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The telomerase inhibitor Gno1p/PINX1 activates the helicase Prp43p during ribosome biogenesis.

Chen YL, Capeyrou R, Humbert O, Mouffok S, Kadri YA, Lebaron S, Henras AK, Henry Y - Nucleic Acids Res. (2014)

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.

View Article: PubMed Central - PubMed

Affiliation: Equipe labellisée Ligue Contre le Cancer, LBME, CNRS and Toulouse University, Toulouse 31062, France.

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Consequences of alterations to the G-patch domain of Gno1p or PINX1 on ribosome biogenesis in yeast. (A) Northern analysis of rRNAs and pre-rRNAs extracted from Δgno1 cells expressing Gno1p or PINX1 variants featuring amino acid substitutions within their G-patch domain. Total RNAs were extracted from Δgno1 cells transformed with yeast vectors directing expression of wild-type Gno1p-HA (lane 1), Gno1pGm1-HA (lane 2), Gno1pGm2-HA (lane 3), PINX1-HA (lane 4), PINX1Gm1-HA (lane 5), PINX1Gm2-HA (lane 6) or the empty parental vector (lane 7) and analyzed by northern as described in the legend of Figure 4A. (B) Northern analysis of pre-rRNAs co-precipitated with HA-tagged Gno1p, PINX1 or G-patch variants thereof. Immunoprecipitation experiments were performed with an anti-HA matrix using extracts from Δgno1 cells expressing the indicated HA-tagged protein or no tagged protein as control. Input (lanes 1 to 7) and co-precipitated (lanes 8 to 14) pre-rRNAs were analyzed as described in the legend of Figure 5A.
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Figure 6: Consequences of alterations to the G-patch domain of Gno1p or PINX1 on ribosome biogenesis in yeast. (A) Northern analysis of rRNAs and pre-rRNAs extracted from Δgno1 cells expressing Gno1p or PINX1 variants featuring amino acid substitutions within their G-patch domain. Total RNAs were extracted from Δgno1 cells transformed with yeast vectors directing expression of wild-type Gno1p-HA (lane 1), Gno1pGm1-HA (lane 2), Gno1pGm2-HA (lane 3), PINX1-HA (lane 4), PINX1Gm1-HA (lane 5), PINX1Gm2-HA (lane 6) or the empty parental vector (lane 7) and analyzed by northern as described in the legend of Figure 4A. (B) Northern analysis of pre-rRNAs co-precipitated with HA-tagged Gno1p, PINX1 or G-patch variants thereof. Immunoprecipitation experiments were performed with an anti-HA matrix using extracts from Δgno1 cells expressing the indicated HA-tagged protein or no tagged protein as control. Input (lanes 1 to 7) and co-precipitated (lanes 8 to 14) pre-rRNAs were analyzed as described in the legend of Figure 5A.

Mentions: If Gno1p exerts its function during ribosome biogenesis mainly, or solely, via its interaction with Prp43p, we expect that disrupting this interaction would have the same effects on ribosome biogenesis as the total absence of the Gno1p protein. Although we still lack direct evidence for this assertion, we assume, based on the results obtained with PINX1, that Gno1p also directly interacts with Prp43p via its G-patch domain. We therefore assessed the effects on ribosome biogenesis of the amino acid substitutions within the G-patch domain of Gno1p or PINX1 which inhibit the Gno1p/Prp43p interactions in yeast and the PINX1/Prp43p interactions both in yeast and in vitro (see Figures 1B, C and 2B). To this end, we transformed the Δgno1 strain with plasmids directing expression of Gno1pGm1-HA, Gno1pGm2-HA, PINX1Gm1-HA or PINX1Gm2-HA. As controls, the Δgno1 strain was also transformed with the empty parental expression vector or vectors directing expression of wild-type HA-tagged Gno1p or PINX1. Western analysis demonstrated that Gno1pGm1-HA and Gno1pGm2-HA accumulate to the same levels as wild-type Gno1p-HA expressed from the plasmid, while the accumulation of PINX1Gm1-HA or PINX1Gm2-HA is somewhat diminished compared to that of PINX1-HA (Supplementary Figure S4A). Northern analysis of the pre-rRNAs and mature rRNAs extracted from these transformed strains demonstrated that expression of Gno1pGm1-HA, Gno1pGm2-HA, PINX1Gm1-HA or PINX1Gm2-HA completely fails to correct the mature rRNA and pre-rRNA accumulation defects (in particular that of 27SB pre-rRNAs) observed in the Δgno1 strain transformed with the empty vector (Figure 6A). Immunoprecipitation experiments of HA-tagged proteins followed by western blot assessment of the co-precipitation efficiency of Prp43p confirm that G-patch alterations strongly weaken the association between Prp43p and Gno1p or PINX1 (Supplementary Figure S4B). Note that for reasons that are not clear, the Gno1pGm2-HA protein is precipitated far better than Gno1p-HA (Supplementary Figure S4B, lanes 9 and 11). Northern analysis of the co-precipitated RNAs shows that the weakened association between Prp43p and the altered versions of Gno1p or PINX1 is coupled with a weakened interaction of the latter proteins with the 35S, 32/33S and 27SA2 pre-rRNAs (Figure 6B). Collectively, these data lead us to conclude that these G-patch alterations completely abrogate the functionality of Gno1p and PINX1 during ribosome biogenesis in yeast.


