Structure and Functional Studies of the CS Domain of the Essential H/ACA Ribonucleoparticle Assembly Protein SHQ1

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 β-sandwich fold that is composed of two β-sheets containing four and three β-strands, respectively, and a short α-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.Modification of uridine to pseudouridine in ribosomal RNA and some spliceosomal RNAs is catalyzed by highly specialized ribonucleoparticle (RNP)³ complexes called box H/ACA RNPs (1-5). Depending on their site of maturation and action H/ACA RNPs are classified into two classes, small nucleolar RNPs (snoRNPs) and small Cajal body RNPs. In Saccharomyces cerevisiae, H/ACA snoRNPs contain four proteins: Nhp2p (L7ae in archaea (6) and Cbf5p, also called dyskerin, in humans (7)), Nop10p, Gar1p, and a single small nucleolar RNA (snoRNA), specific to each snoRNP (8-11). Cbf5p provides the pseudouridylase activity to the complex, and the snoRNA component provides the “guide RNA” for positioning the substrate RNA for modification (8, 10, 12-15). The 3′ end of human telomerase RNA (hTR) contains an H/ACA scaRNA domain that binds the H/ACA proteins and is required for 3′ end processing, accumulation, and localization of hTR to Cajal bodies (16-19). In archaea, the assembly of H/ACA snoRNP appears to proceed by assembly of the protein components, followed by the incorporation of the H/ACA RNA (8, 20-23). In eukaryotes, the assembly and final maturation of the holoenzyme RNP are more complicated, possibly because of subcellular compartmentalization, and require accessory proteins (22, 24). Two proteins specifically found in eukaryotes, Naf1p and Shq1p, were initially identified in yeast as factors involved in the assembly of H/ACA snoRNPs (23-25). Both Shq1p and Naf1p are essential proteins, and their depletion leads to the loss of H/ACA snoRNAs (22, 24). Shq1p and Naf1p interact with the H/ACA RNP components Cbf5p and Nhp2p as shown by high throughput proteomic approaches and by directed protein interaction studies (24, 26-28). Both Naf1p and Gar1p contain a central domain that forms a six-stranded β-barrel fold and interact competitively with Cbf5p using this “core-Gar1” domain (29).Although Shq1p was first identified in yeast (24), orthologues have been found in all eukaryotic genomes investigated, including human (22). Shq1p is not associated with the precursor or mature RNPs and is localized in the nucleoplasm (24) rather than in the nucleolus or Cajal bodies where mature H/ACA RNPs reside. It was therefore proposed that Shq1p is involved in the early biogenesis steps of H/ACA snoRNPs. However, Shq1p does not share any homology with either Naf1p or Gar1p, and its mode of action remains unclear. Based on the Saccharomyces Genome Data base annotations and domain predictions, Shq1p seems to be a modular protein with two predicted domains in its sequence (Fig. 1A). The C-terminal half contains a region of high sequence homology with other Shq1 proteins called the Shq1 domain, but this region shows no identified folding motif. The N-terminal region of the protein has a predicted CS (named after CHORD-containing proteins and SGT1) or HSP20-like domain, which is found in a number of co-chaperones for heat shock protein 90 (Hsp90) (30-34), and hence is presumed to be an Hsp90 (Hsp82p in yeast) binding domain. The CS, HSP20-like, and p23-like domains belong to the HSP20 domain superfamily.Open in a separate windowFIGURE 1.Deletion analysis and stability of Shq1p truncated mutants. A, domain organization of Shq1p and complementation of the shq1Δ strain by the various truncation mutants. Numbers refer to SHQ1 amino acid residues. Cells were grown in plates containing 5-fluoroacetic acid to counterselect the wild-type (wt) SHQ1 copy borne by the URA3-containing plasmid and in the absence of methionine to express Shq1p, and cell growth was assessed. B, example of growth assay used to generate the previous figure. Shown is the growth of strains expressing the indicated deletions of Shq1p on a plate containing 5-fluoroacetic acid to counterselect the wild-type SHQ1 copy borne by the URA3-containing plasmid. C, stability of truncated mutants. Cells containing the endogenous SHQ1 copy and the indicated constructs were grown on liquid medium in the absence of methionine. Proteins were expressed under the control of the Met25 promoter, which is highly induced in the absence of Met. Numbers refer to SHQ1 amino acid residues. Shq1p proteins were detected by immunoblotting with anti-GFP. Hexokinase (Hxk) was used as a loading control.We have determined the x-ray crystal structure of the CS domain of Shq1p and investigated its importance for Shq1p function. The structure consists of a β-sandwich immunoglobulin light chain fold (35). Like other CS domains, the CS domain of Shq1p is primarily composed of two β-sheets (30, 31, 33, 36), but it has an additional short helix at the C terminus. We show that the Shq1p CS domain is essential for growth in S. cerevisiae and that the integrity of the CS domain is required for the function of Shq1p in vivo. Based on the structure and sequence conservation among Shq1 proteins, we investigated the effect of three single point mutations, F6A, Q10A, and K80A, on Shq1p structure and function. All three mutations destabilized the tertiary structure of the CS domain, albeit to differing extents. Incorporation of these mutations in Shq1p resulted in a temperature-sensitive growth phenotype, specific depletion of H/ACA snoRNAs, and rRNA processing defects. NMR chemical shift mapping showed no interaction between the Shq1p CS domain and Hsp82p, suggesting that the Shq1p CS domain does not have a canonical role as an Hsp82p co-chaperone protein.