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

Abstract

Box H/ACA small nucleolar ribonucleoproteins (snoRNPs) 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. The 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 the snoRNA stable expression. 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' (CS) domain of known Hsp90 co-chaperones, 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.

Mechanism of the AAA+ ATPases pontin and reptin in the biogenesis of H/ACA RNPs

“…The SSD was retained by neither construct (Fig. 1H, lanes 2,4), consistent with previous data using yeast orthologs (Godin et al 2009). This indicated that the CS domain of SHQ1, even in the presence of the SSD, required NAP57 for binding.…”

“…4D, lanes 1,2) whereas binding of the SSD was unaffected by incubation with S100 (lanes 3,4) and the CS domain alone was unable to bind (lanes 5,6), as observed previously ( Fig. 1G; Godin et al 2009;Grozdanov et al 2009b). However, when bound in trans, S100 quantitatively removed the CS domain of SHQ1 but barely impacted binding of its SSD (Fig.…”

“…Therefore, release of SHQ1 from MBP-NAP57 apparently occurred independent of ATP and HSP90. The latter is consistent with the failure of HSP90 to interact with the CS domain of SHQ1, which is homologous with the HSP90 cochaperone p23 (Godin et al 2009;Singh et al 2009). …”

“…The SSD of SHQ1 (Fig. 1D) binds independently to NAP57, whereas the CS domain alone fails to bind (Godin et al 2009;Grozdanov et al 2009b), yet is essential for cell viability (Singh et al 2009). This and other puzzles prompted us to further investigate the role of the CS domain of SHQ1 in its interaction with NAP57.…”

“…It consists of the N-terminal HSP90 cochaperone p23-like CS domain (90 4 amino acids) and of the SHQ1-specific domain (SSD, also DCS domain) that accounts for the majority of the protein (487 amino acids; Fig. 1D; Godin et al 2009;Grozdanov et al 2009b;Singh et al 2009). Recently, we and others solved the crystal structures of the yeast orthologs of the SSD alone and in complex with the NAPDcat domain of NAP57, demonstrating that the SSD functions as an RNAmimic hugging the surface of NAP57 that is destined for H/ACA RNAs (Li et al 2011;Walbott et al 2011).…”