Eukaryote specific RNA and protein features facilitate assembly and catalysis of H/ACA snoRNPs

H/ACA Box ribonucleoprotein complexes (RNPs) play a major role in modification of rRNA and snRNA, catalyzing the sequence specific pseudouridylation in eukaryotes and archaea. This enzymatic reaction takes place on a substrate RNA recruited via base pairing to an internal loop of the snoRNA. Eukaryotic snoRNPs contain the four proteins Nop10, Cbf5, Gar1 and Nhp2, with Cbf5 as the catalytic subunit. In contrast to archaeal H/ACA RNPs, eukaryotic snoRNPs contain several conserved features in both the snoRNA as well as the protein components. Here, we reconstituted the eukaryotic H/ACA RNP containing snR81 as a guide RNA in vitro and report on the effects of these eukaryote specific features on complex assembly and enzymatic activity. We compare their contribution to pseudouridylation activity for stand-alone hairpins versus the bipartite RNP. Using single molecule FRET spectroscopy, we investigated the role of the different eukaryote-specific proteins and domains on RNA folding and complex assembly, and assessed binding of substrate RNA to the RNP. Interestingly, we found diverging effects for the two hairpins of snR81, suggesting hairpin-specific requirements for folding and RNP formation. Our results for the first time allow assessing interactions between the individual hairpin RNPs in the context of the full, bipartite snoRNP.     

Utp23p is required for dissociation of snR30 small nucleolar RNP from preribosomal particles

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.     

The vertebrate E1/U17 small nucleolar ribonucleoprotein particle

Each of the many different box H/ACA ribonucleoprotein particles (RNPs) present in eukaryotes and archaea consists of four common core proteins and one specific H/ACA small RNA, which bears the sequence elements H (ANANNA) and ACA. Most of the H/ACA RNPs are small nucleolar RNPs (snoRNPs), which are localized in nucleoli, and are one of the two major classes of snoRNPs. Most H/ACA RNPs direct pseudouridine synthesis in pre-rRNA and other RNAs. One H/ACA small nucleolar RNA (snoRNA), vertebrate E1/U17 (snR30 in yeast), is required for pre-rRNA cleavage processing that generates mature 18S rRNA. E1 snoRNA is encoded in introns of protein-coding genes, and the evidence suggests that human E1 RNA undergoes uridine insertional RNA editing. The vertebrate E1 RNA consensus secondary structure shows several features that are absent in other box H/ACA snoRNAs. The available UV-induced RNA-protein crosslinking results suggest that the E1 snoRNP is asymmetrical in vertebrate cells, in contrast to other H/ACA snoRNPs. The vertebrate E1 snoRNP in cells is surprisingly complex: (i) E1 RNA contacts directly and specifically several proteins which do not appear to be any of the H/ACA RNP four core proteins; and (ii) multiple E1 RNA sites are needed for E1 snoRNP formation, E1 RNA stability, and E1 RNA-protein direct interactions.     

Structural study of the H/ACA snoRNP components Nop10p and the 3' hairpin of U65 snoRNA

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.     

Cbf5p, the putative pseudouridine synthase of H/ACA-type snoRNPs, can form a complex with Gar1p and Nop10p in absence of Nhp2p and box H/ACA snoRNAs

Box C/D and box H/ACA small ribonucleoprotein particles (sRNPs) are found from archaea to humans, and some of these play key roles during the biogenesis of ribosomes or components of the splicing apparatus. The protein composition of the core of both types of particles is well established and the assembly pathway of box C/D sRNPs has been extensively investigated both in archaeal and eukaryotic systems. In contrast, knowledge concerning the mode of assembly and final structure of box H/ACA sRNPs is much more limited. In the present study, we have investigated the protein/protein interactions taking place between the four protein components of yeast box H/ACA small nucleolar RNPs (snoRNPs), Cbf5p, Gar1p, Nhp2p, and Nop10p. We provide evidence that Cbf5p, Gar1p, and Nop10p can form a complex devoid of Nhp2p and small nucleolar RNA (snoRNA) components of the particles and that Cbf5p and Nop10p can directly bind to each other. We also show that the absence of any component necessary for assembly of box H/ACA snoRNPs inhibits accumulation of Cbf5p, Gar1p, or Nop10p, whereas Nhp2p levels are little affected.     

