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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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Hsp90 and Tah1 stabilize Pih1 in vivo. (A) The R2TP complex was isolated from log- and stationary-phase WT and tah1Δ cells expressing a chromosomal copy of PIH1–3FLAG using αFLAG resin. The interaction of Hsp90 with the complex was assessed by Western blot analysis. (B) The change in the levels of Pih1, Rvb2, Rvb1, Hsp90, and Tah1 in different strain backgrounds was monitored by Western blot analysis at three growth stages as indicated by OD600. Cells had a chromosomal copy of PIH1–3FLAG in a WT background (sample 1; PIH1–3FLAG), in a background deleted of TAH1 containing an empty vector (sample 2; PIH1–3FLAG, tah1Δ, and p414), or in a background deleted of TAH1 but expressing Tah1 from a p414 plasmid (sample 3; PIH1-3FLAG, tah1Δ, and p414Tah1). 6.5 μg of total proteins was typically loaded and blotted with specific antibodies. (C) WT or deg-TAH1 yeast cells were exposed to heat shock for 2 h in the presence of 40 μg/ml cycloheximide. For each sample, proteins from cells with equivalent OD600 were loaded onto SDS-PAGE gels and blotted with specific antibodies.
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fig3: Hsp90 and Tah1 stabilize Pih1 in vivo. (A) The R2TP complex was isolated from log- and stationary-phase WT and tah1Δ cells expressing a chromosomal copy of PIH1–3FLAG using αFLAG resin. The interaction of Hsp90 with the complex was assessed by Western blot analysis. (B) The change in the levels of Pih1, Rvb2, Rvb1, Hsp90, and Tah1 in different strain backgrounds was monitored by Western blot analysis at three growth stages as indicated by OD600. Cells had a chromosomal copy of PIH1–3FLAG in a WT background (sample 1; PIH1–3FLAG), in a background deleted of TAH1 containing an empty vector (sample 2; PIH1–3FLAG, tah1Δ, and p414), or in a background deleted of TAH1 but expressing Tah1 from a p414 plasmid (sample 3; PIH1-3FLAG, tah1Δ, and p414Tah1). 6.5 μg of total proteins was typically loaded and blotted with specific antibodies. (C) WT or deg-TAH1 yeast cells were exposed to heat shock for 2 h in the presence of 40 μg/ml cycloheximide. For each sample, proteins from cells with equivalent OD600 were loaded onto SDS-PAGE gels and blotted with specific antibodies.

Mentions: The R2TP complex was isolated from yeast cells in which the constitutive PIH1 gene was C-terminally 3FLAG tagged by immunoprecipitation with anti-FLAG antibody as described for Fig. 2 C. The complex was then immunoblotted with anti-Hsp90 antibody. As shown in Fig. 3 A, Hsp90 was not significantly associated with the R2TP complex purified from log-phase cells; however, more of the chaperone was associated with the complex isolated from stationary-phase cells, which indicates that Hsp90, Tah1, and Pih1 are in the same complex under these conditions. Surprisingly, higher levels of Hsp90 were found to be associated with the R2P complex purified from tah1Δ cells at both log- and stationary-phase cells. Considering that Tah1 might act as a cochaperone of Hsp90 (Zhao et al., 2005), it is likely that Hsp90 compensates for the depletion of Tah1 by increasing its association with R2P to preserve the function of the complex. Furthermore, the increased association of the chaperone with the R2P and R2TP complexes in stationary-phase cells compared with log-phase cells seems to indicate that one or more proteins in the complexes require added chaperoning under stress conditions.


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)

Hsp90 and Tah1 stabilize Pih1 in vivo. (A) The R2TP complex was isolated from log- and stationary-phase WT and tah1Δ cells expressing a chromosomal copy of PIH1–3FLAG using αFLAG resin. The interaction of Hsp90 with the complex was assessed by Western blot analysis. (B) The change in the levels of Pih1, Rvb2, Rvb1, Hsp90, and Tah1 in different strain backgrounds was monitored by Western blot analysis at three growth stages as indicated by OD600. Cells had a chromosomal copy of PIH1–3FLAG in a WT background (sample 1; PIH1–3FLAG), in a background deleted of TAH1 containing an empty vector (sample 2; PIH1–3FLAG, tah1Δ, and p414), or in a background deleted of TAH1 but expressing Tah1 from a p414 plasmid (sample 3; PIH1-3FLAG, tah1Δ, and p414Tah1). 6.5 μg of total proteins was typically loaded and blotted with specific antibodies. (C) WT or deg-TAH1 yeast cells were exposed to heat shock for 2 h in the presence of 40 μg/ml cycloheximide. For each sample, proteins from cells with equivalent OD600 were loaded onto SDS-PAGE gels and blotted with specific antibodies.
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Related In: Results  -  Collection

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fig3: Hsp90 and Tah1 stabilize Pih1 in vivo. (A) The R2TP complex was isolated from log- and stationary-phase WT and tah1Δ cells expressing a chromosomal copy of PIH1–3FLAG using αFLAG resin. The interaction of Hsp90 with the complex was assessed by Western blot analysis. (B) The change in the levels of Pih1, Rvb2, Rvb1, Hsp90, and Tah1 in different strain backgrounds was monitored by Western blot analysis at three growth stages as indicated by OD600. Cells had a chromosomal copy of PIH1–3FLAG in a WT background (sample 1; PIH1–3FLAG), in a background deleted of TAH1 containing an empty vector (sample 2; PIH1–3FLAG, tah1Δ, and p414), or in a background deleted of TAH1 but expressing Tah1 from a p414 plasmid (sample 3; PIH1-3FLAG, tah1Δ, and p414Tah1). 6.5 μg of total proteins was typically loaded and blotted with specific antibodies. (C) WT or deg-TAH1 yeast cells were exposed to heat shock for 2 h in the presence of 40 μg/ml cycloheximide. For each sample, proteins from cells with equivalent OD600 were loaded onto SDS-PAGE gels and blotted with specific antibodies.
Mentions: The R2TP complex was isolated from yeast cells in which the constitutive PIH1 gene was C-terminally 3FLAG tagged by immunoprecipitation with anti-FLAG antibody as described for Fig. 2 C. The complex was then immunoblotted with anti-Hsp90 antibody. As shown in Fig. 3 A, Hsp90 was not significantly associated with the R2TP complex purified from log-phase cells; however, more of the chaperone was associated with the complex isolated from stationary-phase cells, which indicates that Hsp90, Tah1, and Pih1 are in the same complex under these conditions. Surprisingly, higher levels of Hsp90 were found to be associated with the R2P complex purified from tah1Δ cells at both log- and stationary-phase cells. Considering that Tah1 might act as a cochaperone of Hsp90 (Zhao et al., 2005), it is likely that Hsp90 compensates for the depletion of Tah1 by increasing its association with R2P to preserve the function of the complex. Furthermore, the increased association of the chaperone with the R2P and R2TP complexes in stationary-phase cells compared with log-phase cells seems to indicate that one or more proteins in the complexes require added chaperoning under stress conditions.

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