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 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.     

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.     

Unveiling substrate RNA binding to H/ACA RNPs: one side fits all

The H/ACA RNP pseudouridylases function on a large number of extraordinarily complex RNA substrates including pre-ribosomal and small nuclear RNAs. Recent structural data show that H/ACA RNPs capture their RNA substrates via a simple one-sided attachment model. However, the precise placement of each RNA substrate into the active site of the catalytic subunit relies on the essential functions of the RNP proteins. The specific roles of each H/ACA RNP protein are being elucidated by a combination of structural and biochemical studies.     

Pseudouridine-guide RNAs and other Cbf5p-associated RNAs in Euglena gracilis

In eukaryotes, box H/ACA small nucleolar RNAs (snoRNAs) guide sites of pseudouridine (Psi) formation in rRNA. These snoRNAs reside in RNP complexes containing the putative Psi synthase, Cbf5p. In this study we have identified Cbf5p-associated RNAs in Euglena gracilis, an early diverging eukaryote, by immunoprecipitating Cbf5p-containing complexes from cellular extracts. We characterized one box H/ACA-like RNA which, however, does not appear to guide Psi formation in rRNA. We also identified four single Psi-guide box AGA RNAs. We determined target sites for these putative Psi-guide RNAs and confirmed that the predicted Psi modifications do, in fact, occur at these positions in Euglena rRNA. The Cbf5p-associated snoRNAs appear to be encoded by multicopy genes, some of which are clustered in the genome together with methylation-guide snoRNA genes. These modification-guide snoRNAs and snoRNA genes are the first ones to be reported in euglenid protists, the evolutionary sister group to the kinetoplastid protozoa. Unexpectedly, we also found and have partially characterized a selenocysteine tRNA homolog in the anti-Cbf5p-immunoprecipitated sample.     

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.     

A computational screen for mammalian pseudouridylation guide H/ACA RNAs

The box H/ACA RNA gene family is one of the largest non-protein-coding gene families in eukaryotes and archaea. Recently, we developed snoGPS, a computational screening program for H/ACA snoRNAs, and applied it to Saccharomyces cerevisiae. We report here results of extending our method to screen for H/ACA RNAs in multiple large genomes of related species, and apply it to the human, mouse, and rat genomes. Because of the 250-fold larger search space compared to S. cerevisiae, significant enhancements to our algorithms were required. Complementing extensive cloning experiments performed by others, our findings include the detection and experimental verification of seven new mammalian H/ACA RNAs and the prediction of 23 new H/ACA RNA pseudouridine guide assignments. These assignments include four for H/ACA RNAs previously classified as orphan H/ACA RNAs with no known targets. We also determined systematic syntenic conservation among human and mouse H/ACA RNAs. With this work, 82 of 97 ribosomal RNA pseudouridines and 18 of 32 spliceosomal RNA pseudouridines in mammals have been linked to H/ACA guide RNAs.     

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.     

An enhanced H/ACA RNP assembly mechanism for human telomerase RNA

The integral telomerase RNA subunit templates the synthesis of telomeric repeats. The biological accumulation of human telomerase RNA (hTR) requires hTR H/ACA domain assembly with the same proteins that assemble on other human H/ACA RNAs. Despite this shared RNP composition, hTR accumulation is particularly sensitized to disruption by disease-linked H/ACA protein variants. We show that contrary to expectation, hTR-specific sequence requirements for biological accumulation do not act at an hTR-specific step of H/ACA RNP biogenesis; instead, they enhance hTR binding to the shared, chaperone-bound scaffold of H/ACA core proteins that mediates initial RNP assembly. We recapitulate physiological H/ACA RNP assembly with a preassembled NAF1/dyskerin/NOP10/NHP2 scaffold purified from cell extract and demonstrate that distributed sequence features of the hTR 3' hairpin synergize to improve scaffold binding. Our findings reveal that the hTR H/ACA domain is distinguished from other human H/ACA RNAs not by a distinct set of RNA-protein interactions but by an increased efficiency of RNP assembly. Our findings suggest a unifying mechanism for human telomerase deficiencies associated with H/ACA protein variants.     

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.     

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.