The Box H/ACA snoRNP Assembly Factor Shq1p is a Chaperone Protein Homologous to Hsp90 Cochaperones that Binds to the Cbf5p Enzyme

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Abstract

Box H/ACA small nucleolar (sno) ribonucleoproteins (RNPs) are responsible for the formation of pseudouridine in a variety of RNAs and are essential for ribosome biogenesis, modification of spliceosomal RNAs, and telomerase stability. A mature snoRNP has been reconstituted in vitro and is composed of a single RNA and four proteins. However, snoRNP biogenesis in vivo requires multiple factors to coordinate a complex and poorly understood assembly and maturation process. Among the factors required for snoRNP biogenesis in yeast is Shq1p, an essential protein necessary for stable expression of box H/ACA snoRNAs. We have found that Shq1p consists of two independent domains that contain casein kinase 1 phosphorylation sites. We also demonstrate that Shq1p binds the pseudourydilating enzyme Cbf5p through the C-terminal domain, in synergy with the N-terminal domain. The NMR solution structure of the N-terminal domain has striking homology to the ‘Chord and Sgt1’ domain of known Hsp90 cochaperones, yet Shq1p does not interact with the yeast Hsp90 homologue in vitro. Surprisingly, Shq1p has stand-alone chaperone activity in vitro. This activity is harbored by the C-terminal domain, but it is increased by the presence of the N-terminal domain. These results provide the first evidence of a specific biochemical activity for Shq1p and a direct link to the H/ACA snoRNP.

Introduction

Box H/ACA small nucleolar (sno) RNAs are a class of noncoding RNAs that play a fundamental role in ribosome biogenesis, spliceosome assembly, and telomere maintenance (for reviews, see Meier1 and Maxwell and Fournier2). In mammals, each of the ∼ 100 H/ACA snoRNAs combines with four highly conserved protein components to form enzymatically active sno ribonucleoproteins (RNPs): the Cbf5p catalytic subunit (dyskerin in humans or NAP57 in rodents), Nop10p, Nhp2p (L7Ae in archaea), and Gar1p. These enzymes primarily function in the nucleolus to catalyze site-specific pseudouridylation of preribosomal RNA,3, 4 but a fraction modifies small nuclear RNPs when directed to Cajal bodies by Cajal-body-specific RNAs.5, 6 Some H/ACA snoRNPs—including yeast small nuclear R30 (E1/U17 in vertebrates), which participates in preribosomal RNA cleaveage,7, 8 and the telomerase H/ACA Cajal-body-specific RNA motif,9 which is essential in vivo for proper RNA processing, activity, and nucleolar localization—do not have pseudouridylase activity.10, 11, 12

The macromolecular architecture and enzymatic role of each protein have been studied with in-vitro-reconstituted archaeal small RNPs consisting of a single-hairpin small RNA and four evolutionarily conserved protein homologues (for a review, see Li13). However, in eukaryotes, the H/ACA snoRNP biogenesis pathway is surprisingly complex, requiring several more proteins than are present in the mature functional holoenzyme (for a review, see Matera et al.14). Assembly of the mature proteins follows a coordinated stepwise process requiring the formation and disassembly of intermediate structures within specific subnuclear compartments.15, 16 Stable expression of H/ACA snoRNAs requires the cotranscriptional formation of an inactive RNP by the association of Cbf5p, Nop10p, Nhp2p, and the H/ACA assembly factor Naf1p.17, 18 Gar1 is not found at the transcription site with the other snoRNP proteins, but is concentrated in the nucleolus19 and within Cajal bodies;20 this protein likely associates with the RNP late in the maturation process. Cbf5p mediates the interaction of Naf1p with other snoRNP proteins during snoRNA transcription,15, 16, 17, 18 yet Naf1p does not localize with the other snoRNP proteins to the nucleolus or Cajal bodies. Since the specificity of Naf1p for the H/ACA snoRNP arises from its Gar1-like domain,21 which makes it interact with Cbf5p in a manner that is competitive with Gar1, the snoRNP is presumed to undergo a remodeling event to introduce Gar1 and to form mature functional snoRNP particles.

