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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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Mapping the interactions between proteins of the R2TP complex. (A and B) Interactions between Hsp82, Tah1, Pih1, Rvb1, and Rvb2 were examined by SE chromatography on a Superdex 200 10/30 column. The positions of the molecular mass standards (in kilodaltons) are given along the top x axis. Tah1–Pih1 complexes are boxed for clarity. Proteins were detected on SDS-PAGE gels by silver staining. v.v., void volume. (C) A 3xFLAG tag was added to the chromosomal TAH1 or PIH1 genes in WT, tah1Δ, or pih1Δ cells. Tah1–3FLAG or Pih1–3FLAG complexes were isolated using αFLAG antibody resin from cells grown to late log phase at 30°C. The same amount of complexes was loaded on the gels. Proteins were visualized by Coomassie staining and were identified by mass spectrometry. (D) Western blot analysis of equal amounts of total cell lysates from different strains. Note that the Hsp90 antibody does not distinguish between Hsp82 and Hsc82, which are 98% identical. (E) A protein interaction model for the R2TP complex.
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fig2: Mapping the interactions between proteins of the R2TP complex. (A and B) Interactions between Hsp82, Tah1, Pih1, Rvb1, and Rvb2 were examined by SE chromatography on a Superdex 200 10/30 column. The positions of the molecular mass standards (in kilodaltons) are given along the top x axis. Tah1–Pih1 complexes are boxed for clarity. Proteins were detected on SDS-PAGE gels by silver staining. v.v., void volume. (C) A 3xFLAG tag was added to the chromosomal TAH1 or PIH1 genes in WT, tah1Δ, or pih1Δ cells. Tah1–3FLAG or Pih1–3FLAG complexes were isolated using αFLAG antibody resin from cells grown to late log phase at 30°C. The same amount of complexes was loaded on the gels. Proteins were visualized by Coomassie staining and were identified by mass spectrometry. (D) Western blot analysis of equal amounts of total cell lysates from different strains. Note that the Hsp90 antibody does not distinguish between Hsp82 and Hsc82, which are 98% identical. (E) A protein interaction model for the R2TP complex.

Mentions: In the next set of experiments, the protein–protein interactions in the R2TP complex were characterized. Purified Rvb1 (50.5 kD) and Rvb2 (51.6 kD) proteins were analyzed by SE chromatography. Each protein individually eluted at fractions corresponding to an apparent molecular mass of ∼50 kD (Fig. 2 A), suggesting that each component alone exists predominantly as a monomer in the absence or presence of ADP. When Rvb1 and Rvb2 were incubated together for 4 h before being separated on the SE column, the two helicases formed a complex of an apparent molecular mass of ∼300–600 kD in the presence or absence of ADP (Fig. 2 A). The complex formed was found to be a heterohexamer of Rvb1 and Rvb2 (Gribun et al., 2008).


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)

Mapping the interactions between proteins of the R2TP complex. (A and B) Interactions between Hsp82, Tah1, Pih1, Rvb1, and Rvb2 were examined by SE chromatography on a Superdex 200 10/30 column. The positions of the molecular mass standards (in kilodaltons) are given along the top x axis. Tah1–Pih1 complexes are boxed for clarity. Proteins were detected on SDS-PAGE gels by silver staining. v.v., void volume. (C) A 3xFLAG tag was added to the chromosomal TAH1 or PIH1 genes in WT, tah1Δ, or pih1Δ cells. Tah1–3FLAG or Pih1–3FLAG complexes were isolated using αFLAG antibody resin from cells grown to late log phase at 30°C. The same amount of complexes was loaded on the gels. Proteins were visualized by Coomassie staining and were identified by mass spectrometry. (D) Western blot analysis of equal amounts of total cell lysates from different strains. Note that the Hsp90 antibody does not distinguish between Hsp82 and Hsc82, which are 98% identical. (E) A protein interaction model for the R2TP complex.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2234237&req=5

fig2: Mapping the interactions between proteins of the R2TP complex. (A and B) Interactions between Hsp82, Tah1, Pih1, Rvb1, and Rvb2 were examined by SE chromatography on a Superdex 200 10/30 column. The positions of the molecular mass standards (in kilodaltons) are given along the top x axis. Tah1–Pih1 complexes are boxed for clarity. Proteins were detected on SDS-PAGE gels by silver staining. v.v., void volume. (C) A 3xFLAG tag was added to the chromosomal TAH1 or PIH1 genes in WT, tah1Δ, or pih1Δ cells. Tah1–3FLAG or Pih1–3FLAG complexes were isolated using αFLAG antibody resin from cells grown to late log phase at 30°C. The same amount of complexes was loaded on the gels. Proteins were visualized by Coomassie staining and were identified by mass spectrometry. (D) Western blot analysis of equal amounts of total cell lysates from different strains. Note that the Hsp90 antibody does not distinguish between Hsp82 and Hsc82, which are 98% identical. (E) A protein interaction model for the R2TP complex.
Mentions: In the next set of experiments, the protein–protein interactions in the R2TP complex were characterized. Purified Rvb1 (50.5 kD) and Rvb2 (51.6 kD) proteins were analyzed by SE chromatography. Each protein individually eluted at fractions corresponding to an apparent molecular mass of ∼50 kD (Fig. 2 A), suggesting that each component alone exists predominantly as a monomer in the absence or presence of ADP. When Rvb1 and Rvb2 were incubated together for 4 h before being separated on the SE column, the two helicases formed a complex of an apparent molecular mass of ∼300–600 kD in the presence or absence of ADP (Fig. 2 A). The complex formed was found to be a heterohexamer of Rvb1 and Rvb2 (Gribun et al., 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.

Show MeSH