Abstract
H/ACA ribonucleoprotein particles are essential for ribosomal RNA and telomerase RNA processing and metabolism. Shq1p has been identified as an essential eukaryotic H/ACA small nucleolar (sno) ribonucleoparticle (snoRNP) biogenesis and assembly factor. Shq1p is postulated to be involved in the early biogenesis steps of H/ACA snoRNP complexes, and Shq1p depletion leads to a specific decrease in H/ACA small nucleolar RNA levels and to defects in ribosomal RNA processing. Shq1p contains two predicted domains as follows: an N-terminal CS (named after CHORD-containing proteins and SGT1) or HSP20-like domain, and a C-terminal region of high sequence homology called the Shq1 domain. Here we report the crystal structure and functional studies of the Saccharomyces cerevisiae Shq1p CS domain. The structure consists of a compact anti-parallel beta-sandwich fold that is composed of two beta-sheets containing four and three beta-strands, respectively, and a short alpha-helix. Deletion studies showed that the CS domain is required for the essential functions of Shq1p. Point mutations in residues Phe-6, Gln-10, and Lys-80 destabilize Shq1p in vivo and induce a temperature-sensitive phenotype with depletion of H/ACA small nucleolar RNAs and defects in rRNA processing. Although CS domains are frequently found in co-chaperones of the Hsp90 molecular chaperone, no interaction was detected between the Shq1p CS domain and yeast Hsp90 in vitro. These results show that the CS domain is essential for Shq1p function in H/ACA snoRNP biogenesis in vivo, possibly in an Hsp90-independent manner.
Highlights
CS domains are frequently found in co-chaperones of the heat shock protein 90 (Hsp90) molecular chaperone, no interaction was detected between the Shq1p CS domain and yeast Hsp90 in vitro. These results show that the CS domain is essential for Shq1p function in H/ACA small nucleolar RNPs (snoRNPs) biogenesis in vivo, possibly in an Hsp90independent manner
Point Mutations in the CS Domain Result in H/ACA small nucleolar RNA (snoRNA) Depletion and Defects in Pre-ribosomal RNA (rRNA) Processing—We further investigated the effect of the Shq1p CS domain mutations on Shq1p function in vivo by analyzing H/ACA snoRNA accumulation in the CS domain mutants grown at 25 °C or shifted to 37 °C
The snoRNA depletion observed for all mutants at 37 °C is likely due to decreased Shq1p levels triggered by the thermal instability of the mutant proteins, the depletion of H/ACA snoRNAs observed in the Shq1p(K80A) and Q10A strains at 25 °C cannot be attributed to protein depletion, because protein levels were normal for these mutants at this temperature (Fig. 5E)
Summary
Protein Expression and Purification for Structural Studies— CS (residues 1–101) and CS-L (residues 1–145) constructs were PCR-amplified using yeast genomic DNA as a template and cloned into pET46 LIC/Ek (Novagen). Yeast Hsp82p was PCR-amplified and cloned into pET30 LIC/ Xa. Protein was expressed in E. coli at room temperature and purified over a nickel-nitrilotriacetic acid-agarose column followed by gel filtration on Superdex S75 column. NMR titrations were carried out by adding unlabeled purified Hsp82p to 15N-labeled CS or CS-L and recording 1H-15N TROSY-HSQC spectra. All the titrations were carried out at 22 °C at 600 MHz. NMR data were processed using the Bruker program Xwin-NMR version 3.5 and assigned using the Sparky software program Crystallization, Data Collection, and Structure Determination—Crystallization was carried out with a hanging drop, vapor-diffusion method by mixing equal volumes of protein and reservoir solutions (0.1 M BisTris, pH 6.0, 0.2 M ammonium sulfate, 25% w/v PEG 3350). Rfree ϭ ͉Fo Ϫ Fc/͚Fo, calculated using a random set containing 5% reflections that were not included throughout refinement
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