Crystal structure of a Cbf5-Nop10-Gar1 complex and implications in RNA-guided pseudouridylation and dyskeratosis congenita

H/ACA RNA-protein complexes, comprised of four proteins and an H/ACA guide RNA, modify ribosomal and small nuclear RNAs. The H/ACA proteins are also essential components of telomerase in mammals. Cbf5 is the H/ACA protein that catalyzes isomerization of uridine to pseudouridine in target RNAs. Mutations in human Cbf5 (dyskerin) lead to dyskeratosis congenita. Here, we describe the 2.1 A crystal structure of a specific complex of three archaeal H/ACA proteins, Cbf5, Nop10, and Gar1. Cbf5 displays structural properties that are unique among known pseudouridine synthases and are consistent with its distinct function in RNA-guided pseudouridylation. We also describe the previously unknown structures of both Nop10 and Gar1 and the structural basis for their essential roles in pseudouridylation. By using information from related structures, we have modeled the entire ribonucleoprotein complex including both guide and substrate RNAs. We have also identified a dyskeratosis congenita mutation cluster site within a modeled dyskerin structure.     

H/ACA guide RNAs, proteins and complexes

H/ACA guide RNAs direct site-specific pseudouridylation of substrate RNAs by forming ribonucleoprotein (RNP) complexes with pseudouridine synthase Cbf5 and three accessory proteins. Recently determined crystal structures of H/ACA protein complexes and a fully assembled H/ACA RNP complex have provided significant insights into the architecture, assembly and mechanism of action of RNA-guided pseudouridine synthase. The binding of guide RNA is directed by its conserved secondary structure and sequence motifs, which enables guide RNA with different sequences to be incorporated into the same protein complex. Accessory proteins and peripheral domains crucially coordinate the position of guide RNA, and possibly regulate the reaction process.     

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.     

Elucidating the role of H/ACA-like RNAs in trans-splicing and rRNA processing via RNA interference silencing of the Trypanosoma brucei CBF5 pseudouridine synthase

Most pseudouridinylation in eukaryotic rRNA and small nuclear RNAs is guided by H/ACA small nucleolar RNAs. In this study, the Trypanosoma brucei pseudouridine synthase, Cbf5p, a snoRNP protein, was identified and silenced by RNAi. Depletion of this protein destabilized all small nucleolar RNAs of the H/ACA-like family. Following silencing, defects in rRNA processing, such as accumulation of precursors and inhibition of cleavages to generate the mature rRNA, were observed. snR30, an H/ACA RNA involved in rRNA maturation, was identified based on prototypical conserved domains characteristic of this RNA in other eukaryotes. The silencing of CBF5 also eliminated the spliced leader-associated (SLA1) RNA that directs pseudouridylation on the spliced leader RNA (SL RNA), which is the substrate for the trans-splicing reaction. Surprisingly, the depletion of Cbf5p not only eliminated the pseudouridine on the SL RNA but also abolished capping at the fourth cap-4 nucleotide. As a result of defects in the SL RNA and decreased modification on the U small nuclear RNA, trans-splicing was inhibited at the first step of the reaction, providing evidence for the essential role of H/ACA RNAs and the modifications they guide on trans-splicing.     

Determination of protein-RNA interaction sites in the Cbf5-H/ACA guide RNA complex by mass spectrometric protein footprinting

Pseudouridylation is one of the most common forms of RNA modification. In eukaryotes and archaea, these modifications are carried out by H/ACA ribonucleoprotein (RNP) complexes, composed of an H/ACA guide RNA and four proteins, including the pseudouridine synthase, Cbf5. Remarkable progress has been made toward understanding the structure and function of H/ACA RNPs, both through mapping of RNA-protein and protein-protein interactions and the availability of X-ray structures, including that of the entire RNP. The pseudouridine synthase, Cbf5, is also the protein that specifically recognizes the guide RNAs. In this work, we have investigated the molecular basis of this key interaction. A mass spectrometric protein footprinting approach was employed to determine the amino acids of archaeal Cbf5 involved in interaction with the guide RNA. We found amino acid protections along the same RNA binding track observed in the crystal structure of the fully assembled complex, indicating that this interaction is established in the subcomplex. However, in addition, we observed a set of protections in the D2 subdomain of Cbf5 that appear to represent a unique, additional interaction of the guide RNA with the protein in the subcomplex. On the basis of these results, we present a model for the Cbf5-guide RNA complex that also incorporates other recent findings. Our analysis suggests that the assembly or function of H/ACA RNPs may be accompanied by dynamic changes in RNA-protein interactions.     

Dynamic interactions within sub-complexes of the H/ACA pseudouridylation guide RNP

H/ACA RNP complexes change uridines to pseudouridines in target non-coding RNAs in eukaryotes and archaea. H/ACA RNPs are comprised of a guide RNA and four essential proteins: Cbf5 (pseudouridine synthase), L7Ae, Gar1 and Nop10 in archaea. The guide RNA captures the target RNA via two antisense elements brought together to form a contiguous binding site within the pseudouridylation pocket (internal loop) of the guide RNA. Cbf5 and L7Ae interact independently with the guide RNA, and here we have examined the impacts of these proteins on the RNA in nucleotide protection assays. The results indicate that the interactions observed in a fully assembled H/ACA RNP are established in the sub-complexes, but also reveal a unique Cbf5–guide RNA interaction that is displaced by L7Ae. In addition, the results indicate that L7Ae binding at the kink (k)-turn of the guide RNA induces the formation of the upper stem, and thus also the pseudouridylation pocket. Our findings indicate that L7Ae is essential for formation of the substrate RNA binding site in the archaeal H/ACA RNP, and suggest that k-turn-binding proteins may remodel partner RNAs with important effects distant from the protein-binding site.     

