from cell lysates from Rio2TAP (A,C,E) or Rio2TAP, Gal1::Fap7 (B,D,F) cells grown in glucose for 16h. (A,B) Absorbance profiles at 254 nm. (C,D) Western blots for assembly factors. (E,F) Northern blots for rRNAs and precursors. Exposure times were 4–6h, 2h and 1–2 h for 20S, 18S and 25S rRNAs, respectively. See also Figure S1.

Figure 2. 80S-like Ribosomes from Gal1:Fap7 Strains Are Not Translation Initiation Intermediates

(A) 80S ribosomes from Fap7-depleted cells do not contain initiator tRNA. Northern blots for 18S, 25S, and 20S pre-rRNAs as well as for initiator tRNA shows that levels of initiator tRNA do not reflect the levels of 20S rRNA, but are concordant with the levels of mature 18S rRNA. (B) tRNA levels in 80S peaks averaged from six experiments such as those in panel A. (C) 80S-like ribosomes contain Rps10, but Rps26, Rps14 and Rps1 are underrepresented. The LC peak area for each peptide identified by MS/MS in 80S-like ribosomes isolated from Fap7 D82AH84A cells is plotted relative to the LC peak area for the same peptide obtained from 80S ribosomes isolated from wild type cells. Data from four independent experiments were averaged and plotted with error bars representing the standard deviation.

Figure 3. eIF5B Is Required for the Accumulation of 40S Assembly Factors in 80S-like Ribosomes

10–50% sucrose gradients from cell lysates from ΔeIF5B,Gal1::Fap7 cells grown in glucose for 24h. Longer times for Fap7 depletion were required because due to the absence of eIF5B the strain grows slowly even in galactose. (A) Absorbance profile at 254 nm. (B) Western blots for assembly factors. (C) Northern blots for rRNAs and precursors. See Figure S2 for related data.

Figure 4. Rli1•Dom34 Are Required for Dissociation of 80S-like Ribosomes

(A) 10–50% sucrose gradients from cell lysates from Gal1::Rli1 cells grown in glucose for 12h. (B) 10–50% sucrose gradients from cell lysates from cells lacking Dom34. Shown are absorbance profile at 254 nm (top), Western blots for assembly factors (middle) and Northern blots for rRNAs and precursors (bottom). Probe b was used to probe for 20S, 23S and 35S rRNA. The 25S probe was used to detect 25S and 27S rRNAs. See Figures S3 and S4 for related data.

Figure 5. Rli1 Is Required for Release of pre-40S Subunits into the Translating Pool

(A) 10–50% sucrose gradients from cell lysates from Gal1::Nob1 cells grown in glucose for 12h. (B) 10–50% sucrose gradients from cell lysates from Gal1::Nob1,Gal1::Rli1 cells grown in glucose for 12h. Shown are absorbance profile at 254 nm (top), Western blots for assembly factors (middle) and Northern blots for rRNAs and precursors (bottom). Probe b was used to probe for 20S, 23S and 35S rRNA. The 25S probe was used to detect 25S and 27S rRNAs. See Figure S5 for biological replicates.

Figure 6. Model for Late Cytoplasmic Events in 40S Assembly

(A) Summary of the data presented in Figures 1–5 and Figures S1–S5. On the top are cartoon images to show whether pre-40S subunits are free, or bound to 60S subunits, or mRNA in each accumulated case. The complement of assembly factors used to “stage” the assembly intermediates are below. (B) Detailed model of cytoplasmic events during small ribosomal subunit maturation. Assembly factors that stably bind to pre-40S ribosomes are shown in yellow, energy-consuming assembly factors that bind transiently to pre-40S ribosomes are shown in magenta, pre-40S ribosomes and 60S ribosomes are shown in light and dark grey, respectively, ribosomal proteins are shown in white and mRNA is shown in blue. Intermediates defined herein are labeled I through VIII. It is likely that additional intermediates are present, yet these remain unknown.