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1.
Naf1 p is a box H/ACA snoRNP assembly factor   总被引:6,自引:1,他引:5       下载免费PDF全文
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2.
Nhp2p and Nop10p are essential for the function of H/ACA snoRNPs.   总被引:29,自引:3,他引:26  
The small nucleolar ribonucleoprotein particles containing H/ACA-type snoRNAs (H/ACA snoRNPs) are crucial trans-acting factors intervening in eukaryotic ribosome biogenesis. Most of these particles generate the site-specific pseudouridylation of rRNAs while a subset are required for 18S rRNA synthesis. To understand in detail how these particles carry out these functions, all of their protein components have to be characterized. For that purpose, we have affinity-purified complexes containing epitope-tagged Gar1p protein, previously shown to be part of H/ACA snoRNPs. Under the conditions used, three polypeptides of 65, 22 and 10 kDa apparent molecular weight specifically copurify with epitope-tagged Gar1p. The 22 and 10 kDa polypeptides were identified as Nhp2p and a novel protein we termed Nop10p, respectively. Both proteins are conserved, essential and present in the dense fibrillar component of the nucleolus. Nhp2p and Nop10p are specifically associated with all H/ACA snoRNAs and are essential to the function of H/ACA snoRNPs. Cells lacking Nhp2p or Nop10p are impaired in global rRNA pseudouridylation and in the A1 and A2 cleavage steps of the pre-rRNA required for the synthesis of mature 18S rRNA. These phenotypes are probably a direct consequence of the instability of H/ACA snoRNAs and Gar1p observed in cells deprived of Nhp2p or Nop10p. Our results suggest that Nhp2p and Nop10p, together with Cbf5p, constitute the core of H/ACA snoRNPs.  相似文献   

3.
The H/ACA small nucleolar ribonucleoprotein (snoRNP) complexes guide the modification of uridine to pseudouridine at conserved sites in rRNA. The H/ACA snoRNPs each comprise a target-site-specific snoRNA and four core proteins, Nop10p, Nhp2p, Gar1p, and the pseudouridine synthase, Cbf5p, in yeast. The secondary structure of the H/ACA snoRNAs includes two hairpins that each contain a large internal loop (the pseudouridylation pocket), one or both of which are partially complementary to the target RNA(s). We have determined the solution structure of an RNA hairpin derived from the human U65 box H/ACA snoRNA including the pseudouridylation pocket and adjacent stems, providing the first three-dimensional structural information on these H/ACA snoRNAs. We have also determined the structure of Nop10p and investigated its interaction with RNA using NMR spectroscopy. Nop10p contains a structurally well-defined N-terminal region composed of a beta-hairpin, and the rest of the protein lacks a globular structure. Chemical shift mapping of the interaction of RNA constructs of U65 box H/ACA 3' hairpin with Nop10p shows that the beta-hairpin binds weakly but specifically to RNA. The unstructured region of Nop10p likely interacts with Cbf5p.  相似文献   

4.
Box H/ACA small nucleolar ribonucleoprotein particles (H/ACA snoRNPs) play key roles in the synthesis of eukaryotic ribosomes. The ways in which these particles are assembled and correctly localized in the dense fibrillar component of the nucleolus remain largely unknown. Recently, the essential Saccharomyces cerevisiae Naf1p protein (encoded by the YNL124W open reading frame) was found to interact in a two-hybrid assay with two core protein components of mature H/ACA snoRNPs, Cbf5p and Nhp2p (T. Ito, T. Chiba, R. Ozawa, M. Yoshida, M. Hattori, and Y. Sakaki, Proc. Natl. Acad. Sci. USA 98:4569-4574, 2001). Here we show that several H/ACA snoRNP components are weakly but specifically immunoprecipitated with epitope-tagged Naf1p, suggesting that the latter protein is involved in H/ACA snoRNP biogenesis, trafficking, and/or function. Consistent with this, we find that depletion of Naf1p leads to a defect in 18S rRNA accumulation. Naf1p is unlikely to directly assist H/ACA snoRNPs during pre-rRNA processing in the dense fibrillar component of the nucleolus for two reasons. Firstly, Naf1p accumulates predominantly in the nucleoplasm. Secondly, Naf1p sediments in a sucrose gradient chiefly as a free protein or associated in a complex of the size of free snoRNPs, whereas extremely little Naf1p is found in fractions containing preribosomes. These results are more consistent with a role for Naf1p in H/ACA snoRNP biogenesis and/or intranuclear trafficking. Indeed, depletion of Naf1p leads to a specific and dramatic decrease in the steady-state accumulation of all box H/ACA snoRNAs tested and of Cbf5p, Gar1p, and Nop10p. Naf1p is unlikely to be directly required for the synthesis of H/ACA snoRNP components. Naf1p could participate in H/ACA snoRNP assembly and/or transport.  相似文献   

