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Molecular chaperone Hsp90 stabilizes Pih1/Nop17 to maintain R2TP complex activity that regulates snoRNA accumulation.

Zhao R, Kakihara Y, Gribun A, Huen J, Yang G, Khanna M, Costanzo M, Brost RL, Boone C, Hughes TR, Yip CM, Houry WA - J. Cell Biol. (2008)

Bottom Line: Tah1 is a small protein containing tetratricopeptide repeats, whereas Pih1 is found to be an unstable protein.As a consequence, the chaperone is shown to affect box C/D accumulation and maintenance, especially under stress conditions.Hsp90 and R2TP proteins are also involved in the proper accumulation of box H/ACA small nucleolar RNAs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada.

ABSTRACT
Hsp90 is a highly conserved molecular chaperone that is involved in modulating a multitude of cellular processes. In this study, we identify a function for the chaperone in RNA processing and maintenance. This functionality of Hsp90 involves two recently identified interactors of the chaperone: Tah1 and Pih1/Nop17. Tah1 is a small protein containing tetratricopeptide repeats, whereas Pih1 is found to be an unstable protein. Tah1 and Pih1 bind to the essential helicases Rvb1 and Rvb2 to form the R2TP complex, which we demonstrate is required for the correct accumulation of box C/D small nucleolar ribonucleoproteins. Together with the Tah1 cofactor, Hsp90 functions to stabilize Pih1. As a consequence, the chaperone is shown to affect box C/D accumulation and maintenance, especially under stress conditions. Hsp90 and R2TP proteins are also involved in the proper accumulation of box H/ACA small nucleolar RNAs.

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Related in: MedlinePlus

Northern blot analysis of pre-rRNA and snoRNA in different strains. All Northern blot analyses shown in this figure have been repeated at least three times. (A) Accumulation of 35S pre-rRNA in WT and hsp82ts hsc82Δ cells grown to log or stationary phase at 30°C upon temperature upshift to 37°C for the indicated time periods. The levels of the U2 snRNA (a component of the spliceosome) are shown as a loading control. (B) Comparison of 35S pre-rRNA and snoRNAs levels in hsp82ts hsc82Δ cells to those in WT cells. Cells were grown at 30°C to log or stationary phase. Numbers next to each panel give the relative intensity of the bands in the mutant strain compared with WT levels after normalization to loading controls of scR1 RNA (involved in signal recognition particle–dependent cotranslational protein targeting to the membrane). Bottom panels show Western blot analysis using αHsp90 antibodies of total yeast lysates obtained from cells with equal OD600. (C) 35S pre-rRNA and snoRNA accumulation in different strains grown at 30°C during log and stationary phases was assessed by Northern blot analysis. U2 snRNA and scR1 were used as internal controls for RNA loading of rRNA and snoRNA, respectively. (D) Quantification of the bands from Northern analysis experiments similar to those shown in C normalized to loading controls. WT snoRNA levels are arbitrarily set to 1. The errors in these measurements are based on three separate experiments.
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fig6: Northern blot analysis of pre-rRNA and snoRNA in different strains. All Northern blot analyses shown in this figure have been repeated at least three times. (A) Accumulation of 35S pre-rRNA in WT and hsp82ts hsc82Δ cells grown to log or stationary phase at 30°C upon temperature upshift to 37°C for the indicated time periods. The levels of the U2 snRNA (a component of the spliceosome) are shown as a loading control. (B) Comparison of 35S pre-rRNA and snoRNAs levels in hsp82ts hsc82Δ cells to those in WT cells. Cells were grown at 30°C to log or stationary phase. Numbers next to each panel give the relative intensity of the bands in the mutant strain compared with WT levels after normalization to loading controls of scR1 RNA (involved in signal recognition particle–dependent cotranslational protein targeting to the membrane). Bottom panels show Western blot analysis using αHsp90 antibodies of total yeast lysates obtained from cells with equal OD600. (C) 35S pre-rRNA and snoRNA accumulation in different strains grown at 30°C during log and stationary phases was assessed by Northern blot analysis. U2 snRNA and scR1 were used as internal controls for RNA loading of rRNA and snoRNA, respectively. (D) Quantification of the bands from Northern analysis experiments similar to those shown in C normalized to loading controls. WT snoRNA levels are arbitrarily set to 1. The errors in these measurements are based on three separate experiments.

Mentions: The aforementioned data establish a link between the R2TP complex and pre-rRNA processing. Because Hsp90 and Tah1 modulate the stability of Pih1, we expected that Hsp90 will also have an effect on pre-rRNA processing. To this end, heat shock experiments were performed on cells knocked out of HSC82 and expressing a temperature-sensitive mutant allele of HSP82, R0013 (hsp82ts hsc82Δ; Zhao et al., 2005). As can be seen in Fig. 6 A, when cells grown to log or stationary phase at 30°C are shifted to the nonpermissive temperature of 37°C for the indicated time periods, there is an increased accumulation of 35S pre-rRNA in hsp82ts hsc82Δ versus WT cells. The levels of the U2 snRNA (a component of the spliceosome) are shown as a loading control. This seemed to indicate that Hsp90 acts on upstream components that result in 35S accumulation.