A second eukaryotic H/ACA snoRNP assembly factor, Shq1, was found to associate with Naf1p in proteomic, in vitro pull-down, and colocalization studies.22 Genetic depletion of Shq1p results in ribosomal RNA processing defects due to loss of the stable accumulation of H/ACA snoRNAs. Shq1p was also found to associate with in-vitro-translated Nhp2p22 and flag-tagged Cbf5,23 providing an additional and direct link to H/ACA snoRNP biogenesis. Since levels of Nhp2p protein are unaffected in Shq1p-depleted cells, Shq1p probably functions directly in the maturation of H/ACA snoRNPs.

Sequence analysis indicates that Shq1p has two structural domains. We show that these two domains do not interact with each other, yet the interaction between Shq1p and Cbf5 that we report here requires both domains. We report the solution structure of its N-terminal domain, which shows that it adopts a ‘Chord and Sgt1’ (CS) fold related to the Hsp90 cochaperones Sgt1 and Sba1p/p23 proteins; however, as recently reported, this domain does not interact directly with the yeast Hsp90 homologue.24 We further show that the C-terminal domain does not rescue the binding of Shq1p to Hsp90. However, we report that the C-terminal domain has a stand-alone chaperone-like activity that is enhanced in the complete protein. We also observe that both domains of Shq1p are substrates for casein kinase 1 (CK1) phosphorylation.

Section snippets

Shq1p has two structurally independent domains

Yeast Shq1p consists of 507 residues with a very high sequence conservation from yeast to humans (Fig. 1). Comparison of Shq1p with sequences from known domains or motifs using the InterPro database25 suggests that Shq1p contains two domains: a CS domain within the first 100 residues and a C-terminal sequence unique to Shq1. To verify this assumption, purified wild-type Shq1p was digested in three different ways with proteinase K, trypsin, or chymotrypsin. Analysis by SDS-PAGE consistently

Discussion

Stable accumulation of mature H/ACA snoRNAs in eukaryotes requires cotranscriptional association with protein factors that protect the snoRNA from degradation and promote the stepwise assembly of mature RNP.15, 17, 18 This assembly process is assisted by at least two proteins that appear to be specific for this class of snoRNPs: Naf1p and Shq1p.22 A direct role for Naf1p recruitment to Cbf5p during snoRNA transcription and exchange with Gar1p at later stages of the maturation process was

Conclusions

Our studies demonstrate that Shq1p is capable of Cbf5p recognition and that it has a chaperone activity independent of its interaction with the yeast Hsp90 chaperone, suggesting that it may participate in chaperone-assisted maintenance or assembly of the H/ACA snoRNP. Since Shq1p does not interact efficiently with mature H/ACA particles, the interaction with Cbf5p occurs transiently during RNP assembly. The potential phosphorylation sites in both the N-terminal and the C-terminal domains

Plasmids

Full-length Shq1p (Shq1FL), the N-terminal domain consisting of residues Ile2-Asn134 (Shq1Np), and the C-terminal domain (Shq1C) from Ile123 to Gln507 were amplified by PCR from single-yeast colonies of the CRY1 strain and cloned into pET32 (Novagen) using ligation-independent cloning techniques. All pET32-Shq1p constructs contain an N-terminal thioredoxin fusion protein and a 6xHis tag, followed by a TEV protease cleavage site. Cbf5Δp was PCR-amplified from yeast, as previously described,50

Acknowledgements

We would like to thank Drs. Per Widlund and Trisha Davis for the supply of yeast cultures and the initial PCR amplification of Shq1p. We thank Dr. David Waugh for supplying a TEV protease expression line, Drs. Lina Dahlberg and David Kimelman for supplying CK1ɛ, Philippe Meyer for supplying Hsp82p constructs, and Michael Pereckas (Protein and Nucleic Acid Facility at the Medical College of Wisconsin) for performing N-terminal sequencing. NMR experiments collected at the Pacific Northwest

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