5.
The eukaryotic nucleolus contains a large number of small nucleolar RNAs (snoRNAs) that are involved in preribosomal RNA (pre-rRNA) processing. The H box/ACA-motif (H/ACA) class of snoRNAs has recently been demonstrated to function as guide RNAs targeting specific uridines in the pre-rRNA for pseudouridine (psi) synthesis. To characterize the protein components of this class of snoRNPs, we have purified the snR42 and snR30 snoRNP complexes by anti-m3G-immunoaffinity and Mono-Q chromatography of Saccharomyces cerevisiae extracts. Sequence analysis of the individual polypeptides demonstrated that the three proteins Gar1p, Nhp2p, and Cbf5p are common to both the snR30 and snR42 complexes. Nhp2p is a highly basic protein that belongs to a family of putative RNA-binding proteins. Cbf5p has recently been demonstrated to be involved in ribosome biogenesis and also shows striking homology with known prokaryotic psi synthases. The presence of Cbf5p, a putative psi synthase in each H/ACA snoRNP suggests that this class of RNPs functions as individual modification enzymes. Immunoprecipitation studies using either anti-Cbf5p antibodies or a hemagglutinin-tagged Nhp2p demonstrated that both proteins are associated with all H/ACA-motif snoRNPs. In vivo depletion of Nhp2p results in a reduction in the steady-state levels of all H/ACA snoRNAs. Electron microscopy of purified snR42 and snR30 particles revealed that these two snoRNPs possess a similar bipartite structure that we propose to be a major structural determining principle for all H/ACA snoRNPs.  相似文献   

6.
Li S  Duan J  Li D  Ma S  Ye K 《The EMBO journal》2011,30(24):5010-5020
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.  相似文献   

7.
H/ACA small nucleolar and Cajal body ribonucleoproteins (RNPs) function in site-specific pseudouridylation of eukaryotic rRNA and snRNA, rRNA processing, and vertebrate telomerase biogenesis. Nhp2, one of four essential protein components of eukaryotic H/ACA RNPs, forms a core trimer with the pseudouridylase Cbf5 and Nop10 that binds to H/ACA RNAs specifically. Crystal structures of archaeal H/ACA RNPs have revealed how the protein components interact with each other and with the H/ACA RNA. However, in place of Nhp2p, archaeal H/ACA RNPs contain L7Ae, which binds specifically to an RNA K-loop motif absent from eukaryotic H/ACA RNPs, while Nhp2 binds a broader range of RNA structures. We report solution NMR studies of Saccharomyces cerevisiae Nhp2 (Nhp2p), which reveal that Nhp2p exhibits two major conformations in solution due to cis/trans isomerization of the evolutionarily conserved Pro83. The equivalent proline is in the cis conformation in all reported structures of L7Ae and other homologous proteins. Nhp2p has the expected α-β-α fold, but the solution structures of the major conformation of Nhp2p with trans Pro83 and of Nhp2p-S82W with cis Pro83 reveal that Pro83 cis/trans isomerization affects the positions of numerous residues at the Nop10 and RNA binding interface. An S82W substitution, which stabilizes the cis conformation, also stabilizes the association of Nhp2p with H/ACA snoRNPs expressed in vivo. We propose that Pro83 plays a key role in the assembly of the eukaryotic H/ACA RNP, with the cis conformation locking in a stable Cbf5-Nop10-Nhp2 ternary complex and positioning the protein backbone to interact with the H/ACA RNA.  相似文献   