Molecular chaperone Hsp90 stabilizes Pih1/Nop17 to maintain R2TP complex activity that regulates snoRNA accumulation.

Zhao R, Kakihara Y, Gribun A, Huen J, Yang G, Khanna M, Costanzo M, Brost RL, Boone C, Hughes TR, Yip CM, Houry WA - J. Cell Biol. (2008)

Northern blot analysis of pre-rRNA and snoRNA in different strains. All Northern blot analyses shown in this figure have been repeated at least three times. (A) Accumulation of 35S pre-rRNA in WT and hsp82ts hsc82Δ cells grown to log or stationary phase at 30°C upon temperature upshift to 37°C for the indicated time periods. The levels of the U2 snRNA (a component of the spliceosome) are shown as a loading control. (B) Comparison of 35S pre-rRNA and snoRNAs levels in hsp82ts hsc82Δ cells to those in WT cells. Cells were grown at 30°C to log or stationary phase. Numbers next to each panel give the relative intensity of the bands in the mutant strain compared with WT levels after normalization to loading controls of scR1 RNA (involved in signal recognition particle–dependent cotranslational protein targeting to the membrane). Bottom panels show Western blot analysis using αHsp90 antibodies of total yeast lysates obtained from cells with equal OD600. (C) 35S pre-rRNA and snoRNA accumulation in different strains grown at 30°C during log and stationary phases was assessed by Northern blot analysis. U2 snRNA and scR1 were used as internal controls for RNA loading of rRNA and snoRNA, respectively. (D) Quantification of the bands from Northern analysis experiments similar to those shown in C normalized to loading controls. WT snoRNA levels are arbitrarily set to 1. The errors in these measurements are based on three separate experiments.
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Related In: Results  -  Collection

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fig6: Northern blot analysis of pre-rRNA and snoRNA in different strains. All Northern blot analyses shown in this figure have been repeated at least three times. (A) Accumulation of 35S pre-rRNA in WT and hsp82ts hsc82Δ cells grown to log or stationary phase at 30°C upon temperature upshift to 37°C for the indicated time periods. The levels of the U2 snRNA (a component of the spliceosome) are shown as a loading control. (B) Comparison of 35S pre-rRNA and snoRNAs levels in hsp82ts hsc82Δ cells to those in WT cells. Cells were grown at 30°C to log or stationary phase. Numbers next to each panel give the relative intensity of the bands in the mutant strain compared with WT levels after normalization to loading controls of scR1 RNA (involved in signal recognition particle–dependent cotranslational protein targeting to the membrane). Bottom panels show Western blot analysis using αHsp90 antibodies of total yeast lysates obtained from cells with equal OD600. (C) 35S pre-rRNA and snoRNA accumulation in different strains grown at 30°C during log and stationary phases was assessed by Northern blot analysis. U2 snRNA and scR1 were used as internal controls for RNA loading of rRNA and snoRNA, respectively. (D) Quantification of the bands from Northern analysis experiments similar to those shown in C normalized to loading controls. WT snoRNA levels are arbitrarily set to 1. The errors in these measurements are based on three separate experiments.
Mentions: The aforementioned data establish a link between the R2TP complex and pre-rRNA processing. Because Hsp90 and Tah1 modulate the stability of Pih1, we expected that Hsp90 will also have an effect on pre-rRNA processing. To this end, heat shock experiments were performed on cells knocked out of HSC82 and expressing a temperature-sensitive mutant allele of HSP82, R0013 (hsp82ts hsc82Δ; Zhao et al., 2005). As can be seen in Fig. 6 A, when cells grown to log or stationary phase at 30°C are shifted to the nonpermissive temperature of 37°C for the indicated time periods, there is an increased accumulation of 35S pre-rRNA in hsp82ts hsc82Δ versus WT cells. The levels of the U2 snRNA (a component of the spliceosome) are shown as a loading control. This seemed to indicate that Hsp90 acts on upstream components that result in 35S accumulation.

Bottom Line: Tah1 is a small protein containing tetratricopeptide repeats, whereas Pih1 is found to be an unstable protein.As a consequence, the chaperone is shown to affect box C/D accumulation and maintenance, especially under stress conditions.Hsp90 and R2TP proteins are also involved in the proper accumulation of box H/ACA small nucleolar RNAs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada.

ABSTRACT
Hsp90 is a highly conserved molecular chaperone that is involved in modulating a multitude of cellular processes. In this study, we identify a function for the chaperone in RNA processing and maintenance. This functionality of Hsp90 involves two recently identified interactors of the chaperone: Tah1 and Pih1/Nop17. Tah1 is a small protein containing tetratricopeptide repeats, whereas Pih1 is found to be an unstable protein. Tah1 and Pih1 bind to the essential helicases Rvb1 and Rvb2 to form the R2TP complex, which we demonstrate is required for the correct accumulation of box C/D small nucleolar ribonucleoproteins. Together with the Tah1 cofactor, Hsp90 functions to stabilize Pih1. As a consequence, the chaperone is shown to affect box C/D accumulation and maintenance, especially under stress conditions. Hsp90 and R2TP proteins are also involved in the proper accumulation of box H/ACA small nucleolar RNAs.

Show MeSH
Related in: MedlinePlus