The telomerase inhibitor Gno1p/PINX1 activates the helicase Prp43p during ribosome biogenesis.
Bottom Line: In yeast, lack of Gno1p leads to a decrease in the levels of pre-40S and intermediate pre-60S pre-ribosomal particles, defects that can be corrected by PINX1 expression.G-patch alterations in Gno1p or PINX1 that inhibit their interactions with Prp43p completely abolish their function in yeast ribosome biogenesis.Altogether, our results suggest that activation of Prp43p by Gno1p/PINX1 within early pre-ribosomal particles is crucial for their subsequent maturation.
Affiliation: Equipe labellisée Ligue Contre le Cancer, LBME, CNRS and Toulouse University, Toulouse 31062, France.Show MeSH
Mentions: To help narrow down at which stage Gno1p acts during ribosome biogenesis in yeast, we precipitated HA-tagged Gno1p from total yeast extracts using anti-HA antibodies and identified the co-precipitated pre-rRNAs by northern analysis (Figure 5A). The most efficiently co-precipitated pre-rRNAs were the 33/32S and 27SA2 pre-rRNAs. 35S pre-rRNA was also significantly co-precipitated, while the 27SB and even more so the 20S pre-rRNAs were precipitated to a far lesser extent. We also assessed whether HA-tagged Gno1p can be co-precipitated with marker proteins of specific pre-ribosomal particles. Immunoprecipitation experiments were performed using IgG-sepharose and extracts from strains expressing HA-tagged Gno1p and a TAP-tagged component of a given subset of pre-ribosomal particles. Western analysis of the precipitated material using anti-HA antibodies shows that Gno1p-HA is co-precipitated together with components of the UTPA, UTPB and UTPC subcomplexes that sequentially assemble on the nascent pre-rRNA to nucleate assembly of the 90S particles (Figure 5B, lanes 3 to 16). Using the same approach, we could show that Gno1p-HA associates with Npa1p-ZZ and Ssf1p-TAP (Figure 5B, lanes 19 and 22), components of very early pre-60S particles containing 27SA2 pre-rRNA. In contrast, Gno1p-HA is not co-precipitated with Nog2p-TAP (Figure 5B, lanes 23 and 24), a protein that assembles within intermediate pre-60S particles containing 27SB pre-rRNA, nor is it associated with Rix1p-TAP (Figure 5B, lanes 25 and 26) or Arx1p-TAP (Figure 5B, lanes 27 and 28), components of late nucleoplasmic or cytoplasmic pre-60S particles. Gno1p-HA also failed to be co-precipitated with Rio2p-TAP, a component of late pre-40S particles (Figure 5B, lanes 17 and 18). Altogether, our data suggest that Gno1p is recruited within 90S pre-ribosomal particles and remains associated with early 27SA2 pre-rRNA-containing pre-60S pre-ribosomal particles. Our data further suggest that Gno1p displays only a transient association with pre-40S particles and that it dissociates from 27SB-containing intermediate pre-60S particles before the recruitment of Nog2p.
Affiliation: Equipe labellisée Ligue Contre le Cancer, LBME, CNRS and Toulouse University, Toulouse 31062, France.