The telomerase inhibitor Gno1p/PINX1 activates the helicase Prp43p during ribosome biogenesis.
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.
- DEAD-box RNA Helicases/metabolism*
- RNA Helicases/metabolism*
- RNA-Binding Proteins/physiology*
- Saccharomyces cerevisiae Proteins/metabolism*
- Tumor Suppressor Proteins/chemistry/metabolism*
- Adenosine Triphosphatases/metabolism
- Enzyme Activation
- HeLa Cells
- Protein Structure, Tertiary
- Telomerase/antagonists & inhibitors
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Figure 1: Interactions between Prp43p or DHX15 and the G-patch proteins Gno1p or PINX1. (A) Gno1p-HA associates with Prp43p-TAP. Immunoprecipitations have been performed using IgG sepharose beads and cells expressing Gno1p-HA (lanes 1 and 2), Prp43p-TAP and Gno1p-HA (lanes 3 and 4) or Pfa1p-TAP and Gno1p-HA (lanes 5 and 6). Proteins have been extracted from the pellets (lanes IP) or from 1/320th of the input extracts (lanes Tot.), separated by SDS-PAGE and transferred to nitrocellulose. TAP-tagged proteins have been detected using rabbit PAP and HA-tagged Gno1p using anti-HA antibodies. (B) Human PINX1-HA interacts with Prp43p-TAP in yeast and the integrity of PINX1 G patch is important for this interaction. Immunoprecipitations have been performed as described in (A) using yeast cells expressing PINX1-HA (lanes 1 and 2), Prp43p-TAP and PINX1-HA (lanes 3 and 4), PINX1Gm1-HA (lanes 5 and 6), Prp43p-TAP and PINX1Gm1-HA (lanes 7 and 8), PINX1Gm2-HA (lanes 9 and 10) and Prp43p-TAP and PINX1Gm2-HA (lanes 11 and 12). PINX1Gm1-HA features the L34 to A and L45 to A substitutions, while PINX1Gm2-HA features the L34 to A, G38 to E, G42 to E, G44 to E, L45 to A and G46 to E substitutions. (C) Gno1p-HA interacts with Prp43p-TAP in yeast and the integrity of Gno1p G patch is important for this interaction. Immunoprecipitations have been performed as described in (A) using yeast cells expressing Gno1p-HA (lanes 1 and 2), Prp43p-TAP and Gno1p-HA (lanes 3 and 4), Gno1pGm1-HA (lanes 5 and 6), Prp43p-TAP and Gno1pGm1-HA (lanes 7 and 8), Gno1pGm2-HA (lanes 9 and 10) and Prp43p-TAP and Gno1pGm2-HA (lanes 11 and 12). Gno1pGm1-HA features the L33 to A, L44 to A and L67 to A substitutions, while Gno1pGm2-HA features the L33 to A, G37 to E, G41 to E, G43 to E, L44 to A, G45 to E and L67 to A substitutions. (D) PINX1 interacts with DHX15 in HeLa cells. Immunoprecipitations have been performed with HeLa cell extracts using an anti-DHX15 serum (lanes 3 and 4), an anti-PINX1 serum (lanes 5 and 6) or no serum (lanes 1 and 2). Proteins extracted from the pellets (lanes IP) or from 1/30th (top lane) or 1/250th (bottom lane) of the input extracts (lanes Tot.) have been processed as described in (A) and detected using the anti-DHX15 or anti-PINX1 sera.
We have previously shown that the yeast G-patch protein Gno1p is physically associated with Prp43p. Indeed Gno1p exhibits a positive double-hybrid interaction with yeast Prp43p and Gno1p is found associated with Prp43p after its TAP (20). We have now further confirmed these results by showing that HA-tagged Gno1p expressed in yeast is co-precipitated with Prp43p-TAP using IgG-sepharose beads (Figure 1A, lanes 3 and 4). In contrast, Gno1p-HA is not co-precipitated with Pfa1p-TAP, the other G-patch partner of Prp43p implicated in ribosome biogenesis (Figure 1A, lanes 5 and 6). This latter result suggests that the interactions between Prp43p and Gno1p on the one hand and between Prp43p and Pfa1p on the other hand are mutually exclusive. It has previously been shown that the putative human ortholog of yeast Gno1p, the telomerase inhibitor PINX1, can restore the growth and normal rRNA accumulation of cells lacking Gno1p (29). We have now assessed whether PINX1 expressed in yeast can also interact with yeast Prp43p. Indeed this is the case, since we find that HA-tagged PINX1 expressed in Δgno1 cells is efficiently co-precipitated with Prp43p-TAP from total yeast extracts (Figure 1B, lanes 3 and 4).