8.
Telomerase is a ribonucleoprotein (RNP) particle required for the replication of telomeres. The RNA component, termed hTR, of human telomerase contains a domain structurally and functionally related to box H/ACA small nucleolar RNAs (snoRNAs). Furthermore, hTR is known to be associated with two core components of H/ACA snoRNPs, hGar1p and Dyskerin (the human counterpart of yeast Cbf5p). To assess the functional importance of the association of hTR with H/ACA snoRNP core proteins, we have attempted to express hTR in a genetically tractable system, Saccharomyces cerevisiae. Both mature non-polyadenylated and polyadenylated forms of hTR accumulate in yeast. The former is associated with all yeast H/ACA snoRNP core proteins, unlike TLC1 RNA, the endogenous RNA component of yeast telomerase. We show that the presence of the H/ACA snoRNP proteins Cbf5p, Nhp2p and Nop10p, but not Gar1p, is required for the accumulation of mature non-polyadenylated hTR in yeast, while accumulation of TLC1 RNA is not affected by the absence of any of these proteins. Our results demonstrate that yeast telomerase is unrelated to H/ACA snoRNPs. In addition, they show that the accumulation in yeast of the mature RNA component of human telomerase depends on its association with three of the four core H/ACA snoRNP proteins. It is likely that this is the case in human cells as well.  相似文献   

9.
Small nucleolar RNAs (designated as snoRNAs in Eukarya or sRNAs in Archaea) can be grouped into H/ACA or C/D box snoRNA (sRNA) subclasses. In Eukarya, H/ACA snoRNAs assemble into a ribonucleoprotein (RNP) complex comprising four proteins: Cbf5p, Gar1p, Nop10p and Nhp2p. A homolog for the Nhp2p protein has not been identified within archaeal H/ACA RNPs thus far, while potential orthologs have been identified for the other three proteins. Nhp2p is related, particularly in the middle portion of the protein sequence, to the archaeal ribosomal protein and C/D box protein L7Ae. This finding suggests that L7Ae may be able to substitute for the Nhp2p protein in archaeal H/ACA sRNAs. By band shift assays, we have analyzed in vitro the interaction between H/ACA box sRNAs and protein L7Ae from the archaeon Archaeoglobus fulgidus. We present evidence that L7Ae forms specific complexes with three different H/ACA sRNAs, designated as Afu-4, Afu-46 and Afu-190 with an apparent K(d) ranging from 28 to 100 nM. By chemical and enzymatic probing we show that distinct bases located within bulges or loops of H/ACA sRNAs interact with the L7Ae protein. These findings are corroborated by mutational analysis of the L7Ae binding site. Thereby, the RNA motif required for L7Ae binding exhibits a structure, designated as the K-turn, which is present in all C/D box sRNAs. We also identified four H/ACA RNAs from the archaeal species Pyrococcus which exhibit the K-turn motif at a similar position in their structure. These findings suggest a triple role for L7Ae protein in Archaea, e.g. in ribosomes as well as H/ACA and C/D box sRNP biogenesis and function by binding to the K-turn motif.  相似文献   

