Naf1p is a box H/ACA snoRNP assembly factor

Wellcome Trust Centre for Cell Biology, Swann Building, King's Buildings, University of Edinburgh, Edinburgh EH9 3JR, UK.
RNA (Impact Factor: 4.94). 01/2003; 8(12):1502-14.
Source: PubMed


Box H/ACA small nucleolar ribonucleoprotein particles (snoRNPs) contain four essential proteins, Cbf5p, Gar1p, Nhp2p, and Nop10p, each of which, with the exception of Gar1p, is required for box H/ACA snoRNA accumulation. Database searches identified a novel essential protein, which we termed Naf1p, with a region of homology to the RNA-binding domain of Gar1p and other features in common with hnRNP-like proteins. Naf1p is localized to the nucleus and is not a stable component of the H/ACA snoRNPs, but it is required for the accumulation of all box H/ACA snoRNAs. This requirement is not at the level of snoRNA transcription initiation or termination. Naf1 p shows in vitro RNA-binding activity and also binds directly to Cbf5p and Nhp2p. Naf1p was shown to bind to the CTD in vivo in a two-hybrid assay, and the phosphorylated CTD, but not the nonphosphorylated CTD, was shown to precipitate tagged Naf1p from a cell lysate. We propose that Naf1 p is recruited to the CTD of RNA polymerase II and binds to nascent box H/ACA snoRNAs promoting snoRNP assembly.

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Available from: David Tollervey, Sep 30, 2014
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    • "Two conserved proteins, Naf1 and Shq1, are specifically required for H/ACA RNP formation in yeast Saccharomyces cerevisiae (Dez et al, 2002; Fatica et al, 2002; Yang et al, 2002) and in mammalian cells (Darzacq et al, 2006; Hoareau-Aveilla et al, 2006; Grozdanov et al, 2009b). Naf1 and Shq1 are localized in the nucleoplasm and excluded from nucleoli and Cajal bodies, where mature H/ACA RNPs reside (Dez et al, 2002; Fatica et al, 2002; Yang et al, 2002; Darzacq et al, 2006; Grozdanov et al, 2009b). "
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    ABSTRACT: Shq1 is a conserved protein required for the biogenesis of eukaryotic H/ACA ribonucleoproteins (RNPs), including human telomerase. We report the structure of the Shq1-specific domain alone and in complex with H/ACA RNP proteins Cbf5, Nop10 and Gar1. The Shq1-specific domain adopts a novel helical fold and primarily contacts the PUA domain and the otherwise disordered C-terminal extension (CTE) of Cbf5. The structure shows that dyskeratosis congenita mutations found in the CTE of human Cbf5 likely interfere with Shq1 binding. However, most mutations in the PUA domain are not located at the Shq1-binding surface and also have little effect on the yeast Cbf5-Shq1 interaction. Shq1 binds Cbf5 independently of the H/ACA RNP proteins Nop10, Gar1 and Nhp2 and the assembly factor Naf1, but shares an overlapping binding surface with H/ACA RNA. Shq1 point mutations that disrupt Cbf5 interaction suppress yeast growth particularly at elevated temperatures. Our results suggest that Shq1 functions as an assembly chaperone that protects the Cbf5 protein complexes from non-specific RNA binding and aggregation before assembly of H/ACA RNA.
    The EMBO Journal 11/2011; 30(24):5010-20. DOI:10.1038/emboj.2011.427 · 10.43 Impact Factor
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    • "These systems depend on in vivo preassembled protein complexes and have limited utility in structural analysis . Moreover, the in vivo assembly of eukaryotic H/ACA RNPs requires at least two specific assembly factors: Naf1 and Shq1 (Dez et al. 2002; Fatica et al. 2002; Yang et al. 2002). It remains unclear whether eukaryotic H/ACA RNPs also require these factors for in vitro assembly. "
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    ABSTRACT: Box H/ACA ribonucleoprotein particles (RNPs) mediate pseudouridine synthesis, ribosome formation, and telomere maintenance. The structure of eukaryotic H/ACA RNPs remains poorly understood. We reconstituted functional Saccharomyces cerevisiae H/ACA RNPs with recombinant proteins Cbf5, Nop10, Gar1, and Nhp2 and a two-hairpin H/ACA RNA; determined the crystal structure of a Cbf5, Nop10, and Gar1 ternary complex at 1.9 Å resolution; and analyzed the structure-function relationship of the yeast complex. Although eukaryotic H/ACA RNAs have a conserved two-hairpin structure, isolated single-hairpin RNAs are also active in guiding pseudouridylation. Nhp2, unlike its archaeal counterpart, is largely dispensable for the activity, reflecting a functional adaptation of eukaryotic H/ACA RNPs to the variable RNA structure that Nhp2 binds. The N-terminal extension of Cbf5, a hot spot for dyskeratosis congenita mutation, forms an extra structural layer on the PUA domain. Gar1 is distinguished from the assembly factor Naf1 by containing a C-terminal extension that controls substrate turnover and the Gar1-Naf1 exchange during H/ACA RNP maturation. Our results reveal significant novel features of eukaryotic H/ACA RNPs.
    Genes & development 11/2011; 25(22):2409-21. DOI:10.1101/gad.175299.111 · 10.80 Impact Factor
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    • "Indeed, mature snoRNPs contain only the four core proteins, whereas additional proteins are required for snoRNA maturation and snoRNP assembly. For example, Naf1 and Shq1 are required for H/ACA snoRNP assembly but are absent in mature snoRNP (48–50). It has been shown that Naf1 interacts with pre-snoRNP co-transcriptionally, and after splicing, this protein is replaced by a H/ACA snoRNP core protein, Gar1 (51–53). "
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    ABSTRACT: Hundreds of small nuclear non-coding RNAs, including small nucleolar RNAs (snoRNAs), have been identified in different organisms, with important implications in regulating gene expression and in human diseases. However, functionalizing these nuclear RNAs in mammalian cells remains challenging, due to methodological difficulties in depleting these RNAs, especially snoRNAs. Here we report a convenient and efficient approach to deplete snoRNA, small Cajal body RNA (scaRNA) and small nuclear RNA in human and mouse cells by conventional transfection of chemically modified antisense oligonucleotides (ASOs) that promote RNaseH-mediated cleavage of target RNAs. The levels of all seven tested snoRNA/scaRNAs and four snRNAs were reduced by 80-95%, accompanied by impaired endogenous functions of the target RNAs. ASO-targeting is highly specific, without affecting expression of the host genes where snoRNAs are embedded in the introns, nor affecting the levels of snoRNA isoforms with high sequence similarities. At least five snoRNAs could be depleted simultaneously. Importantly, snoRNAs could be dramatically depleted in mice by systematic administration of the ASOs. Together, our findings provide a convenient and efficient approach to characterize nuclear non-coding RNAs in mammalian cells, and to develop antisense drugs against disease-causing non-coding RNAs.
    Nucleic Acids Research 11/2010; 39(3):e13. DOI:10.1093/nar/gkq1121 · 9.11 Impact Factor
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