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.     

Revisiting the structure/function relationships of H/ACA(-like) RNAs: a unified model for Euryarchaea and Crenarchaea

A structural and functional classification of H/ACA and H/ACA-like motifs is obtained from the analysis of the H/ACA guide RNAs which have been identified previously in the genomes of Euryarchaea (Pyrococcus) and Crenarchaea (Pyrobaculum). A unified structure/function model is proposed based on the common structural determinants shared by H/ACA and H/ACA-like motifs in both Euryarchaea and Crenarchaea. Using a computational approach, structural and energetic rules for the guide:target RNA-RNA interactions are derived from structural and functional data on the H/ACA RNP particles. H/ACA(-like) motifs found in Pyrococcus are evaluated through the classification and their biological relevance is discussed. Extra-ribosomal targets found in both Pyrococcus and Pyrobaculum might support the hypothesis of a gene regulation mediated by H/ACA(-like) guide RNAs in archaea.     

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.     

RNomics: an experimental approach that identifies 201 candidates for novel, small, non-messenger RNAs in mouse

In mouse brain cDNA libraries generated from small RNA molecules we have identified a total of 201 different expressed RNA sequences potentially encoding novel small non-messenger RNA species (snmRNAs). Based on sequence and structural motifs, 113 of these RNAs can be assigned to the C/D box or H/ACA box subclass of small nucleolar RNAs (snoRNAs), known as guide RNAs for rRNA. While 30 RNAs represent mouse homologues of previously identified human C/D or H/ACA snoRNAs, 83 correspond to entirely novel snoRNAS: Among these, for the first time, we identified four C/D box snoRNAs and four H/ACA box snoRNAs predicted to direct modifications within U2, U4 or U6 small nuclear RNAs (snRNAs). Furthermore, 25 snoRNAs from either class lacked antisense elements for rRNAs or snRNAS: Therefore, additional snoRNA targets have to be considered. Surprisingly, six C/D box snoRNAs and one H/ACA box snoRNA were expressed exclusively in brain. Of the 88 RNAs not belonging to either snoRNA subclass, at least 26 are probably derived from truncated heterogeneous nuclear RNAs (hnRNAs) or mRNAS: Short interspersed repetitive elements (SINEs) are located on five RNA sequences and may represent rare examples of transcribed SINES: The remaining RNA species could not as yet be assigned either to any snmRNA class or to a part of a larger hnRNA/mRNA. It is likely that at least some of the latter will represent novel, unclassified snmRNAS:     

Inducing nonsense suppression by targeted pseudouridylation

Isomerization from uridine to pseudouridine (pseudouridylation) is largely catalyzed by a family of small ribonucleoproteins called box H/ACA RNPs, each of which contains one unique small RNA-the box H/ACA RNA. The specificity of the pseudouridylation reaction is determined by the base-pairing interactions between the guide sequence of the box H/ACA RNA and the target sequence within an RNA substrate. Thus, by creating a new box H/ACA RNA harboring an artificial guide sequence that base-pairs with the substrate sequence, one can site-specifically introduce pseudouridines into virtually any RNA (e.g., mRNA, ribosomal RNA, small nuclear RNA, telomerase RNA and so on). Pseudouridylation changes the properties of a uridine residue and is likely to alter the role of its corresponding RNA in certain cellular processes, thereby enabling basic research into the effects of RNA modifications. Here we take a TRM4 reporter gene (also known as NCL1) as an example, and we present a protocol for designing a box H/ACA RNA to site-specifically pseudouridylate TRM4 mRNA. Disease-related mutation can result in early termination of translation by creating a premature termination codon (PTC); however, pseudouridylation at the PTC can suppress this translation termination (nonsense suppression). Thus, the experimental procedures described in this protocol may provide a novel way to treat PTC-related diseases. This protocol takes 10-13 d to complete.     

A search for H/ACA snoRNAs in yeast using MFE secondary structure prediction

MOTIVATION: Noncoding RNA genes produce functional RNA molecules rather than coding for proteins. One such family is the H/ACA snoRNAs. Unlike the related C/D snoRNAs these have resisted automated detection to date. RESULTS: We develop an algorithm to screen the yeast genome for novel H/ACA snoRNAs. To achieve this, we introduce some new methods for facilitating the search for noncoding RNAs in genomic sequences which are based on properties of predicted minimum free-energy (MFE) secondary structures. The algorithm has been implemented and can be generalized to enable screening of other eukaryote genomes. We find that use of primary sequence alone is insufficient for identifying novel H/ACA snoRNAs. Only the use of secondary structure filters reduces the number of candidates to a manageable size. From genomic context, we identify three strong H/ACA snoRNA candidates. These together with a further 47 candidates obtained by our analysis are being experimentally screened.