10.
The H/ACA small nucleolar RNAs (snoRNAs) are involved in pseudouridylation of pre-rRNAs. In the yeast Saccharomyces cerevisiae, four common proteins are associated with H/ACA snoRNAs: Gar1p, Cbf5p, Nhp2p, and Nop10p. In vitro reconstitution studies showed that four proteins also specifically interact with H/ACA snoRNAs in mammalian cell extracts. Two mammalian proteins, NAP57/dyskerin (the ortholog of Cbf5p) and hGAR1, have been characterized. In this work we describe properties of hNOP10 and hNHP2, human orthologs of yeast Nop10p and Nhp2p, respectively, and further characterize hGAR1. hNOP10 and hNHP2 complement yeast cells depleted of Nhp2p and Nop10p, respectively. Immunoprecipitation experiments with extracts from transfected HeLa cells indicated that epitope-tagged hNOP10 and hNHP2 specifically associate with hGAR1 and H/ACA RNAs; they also interact with the RNA subunit of telomerase, which contains an H/ACA-like domain in its 3' moiety. Immunofluorescence microscopy experiments showed that hGAR1, hNOP10, and hNHP2 are localized in the dense fibrillar component of the nucleolus and in Cajal (coiled) bodies. Deletion analysis of hGAR1 indicated that its evolutionarily conserved core domain contains all the signals required for localization, but progressive deletions from either the N or the C terminus of the core domain abolish localization in the nucleolus and/or the Cajal bodies.  相似文献   

11.
Yeast snR30 is an essential box H/ACA small nucleolar RNA (snoRNA) that promotes 18S rRNA processing through forming transient base-pairing interactions with the newly synthesized 35S pre-rRNA. By using a novel tandem RNA affinity selection approach, followed by coimmunoprecipitation and in vivo cross-linking experiments, we demonstrate that in addition to the four H/ACA core proteins, Cbf5p, Nhp2p, Nop10p and Gar1p, a fraction of snR30 specifically associates with the Utp23p and Kri1p nucleolar proteins. Depletion of Utp23p and Kri1p has no effect on the accumulation and recruitment of snR30 to the nascent pre-ribosomes. However, in the absence of Utp23p, the majority of snR30 accumulates in large pre-ribosomal particles. The retained snR30 is not base-paired with the 35S pre-rRNA, indicating that its aberrant tethering to nascent preribosomes is likely mediated by pre-ribosomal protein(s). Thus, Utp23p may promote conformational changes of the pre-ribosome, essential for snR30 release. Neither Utp23p nor Kri1p is required for recruitment of snR30 to the nascent pre-ribosome. On the contrary, depletion of snR30 prevents proper incorporation of both Utp23p and Kri1p into the 90S pre-ribosome containing the 35S pre-rRNA, indicating that snR30 plays a central role in the assembly of functionally active small subunit processome.  相似文献   

12.
Reichow SL  Varani G 《Biochemistry》2008,47(23):6148-6156
The H/ACA class of small nucleolar ribonucleoproteins (snoRNPs) is primarily responsible for catalyzing the isomerization of uridine to pseudouridine (Psi) in ribosomal and other cellular RNAs. Each H/ACA snoRNP consist of four conserved proteins, Cbf5 (the Psi-synthase), Gar1, Nhp2 (L7Ae in archaea) and Nop10, that assemble onto a unique RNA component (the snoRNA). The smallest of these proteins, Nop10 ( approximately 7 kDa), has an essential role in the assembly and activity of these particles and binds directly to the Psi-synthase to form the minimal active enzyme in archaea. To better understand the conserved function of this protein, we characterized the NMR structure and dynamics of Nop10 proteins from both archaea and yeast. We show that archaeal Nop10 contains a highly stable Zn2+ binding motif that is replaced in eukaryotes by a smaller meta-stable beta-hairpin, while a highly conserved and conformationally dynamic linker connects these motifs to a nascent alpha-helical structure. Our structural analysis and NMR relaxation data show that these motifs do not interact with each other and tumble independently in solution. Several residues within the archaeal Nop10 Zn2+ binding motif have clear structural and functional roles and are conserved in eukaryotes, yet remain disordered in the free yeast Nop10. We propose that the dynamic structure of Nop10 facilitates an induced-fit recognition with the H/ACA Psi-synthase and allows it to act as a molecular adaptor for guiding snoRNP assembly in similar fashion in all archaea and eukaryotic organisms.  相似文献   

