An H/ACA guide RNA directs U2 pseudouridylation at two different sites in the branchpoint recognition region in Xenopus oocytes

U2 is the most extensively modified of all spliceosomal snRNAs. We previously showed that at least some of the internally modified nucleotides in U2 snRNA are required for snRNP biogenesis and pre-mRNA splicing. Recent work from several laboratories suggests that nuclear guide RNAs facilitate U2 snRNA internal modification, including pseudouridylation and 2'-O-methylation. Here, we present a novel approach to identifying guide RNAs for U2 pseudouridylation. Several Xenopus oocyte nuclear RNAs were affinity selected with U2 snRNA substituted with 5-fluorouridine, a pseudouridylation inhibitor that sequesters pseudouridylases. One of these RNAs was sequenced and found to be a novel RNA of 134 nt. This small RNA contains an H/ACA motif and folds into a typical H/ACA RNA structure, and its authenticity as an H/ACA RNA was confirmed by immunoprecipitation analysis. The RNA contains two guide sequences for pseudouridylation (psi) of U2 snRNA at positions 34 and 44 in the branch-site recognition region, and we demonstrate that this RNA indeed guides the formation of psi34 and psi44 in U2 using a Xenopus oocyte reconstitution system. Therefore, this novel RNA was designated pugU2-34/44, for pseudouridylation guide for U2 snRNA U34 and U44. Intranuclear localization analyses indicate that pugU2-34/44 resides within the nucleoplasm rather than nucleoli or Cajal bodies where other guide RNAs have been localized. Our results clarify the mechanism of U2 snRNA pseudouridylation in Xenopus oocytes, and have interesting implications with regard to the intranuclear localization of U2 snRNA pseudouridylation.     

Role of the box C/D motif in localization of small nucleolar RNAs to coiled bodies and nucleoli.

Small nucleolar RNAs (snoRNAs) are a large family of eukaryotic RNAs that function within the nucleolus in the biogenesis of ribosomes. One major class of snoRNAs is the box C/D snoRNAs named for their conserved box C and box D sequence elements. We have investigated the involvement of cis-acting sequences and intranuclear structures in the localization of box C/D snoRNAs to the nucleolus by assaying the intranuclear distribution of fluorescently labeled U3, U8, and U14 snoRNAs injected into Xenopus oocyte nuclei. Analysis of an extensive panel of U3 RNA variants showed that the box C/D motif, comprised of box C', box D, and the 3' terminal stem of U3, is necessary and sufficient for the nucleolar localization of U3 snoRNA. Disruption of the elements of the box C/D motif of U8 and U14 snoRNAs also prevented nucleolar localization, indicating that all box C/D snoRNAs use a common nucleolar-targeting mechanism. Finally, we found that wild-type box C/D snoRNAs transiently associate with coiled bodies before they localize to nucleoli and that variant RNAs that lack an intact box C/D motif are detained within coiled bodies. These results suggest that coiled bodies play a role in the biogenesis and/or intranuclear transport of box C/D snoRNAs.     

Nuclear retention elements of U3 small nucleolar RNA

The processing and methylation of precursor rRNA is mediated by the box C/D small nucleolar RNAs (snoRNAs). These snoRNAs differ from most cellular RNAs in that they are not exported to the cytoplasm. Instead, these RNAs are actively retained in the nucleus where they assemble with proteins into mature small nucleolar ribonucleoprotein particles and are targeted to their intranuclear site of action, the nucleolus. In this study, we have identified the cis-acting sequences responsible for the nuclear retention of U3 box C/D snoRNA by analyzing the nucleocytoplasmic distributions of an extensive panel of U3 RNA variants after injection of the RNAs into Xenopus oocyte nuclei. Our data indicate the importance of two conserved sequence motifs in retaining U3 RNA in the nucleus. The first motif is comprised of the conserved box C' and box D sequences that characterize the box C/D family. The second motif contains conserved box sequences B and C. Either motif is sufficient for nuclear retention, but disruption of both motifs leads to mislocalization of the RNAs to the cytoplasm. Variant RNAs that are not retained also lack 5' cap hypermethylation and fail to associate with fibrillarin. Furthermore, our results indicate that nuclear retention of U3 RNA does not simply reflect its nucleolar localization. A fragment of U3 containing the box B/C motif is not localized to nucleoli but retained in coiled bodies. Thus, nuclear retention and nucleolar localization are distinct processes with differing sequence requirements.     