The telomerase inhibitor Gno1p/PINX1 activates the helicase Prp43p during ribosome biogenesis.

Chen YL, Capeyrou R, Humbert O, Mouffok S, Kadri YA, Lebaron S, Henras AK, Henry Y - Nucleic Acids Res. (2014)

Consequences of alterations to the G-patch domain of Gno1p or PINX1 on ribosome biogenesis in yeast. (A) Northern analysis of rRNAs and pre-rRNAs extracted from Δgno1 cells expressing Gno1p or PINX1 variants featuring amino acid substitutions within their G-patch domain. Total RNAs were extracted from Δgno1 cells transformed with yeast vectors directing expression of wild-type Gno1p-HA (lane 1), Gno1pGm1-HA (lane 2), Gno1pGm2-HA (lane 3), PINX1-HA (lane 4), PINX1Gm1-HA (lane 5), PINX1Gm2-HA (lane 6) or the empty parental vector (lane 7) and analyzed by northern as described in the legend of Figure 4A. (B) Northern analysis of pre-rRNAs co-precipitated with HA-tagged Gno1p, PINX1 or G-patch variants thereof. Immunoprecipitation experiments were performed with an anti-HA matrix using extracts from Δgno1 cells expressing the indicated HA-tagged protein or no tagged protein as control. Input (lanes 1 to 7) and co-precipitated (lanes 8 to 14) pre-rRNAs were analyzed as described in the legend of Figure 5A.
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Figure 6: Consequences of alterations to the G-patch domain of Gno1p or PINX1 on ribosome biogenesis in yeast. (A) Northern analysis of rRNAs and pre-rRNAs extracted from Δgno1 cells expressing Gno1p or PINX1 variants featuring amino acid substitutions within their G-patch domain. Total RNAs were extracted from Δgno1 cells transformed with yeast vectors directing expression of wild-type Gno1p-HA (lane 1), Gno1pGm1-HA (lane 2), Gno1pGm2-HA (lane 3), PINX1-HA (lane 4), PINX1Gm1-HA (lane 5), PINX1Gm2-HA (lane 6) or the empty parental vector (lane 7) and analyzed by northern as described in the legend of Figure 4A. (B) Northern analysis of pre-rRNAs co-precipitated with HA-tagged Gno1p, PINX1 or G-patch variants thereof. Immunoprecipitation experiments were performed with an anti-HA matrix using extracts from Δgno1 cells expressing the indicated HA-tagged protein or no tagged protein as control. Input (lanes 1 to 7) and co-precipitated (lanes 8 to 14) pre-rRNAs were analyzed as described in the legend of Figure 5A.
Mentions: If Gno1p exerts its function during ribosome biogenesis mainly, or solely, via its interaction with Prp43p, we expect that disrupting this interaction would have the same effects on ribosome biogenesis as the total absence of the Gno1p protein. Although we still lack direct evidence for this assertion, we assume, based on the results obtained with PINX1, that Gno1p also directly interacts with Prp43p via its G-patch domain. We therefore assessed the effects on ribosome biogenesis of the amino acid substitutions within the G-patch domain of Gno1p or PINX1 which inhibit the Gno1p/Prp43p interactions in yeast and the PINX1/Prp43p interactions both in yeast and in vitro (see Figures 1B, C and 2B). To this end, we transformed the Δgno1 strain with plasmids directing expression of Gno1pGm1-HA, Gno1pGm2-HA, PINX1Gm1-HA or PINX1Gm2-HA. As controls, the Δgno1 strain was also transformed with the empty parental expression vector or vectors directing expression of wild-type HA-tagged Gno1p or PINX1. Western analysis demonstrated that Gno1pGm1-HA and Gno1pGm2-HA accumulate to the same levels as wild-type Gno1p-HA expressed from the plasmid, while the accumulation of PINX1Gm1-HA or PINX1Gm2-HA is somewhat diminished compared to that of PINX1-HA (Supplementary Figure S4A). Northern analysis of the pre-rRNAs and mature rRNAs extracted from these transformed strains demonstrated that expression of Gno1pGm1-HA, Gno1pGm2-HA, PINX1Gm1-HA or PINX1Gm2-HA completely fails to correct the mature rRNA and pre-rRNA accumulation defects (in particular that of 27SB pre-rRNAs) observed in the Δgno1 strain transformed with the empty vector (Figure 6A). Immunoprecipitation experiments of HA-tagged proteins followed by western blot assessment of the co-precipitation efficiency of Prp43p confirm that G-patch alterations strongly weaken the association between Prp43p and Gno1p or PINX1 (Supplementary Figure S4B). Note that for reasons that are not clear, the Gno1pGm2-HA protein is precipitated far better than Gno1p-HA (Supplementary Figure S4B, lanes 9 and 11). Northern analysis of the co-precipitated RNAs shows that the weakened association between Prp43p and the altered versions of Gno1p or PINX1 is coupled with a weakened interaction of the latter proteins with the 35S, 32/33S and 27SA2 pre-rRNAs (Figure 6B). Collectively, these data lead us to conclude that these G-patch alterations completely abrogate the functionality of Gno1p and PINX1 during ribosome biogenesis in yeast.

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.

View Article: PubMed Central - PubMed

Affiliation: Equipe labellisée Ligue Contre le Cancer, LBME, CNRS and Toulouse University, Toulouse 31062, France.

Show MeSH
Related in: MedlinePlus