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Small nucleolar ribonucleoprotein particles (snoRNPs) are essential cofactors in ribosomal RNA metabolism. Although snoRNP composition has been thoroughly characterized, the biogenesis process of these particles is poorly understood. We have identified two proteins from the yeast Saccharomyces cerevisiae, Yil104c/Shq1p and Ynl124w/Naf1p, which are essential and required for the stability of box H/ACA snoRNPs. Depletion of either Shq1p or Naf1p leads to a dramatic and specific decrease in box H/ACA snoRNA levels in vivo. A severe concomitant defect in ribosomal RNA processing is observed, consistent with the depletion of this family of snoRNAs. Shq1p and Naf1p show nuclear localization and interact with Nhp2p and Cbf5p, two core proteins of mature box H/ACA snoRNPs. Shq1p and Naf1p form a complex, but they are not strongly associated with box H/ACA snoRNPs. We propose that Shq1p and Naf1p are involved in the early biogenesis steps of box H/ACA snoRNP assembly.  相似文献   

16.
H/ACA small nucleolar RNPs (snoRNPs) that guide pseudouridylation reactions are comprised of one small nucleolar RNA (snoRNA) and four common proteins (Cbf5, Gar1, Nhp2 and Nop10). Unlike other H/ACA snoRNPs, snR30 is essential for the early processing reactions that lead to the production of 18S ribosomal RNA in the yeast Saccharomyces cerevisiae. To determine whether snR30 RNP contains specific proteins that contribute to its unique functional properties, we devised an affinity purification strategy using TAP-tagged Gar1 and an RNA aptamer inserted in snR30 snoRNA to selectively purify the RNP. Northern blotting and pCp labeling experiments showed that S1-tagged snR30 snoRNA can be selectively purified with streptavidin beads. Protein analysis revealed that aptamer-tagged snR30 RNA was associated with the four H/ACA proteins and a number of additional proteins: Nop6, ribosomal proteins S9 and S18 and histones H2B and H4. Using antibodies raised against Nop6 we show that endogenous Nop6 localizes to the nucleolus and that it cosediments with snR30 snoRNA in sucrose density gradients. We demonstrate through primer extension experiments that snR30 snoRNA is required for cleavages at site A0, A1 and A2, and that the absence of Nop6 decreases the efficiency of cleavage at site A2. Finally, electron microscopy analyses of chromatin spreads from cells depleted of snR30 snoRNA show that it is required for SSU processome assembly.  相似文献   

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Box H/ACA small nucleolar ribonucleoprotein particles (H/ACA snoRNPs) play key roles in the synthesis of eukaryotic ribosomes. How box H/ACA snoRNPs are assembled remains unknown. Here we show that yeast Nhp2p, a core component of these particles, directly binds RNA. In vitro, Nhp2p interacts with high affinity with RNAs containing irregular stem–loop structures but shows weak affinity for poly(A), poly(C) or for double-stranded RNAs. The central region of Nhp2p is believed to function as an RNA-binding domain, since it is related to motifs found in various RNA-binding proteins. Removal of two amino acids that shortens a putative β-strand element within Nhp2p central domain impairs the ability of the protein to interact with H/ACA snoRNAs in cell extracts. In vivo, this deletion prevents cell viability and leads to a strong defect in the accumulation of H/ACA snoRNAs and Gar1p. These data suggest that proper direct binding of Nhp2p to H/ACA snoRNAs is required for the assembly of H/ACA snoRNPs and hence for the stability of some of their components. In addition, we show that converting a highly conserved glycine residue (G59) within Nhp2p central domain to glutamate significantly reduces cell growth at 30 and 37°C. Remarkably, this modification affects the steady-state levels of H/ACA snoRNAs and the strength of Nhp2p association with these RNAs to varying degrees, depending on the nature of the H/ACA snoRNA. Finally, we show that the modified Nhp2p protein whose interaction with H/ACA snoRNAs is impaired cannot accumulate in the nucleolus, suggesting that only the assembled H/ACA snoRNP particles can be efficiently retained in the nucleolus.  相似文献   

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