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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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Nucleotide-dependent disaggregation of Pih1agg by Hsp82. (A) SE chromatography of 100 μg (10 μM) Pih1agg preincubated with equal molar amounts of Hsp82 (210 μg) and Tah1 (30 μg) for 1 h at 30°C with or without ATP. The positions of the molecular mass standards (in kilodaltons) are given along the top x axis. Proteins were detected on SDS-PAGE gels by silver staining. v.v., void volume. (B) Disaggregation of Pih1agg by Hsp82 and Tah1 was monitored by light scattering at 350 nm. 120 μg Pih1agg was incubated with 84 μg Hsp82 and 12 μg Tah1 in the presence or absence of ATP. (C) AFM images of 60 μg Pih1agg incubated with 42 μg Hsp82 and 4 mM ATP monitored as a function of time. Control samples of Pih1agg + 4 mM ATP and of Hsp82 + 4 mM ATP after 60 min of incubation are also shown. Bar, 0.5 μm. (D) Change in particle volume distribution of Pih1agg in the presence of Hsp82 and ATP monitored as a function of time by AFM. Particle shapes were assumed to be partially filled spheres. Errors are derived from three measurements. Error bars represent SD.
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fig4: Nucleotide-dependent disaggregation of Pih1agg by Hsp82. (A) SE chromatography of 100 μg (10 μM) Pih1agg preincubated with equal molar amounts of Hsp82 (210 μg) and Tah1 (30 μg) for 1 h at 30°C with or without ATP. The positions of the molecular mass standards (in kilodaltons) are given along the top x axis. Proteins were detected on SDS-PAGE gels by silver staining. v.v., void volume. (B) Disaggregation of Pih1agg by Hsp82 and Tah1 was monitored by light scattering at 350 nm. 120 μg Pih1agg was incubated with 84 μg Hsp82 and 12 μg Tah1 in the presence or absence of ATP. (C) AFM images of 60 μg Pih1agg incubated with 42 μg Hsp82 and 4 mM ATP monitored as a function of time. Control samples of Pih1agg + 4 mM ATP and of Hsp82 + 4 mM ATP after 60 min of incubation are also shown. Bar, 0.5 μm. (D) Change in particle volume distribution of Pih1agg in the presence of Hsp82 and ATP monitored as a function of time by AFM. Particle shapes were assumed to be partially filled spheres. Errors are derived from three measurements. Error bars represent SD.

Mentions: When 100 μg of Pih1agg was injected onto the SE column in the absence or presence of ATP, most Pih1agg did not enter the column because it was bound to the precolumn filter (Fig. 4 A). Addition of Tah1 ± ATP, Hsp82 2 ATP, or Hsp82 + Tah1 2 ATP had no effect on the behavior of Pih1agg. However, incubation of Pih1agg with an equimolar amount of Hsp82 and ATP for 1 h at 30°C and subsequent loading of the mixture onto the SE column resulted in the appearance of smaller Pih1 oligomers that entered the column. The formation of smaller Pih1 oligomers seemed to be more pronounced when Pih1agg was incubated with Hsp82, Tah1, and ATP. These results indicate that Hsp82 and Tah1 can break down the soluble Pih1 aggregates to smaller oligomers in an ATP-dependent manner.


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)

Nucleotide-dependent disaggregation of Pih1agg by Hsp82. (A) SE chromatography of 100 μg (10 μM) Pih1agg preincubated with equal molar amounts of Hsp82 (210 μg) and Tah1 (30 μg) for 1 h at 30°C with or without ATP. The positions of the molecular mass standards (in kilodaltons) are given along the top x axis. Proteins were detected on SDS-PAGE gels by silver staining. v.v., void volume. (B) Disaggregation of Pih1agg by Hsp82 and Tah1 was monitored by light scattering at 350 nm. 120 μg Pih1agg was incubated with 84 μg Hsp82 and 12 μg Tah1 in the presence or absence of ATP. (C) AFM images of 60 μg Pih1agg incubated with 42 μg Hsp82 and 4 mM ATP monitored as a function of time. Control samples of Pih1agg + 4 mM ATP and of Hsp82 + 4 mM ATP after 60 min of incubation are also shown. Bar, 0.5 μm. (D) Change in particle volume distribution of Pih1agg in the presence of Hsp82 and ATP monitored as a function of time by AFM. Particle shapes were assumed to be partially filled spheres. Errors are derived from three measurements. Error bars represent SD.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2234237&req=5

fig4: Nucleotide-dependent disaggregation of Pih1agg by Hsp82. (A) SE chromatography of 100 μg (10 μM) Pih1agg preincubated with equal molar amounts of Hsp82 (210 μg) and Tah1 (30 μg) for 1 h at 30°C with or without ATP. The positions of the molecular mass standards (in kilodaltons) are given along the top x axis. Proteins were detected on SDS-PAGE gels by silver staining. v.v., void volume. (B) Disaggregation of Pih1agg by Hsp82 and Tah1 was monitored by light scattering at 350 nm. 120 μg Pih1agg was incubated with 84 μg Hsp82 and 12 μg Tah1 in the presence or absence of ATP. (C) AFM images of 60 μg Pih1agg incubated with 42 μg Hsp82 and 4 mM ATP monitored as a function of time. Control samples of Pih1agg + 4 mM ATP and of Hsp82 + 4 mM ATP after 60 min of incubation are also shown. Bar, 0.5 μm. (D) Change in particle volume distribution of Pih1agg in the presence of Hsp82 and ATP monitored as a function of time by AFM. Particle shapes were assumed to be partially filled spheres. Errors are derived from three measurements. Error bars represent SD.
Mentions: When 100 μg of Pih1agg was injected onto the SE column in the absence or presence of ATP, most Pih1agg did not enter the column because it was bound to the precolumn filter (Fig. 4 A). Addition of Tah1 ± ATP, Hsp82 2 ATP, or Hsp82 + Tah1 2 ATP had no effect on the behavior of Pih1agg. However, incubation of Pih1agg with an equimolar amount of Hsp82 and ATP for 1 h at 30°C and subsequent loading of the mixture onto the SE column resulted in the appearance of smaller Pih1 oligomers that entered the column. The formation of smaller Pih1 oligomers seemed to be more pronounced when Pih1agg was incubated with Hsp82, Tah1, and ATP. These results indicate that Hsp82 and Tah1 can break down the soluble Pih1 aggregates to smaller oligomers in an ATP-dependent manner.

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