SHQ1 is required prior to NAF1 for assembly of H/ACA small nucleolar and telomerase RNPs

Abstract

Assembly of H/ACA RNPs in yeast is aided by at least two accessory factors, Naf1p and Shq1p. Although the function of Naf1p and its human ortholog NAF1 has been delineated in detail, that of Shq1p and its putative human ortholog SHQ1 remains obscure. We demonstrate that SHQ1 indeed functions in the biogenesis of human H/ACA RNPs and we dissect its mechanism of action. Like NAF1, SHQ1 binds the major H/ACA core protein and pseudouridine synthase NAP57 (aka dyskerin) but precedes the assembly role of NAF1 at nascent H/ACA RNAs because the interaction of SHQ1 with NAP57 in vivo and in vitro precludes that of NAF1 and of the other H/ACA core proteins that are present at the sites of H/ACA RNA transcription. The N-terminal heat shock protein 20-like CS domain of SHQ1 is dispensable for NAP57 binding. Consistent with its role as an assembly factor, SHQ1 localizes to the nucleoplasm and is excluded from nucleoli and Cajal bodies, the sites of mature H/ACA RNPs. In an in vitro assembly system of functional H/ACA RNPs that is dependent on NAF1, excess recombinant SHQ1 interferes with assembly. Importantly, knockdown of cellular SHQ1 prevents accumulation of a newly synthesized H/ACA reporter RNA and generally reduces the levels of endogenous H/ACA RNAs including telomerase RNA. In summary, the sequential action of SHQ1 and NAF1 is required for functional assembly of H/ACA RNPs in vivo and in vitro. This step-wise process could serve as an efficient means of quality control during H/ACA RNP assembly.

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

“…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.…”

“…A rate-limiting early step in RNP assembly is the interaction of NAP57 with SHQ1, which is the focus of this study (Yang et al 2002;Grozdanov et al 2009b). NAP57 consists of a central catalytic domain (cat) and N and C termini that wrap around each other to form the NAPDcat domain ( Fig.…”

“…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).…”

The R2TP chaperone complex: its involvement in snoRNP assembly and tumorigenesis

“…For dyskerin, although downregulation of the expression has been observed in a subset of tumors (70), upregulated expression was determined in a number of human cancer cells and also its overexpression is correlated with aggressive proliferation of the tumor cells such as hepatocellular (71), colon (72), prostate (73,74), head and neck carcinomas (75). Given that the both Nop58 and dyskerin are unstable proteins and associated/stabilized by R2TP (16,17,76), the expression level of R2TP components also might be upregulated to maintain the target proteins level in above-mentioned cancers. In summary, it is shown how R2TP function is related to tumorigenesis through snoRNP biogenesis (Figure 3).…”

Genetic Variations in Telomere Maintenance, with Implications on Tissue Renewal Capacity and Chronic Disease Pathologies

“…TER is found in most human tissues and its in vivo stability is contingent on its stable association with a chaperone, the H/ACA protein complex [58,59]. Recently, a chain of biogenesis events has been identified: protein factors NAF1 [60,61] and Shq1 [62,63] load the core H/ACA protein complexonto their cognate RNA binding partners, followed by Gar1 protein association with the mature H/ACA complex to determine intracellular location [64]. The core H/ACA proteins, dyskerin, Nhp2 and Nop10, form a complex that binds to and remains in association with telomerase RNA throughout its cellular lifespan [63,65].…”