Fig. 3

Removal of ITS2-associated factors from the pre-60S particles upon in vitro 7S→5.8S pre-rRNA processing. a Glycerol gradient centrifugation of pre-60S particles (affinity-purified via TAPF-Nop53) after the in vitro processing reaction using endogenous yeast exosome (affinity-purified via Rrp6-FTpA) and recombinant wt Mtr4 or mutant Mtr4 (K176A) in the presence of ATP. As a control, the pre-60S particle was also incubated only with ATP (mock). After centrifugation, the linear glycerol gradient was fractionated into fractions 1–9, which were TCA-precipitated and analysed by SDS-PAGE and Coomassie staining (upper panel) or western blotting using the indicated antibodies (lower panel), with Rpl3 serving to identify the particle fractions. The bottom fractions (7–9) contained the pre-60S particles, whereas the top fractions (1–2) contained the released factors such as Cic1 and Nop7. Ribosome assembly factor Nog1 not released during the in vitro reaction is indicated by a dot (fraction 8). b Semi-quantitative mass spectrometry of pre-60S particle glycerol gradient fraction 8 after 7S→5.8S pre-rRNA processing by recombinant nuclear exosome (see Supplementary Fig. 3b). Label-free quantification (LFQ) values between the different experiments (7S→5.8S pre-rRNA processing, 7S→5.8S + 30 pre-rRNA processing, no 7S pre-rRNA processing) were normalised to Nog1. Afterwards, pairs of LFQ values of ribosome biogenesis factors obtained from different processing stages were compared by plotting them against each other. Left: '7S→5.8S pre-rRNA processing' (pre-60S particle + Exo(14)^wt + ATP) vs. 'no 7S pre-rRNA processing' (pre-60S particle + Exo(14)Rrp44^exo/endo + ATP). Right: '7S→5.8S + 30 pre-rRNA processing' (pre-60S particle + Exo(14)Rrp6^exo + ATP) vs. 'no 7S pre-rRNA processing' (pre-60S particle + Exo(14)Rrp44^exo/endo + ATP). Labelled in colour are ribosome assembly factors associated with ITS2, which significantly dropped below the dashed reference line (x = y), indicating release from the 60S particles. In contrast, most of the other pre-60S factors remained close the reference line, indicating no significant release from the pre-60S particles. All experiments were performed twice with similar outcomes (see Supplementary Table 3)