Nhp2p and Nop10p are essential for the function of H/ACA snoRNPs.

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.     

Cbf5p,a potential pseudouridine synthase,and Nhp2p,a putative RNA-binding protein,are present together with Gar1p in all H BOX/ACA-motif snoRNPs and constitute a common bipartite structure.

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.     

Nop10 is a conserved H/ACA snoRNP molecular adaptor

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.     

Stable expression in yeast of the mature form of human telomerase RNA depends on its association with the box H/ACA small nucleolar RNP proteins Cbf5p, Nhp2p and Nop10p

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.     

The Shq1p.Naf1p complex is required for box H/ACA small nucleolar ribonucleoprotein particle biogenesis

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.     

Structural and functional evidence of high specificity of Cbf5 for ACA trinucleotide

Cbf5 is the catalytic subunit of the H/ACA small nucleolar/Cajal body ribonucleoprotein particles (RNPs) responsible for site specific isomerization of uridine in ribosomal and small nuclear RNA. Recent evidence from studies on archaeal Cbf5 suggests its second functional role in modifying tRNA U55 independent of guide RNA. In order to act both as a stand-alone and a RNP pseudouridine synthase, Cbf5 must differentiate features in H/ACA RNA from those in tRNA or rRNA. Most H/ACA RNAs contain a hallmark ACA trinucleotide downstream of the H/ACA motif. Here we challenged an archaeal Cbf5 (in the form of a ternary complex with its accessory proteins Nop10 and Gar1) with T-stem-loop RNAs with or without ACA trinucleotide in the stem. Although these substrates were previously shown to be substrates for the bacterial stand-alone pseudouridine synthase TruB, the Cbf5-Nop10-Gar1 complex was only able to modify those without ACA trinucleotide. A crystal structure of Cbf5-Nop10-Gar1 trimer bound with an ACA-containing T-stem-loop revealed that the ACA trinucleotide detracted Cbf5 from the stand-alone binding mode, thereby suggesting that the H/ACA RNP-associated function of Cbf5 likely supersedes its stand-alone function.     

The helicase Has1p is required for snoRNA release from pre-rRNA

Synthesis of rRNA in eukaryotes involves the action of a large population of snoRNA-protein complexes (snoRNPs), which create modified nucleotides and participate in cleavage of pre-rRNA. The snoRNPs mediate these functions through direct base pairing, in many cases through long complementary sequences. This feature suggests that RNA helicases may be involved in the binding and release of snoRNPs from pre-rRNA. In this study, we determined that the DEAD box helicase Has1p, a nucleolar protein required for the production of 18S rRNA, copurifies with the snR30/U17 processing snoRNP but is also present with other snoRNPs. Blocking Has1p expression causes a substantial increase in snoRNPs associated with 60S-90S preribosomal RNP complexes, including the U3 and U14 processing snoRNPs and several modifying snoRNPs examined. Cosedimentation persisted even after deproteinization. This effect was not observed with depletion of two nonhelicase proteins, Esf1p and Dim2p, that are also required for 18S rRNA production. Point mutations in ATPase and helicase motifs of Has1p block U14 release from pre-rRNA. Surprisingly, depletion of Has1p causes a reduction in the level of free U6 snRNP. The results indicate that the Has1p helicase is required for snoRNA release from pre-rRNA and production of the U6 snRNP.     

