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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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Microarray and pull-down experiments link Hsp90 and R2TP to snoRNA accumulation. (A and B) Microarray analysis of changes in the levels of snoRNAs in pih1Δ, hsp82ts hsc82Δ, and tah1Δ strains compared with levels in the WT strain. Cells were grown to stationary phase at 30°C. The figures include all of the probes on the arrays showing statistically significant changes with respect to WT. Colors reflect intensity changes expressed as log2(mutant/WT), with up-regulated probes in red and down-regulated probes in green. (C) Nop56–3FLAG protein complexes were purified from WT, pih1Δ, and hsp82ts hsc82Δ strains grown to stationary phase. The proteins were separated on SDS-PAGE gels and were visualized by silver staining (top) or immunoblotted with αHsp90 and αFLAG antibodies (middle and bottom, respectively). Nop58, Nop56, Nop1, and Snu13 were identified by mass spectrometry.
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fig7: Microarray and pull-down experiments link Hsp90 and R2TP to snoRNA accumulation. (A and B) Microarray analysis of changes in the levels of snoRNAs in pih1Δ, hsp82ts hsc82Δ, and tah1Δ strains compared with levels in the WT strain. Cells were grown to stationary phase at 30°C. The figures include all of the probes on the arrays showing statistically significant changes with respect to WT. Colors reflect intensity changes expressed as log2(mutant/WT), with up-regulated probes in red and down-regulated probes in green. (C) Nop56–3FLAG protein complexes were purified from WT, pih1Δ, and hsp82ts hsc82Δ strains grown to stationary phase. The proteins were separated on SDS-PAGE gels and were visualized by silver staining (top) or immunoblotted with αHsp90 and αFLAG antibodies (middle and bottom, respectively). Nop58, Nop56, Nop1, and Snu13 were identified by mass spectrometry.

Mentions: Subsequently, microarrays were used to gain a more global view of snoRNA accumulation in hsp82ts hsc82Δ, pih1Δ, and tah1Δ stationary-phase cells. As shown in Fig. 7 A, there was generally a reduction in the accumulation of several box C/D snoRNAs in all three strains, with the greatest reduction evident in pih1Δ cells. The pih1Δ strain showed an overall accumulation of box H/ACA snoRNAs, whereas in tah1Δ and hsp82ts hsc82Δ cells, levels of these snoRNAs varied (Fig. 7 B). There is general agreement between the trends obtained from the microarray analysis and the Northern analysis (Fig. S4, available at http://www.jcb.org/cgi/content/full/jcb.200709061/DC1).


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)

Microarray and pull-down experiments link Hsp90 and R2TP to snoRNA accumulation. (A and B) Microarray analysis of changes in the levels of snoRNAs in pih1Δ, hsp82ts hsc82Δ, and tah1Δ strains compared with levels in the WT strain. Cells were grown to stationary phase at 30°C. The figures include all of the probes on the arrays showing statistically significant changes with respect to WT. Colors reflect intensity changes expressed as log2(mutant/WT), with up-regulated probes in red and down-regulated probes in green. (C) Nop56–3FLAG protein complexes were purified from WT, pih1Δ, and hsp82ts hsc82Δ strains grown to stationary phase. The proteins were separated on SDS-PAGE gels and were visualized by silver staining (top) or immunoblotted with αHsp90 and αFLAG antibodies (middle and bottom, respectively). Nop58, Nop56, Nop1, and Snu13 were identified by mass spectrometry.
© Copyright Policy
Related In: Results  -  Collection

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

fig7: Microarray and pull-down experiments link Hsp90 and R2TP to snoRNA accumulation. (A and B) Microarray analysis of changes in the levels of snoRNAs in pih1Δ, hsp82ts hsc82Δ, and tah1Δ strains compared with levels in the WT strain. Cells were grown to stationary phase at 30°C. The figures include all of the probes on the arrays showing statistically significant changes with respect to WT. Colors reflect intensity changes expressed as log2(mutant/WT), with up-regulated probes in red and down-regulated probes in green. (C) Nop56–3FLAG protein complexes were purified from WT, pih1Δ, and hsp82ts hsc82Δ strains grown to stationary phase. The proteins were separated on SDS-PAGE gels and were visualized by silver staining (top) or immunoblotted with αHsp90 and αFLAG antibodies (middle and bottom, respectively). Nop58, Nop56, Nop1, and Snu13 were identified by mass spectrometry.
Mentions: Subsequently, microarrays were used to gain a more global view of snoRNA accumulation in hsp82ts hsc82Δ, pih1Δ, and tah1Δ stationary-phase cells. As shown in Fig. 7 A, there was generally a reduction in the accumulation of several box C/D snoRNAs in all three strains, with the greatest reduction evident in pih1Δ cells. The pih1Δ strain showed an overall accumulation of box H/ACA snoRNAs, whereas in tah1Δ and hsp82ts hsc82Δ cells, levels of these snoRNAs varied (Fig. 7 B). There is general agreement between the trends obtained from the microarray analysis and the Northern analysis (Fig. S4, available at http://www.jcb.org/cgi/content/full/jcb.200709061/DC1).

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