U17/snR30 is a ubiquitous snoRNA with two conserved sequence motifs essential for 18S rRNA production

Saccharomyces cerevisiae snR30 is an essential box H/ACA small nucleolar RNA (snoRNA) required for the processing of 18S rRNA. Here, we show that the previously characterized human, reptilian, amphibian, and fish U17 snoRNAs represent the vertebrate homologues of yeast snR30. We also demonstrate that U17/snR30 is present in the fission yeast Schizosaccharomyces pombe and the unicellular ciliated protozoan Tetrahymena thermophila. Evolutionary comparison revealed that the 3'-terminal hairpins of U17/snR30 snoRNAs contain two highly conserved sequence motifs, the m1 (AUAUUCCUA) and m2 (AAACCAU) elements. Mutation analysis of yeast snR30 demonstrated that the m1 and m2 elements are essential for early cleavages of the 35S pre-rRNA and, consequently, for the production of mature 18S rRNA. The m1 and m2 motifs occupy the opposite strands of an internal loop structure, and they are located invariantly 7 nucleotides upstream from the ACA box of U17/snR30 snoRNAs. U17/snR30 is the first identified box H/ACA snoRNA that possesses an evolutionarily conserved role in the nucleolytic processing of eukaryotic pre-rRNA.     

Rrp8p is a yeast nucleolar protein functionally linked to Gar1p and involved in pre-rRNA cleavage at site A2

Chemical modifications and processing of the 18S, 5.8S, and 25S ribosomal RNAs from the 35S pre-ribosomal RNA depend on an important set of small nucleolar ribonucleoprotein particles (snoRNPs). Genetic depletion of yeast Gar1p, an essential common component of H/ACA snoRNPs, leads to inhibition of uridine isomerizations to pseudo-uridines on the 35S pre-rRNA and of the early pre-rRNA cleavages at sites A1 and A2, resulting in a loss of mature 18S rRNA synthesis. To identify Gar1p functional partners, we screened for mutations that are synthetically lethal with a gar1 mutant allele encoding a Gar1p mutant protein lacking its two glycine/arginine-rich (GAR) domains. We identified a previously uncharacterized Saccharomyces cerevisiae open reading frame, YDR083W (now designated RRP8), that encodes a highly conserved protein containing motifs found in methyltransferases. Rrp8p localizes to the nucleolus. A yeast strain lacking this protein is viable at 30 degrees C but displays strong growth impairment at lower temperatures. In this strain, cleavage of the pre-rRNA at site A2 is strongly affected whereas cleavages at sites A0 and A1 are only slightly inhibited or delayed.     

Inactivation of cleavage factor I components Rna14p and Rna15p induces sequestration of small nucleolar ribonucleoproteins at discrete sites in the nucleus

Small nucleolar RNAs (snoRNAs) associate with specific proteins forming small nucleolar ribonucleoprotein (snoRNP) particles, which are essential for ribosome biogenesis. The snoRNAs are transcribed, processed, and assembled in snoRNPs in the nucleoplasm. Mature particles are then transported to the nucleolus. In yeast, 3'-end maturation of snoRNAs involves the activity of Rnt1p endonuclease and cleavage factor IA (CFIA). We report that after inhibition of CFIA components Rna14p and Rna15p, the snoRNP proteins Nop1p, Nop58p, and Gar1p delocalize from the nucleolus and accumulate in discrete nucleoplasmic foci. The U14 snoRNA, but not U3 snoRNA, similarly redistributes from the nucleolus to the nucleoplasmic foci. Simultaneous depletion of either Rna14p or Rna15p and the nuclear exosome component Rrp6p induces accumulation of poly(A)(+) RNA at the snoRNP-containing foci. We propose that the foci detected after CFIA inactivation correspond to quality control centers in the nucleoplasm.     

Structure of the Shq1-Cbf5-Nop10-Gar1 complex and implications for H/ACA RNP biogenesis and dyskeratosis congenita

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.     

Binding of L7Ae protein to the K-turn of archaeal snoRNAs: a shared RNA binding motif for C/D and H/ACA box snoRNAs in Archaea

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.