Nucleolar localization signals of box H/ACA small nucleolar RNAs.

The two major families of small nucleolar RNAs (snoRNAs), Box C/D and Box H/ACA, are generated in the nucleoplasm and transported to the nucleolus where they function in rRNA processing and modification. We have investigated the sequences involved in the intranuclear transport of Box H/ACA snoRNAs by assaying the localization of injected fluorescent RNAs in Xenopus oocyte nuclear spreads. Our analysis of U17, U64 and U65 has revealed that disruption of either of the conserved sequence elements, Box H or Box ACA, eliminates nucleolar localization. In addition, the stem present at the base of the 3' hairpin is required for efficient nucleolar localization of U65. Fragments or rearrangements of U65 that consist of Box H and Box ACA flanking either the 5' or 3' hairpin are targeted to the nucleolus. The targeting is dependent on the presence of the Box sequences, but not on their orientation. Our results indicate that in each of the two major families of snoRNAs, a motif composed of the signature conserved sequences and an adjacent structural element that tethers the sequence elements directs the nucleolar localization of the RNAs. We demonstrate that telomerase RNA is also targeted to the nucleolus by a Box ACA-dependent mechanism.     

The many facets of H/ACA ribonucleoproteins

The H/ACA ribonucleoproteins (RNPs) are known as one of the two major classes of small nucleolar RNPs. They predominantly guide the site-directed pseudouridylation of target RNAs, such as ribosomal and spliceosomal small nuclear RNAs. In addition, they process ribosomal RNA and stabilize vertebrate telomerase RNA. Taken together, the function of H/ACA RNPs is essential for ribosome biogenesis, pre-mRNA splicing, and telomere maintenance. Every cell contains 100–200 different species of H/ACA RNPs, each consisting of the same four core proteins and one function-specifying H/ACA RNA. Most of these RNPs reside in nucleoli and Cajal bodies and mediate the isomerization of specific uridines to pseudouridines. Catalysis of the reaction is mediated by the putative pseudouridylase NAP57 (dyskerin, Cbf5p). Unexpectedly, mutations in this housekeeping enzyme are the major determinants of the inherited bone marrow failure syndrome dyskeratosis congenita. This review details the many diverse functions of H/ACA RNPs, some yet to be uncovered, with an emphasis on the role of the RNP proteins. The multiple functions of H/ACA RNPs appear to be reflected in the complex phenotype of dyskeratosis congenita.     

Structure of H/ACA RNP protein Nhp2p reveals cis/trans isomerization of a conserved proline at the RNA and Nop10 binding interface

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.     

Small nucleolar RNAs

Small nucleolar RNAs (snoRNAs) are an abundant class of non-protein-coding RNAs. In association with proteins they perform two most frequent nucleotide modifications in rRNAs and some other cellular RNAs: 2'-O-ribose methylation and pseudouridylation. SnoRNAs also participate in pre-rRNA cleavage and telomerase functions. Most snoRNAs fall into two families, box C/D and H/ACA, distinguished by the presence of conserved sequence boxes. Although C/D and H/ACA snoRNP proteins contain homologous regions, the assembly of these RNPs significantly differ. In addition, snoRNAs include the RNA component of RNAses P and MRP. The structure and function of small RNPs from Cajal bodies (small organelles associated with nucleoli) similar to snoRNP are also discussed.     

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.     

RNA structure and function in C/D and H/ACA s(no)RNPs

From archaea to humans, C/D- and H/ACA-type small ribonucleoprotein particles play key roles in crucial RNA processing events. Various such particles are required for pre-rRNA cleavage steps and/or for chemical modification of rRNAs, spliceosomal small nuclear RNAs, tRNAs and perhaps even mRNAs. Each C/D-type particle contains a small RNA possessing conserved C and D, as well as related C' and D', sequence motifs, whereas each H/ACA-type particle contains a small RNA featuring conserved H and ACA sequence elements. Recently published studies highlight the importance of sequence and structural elements of these RNAs in the localization, activity and assembly of the ribonucleoprotein particles. A novel sequence element, the Cajal body box, found at the apex of stem structures within a subset of H/ACA small RNAs, mediates the specific retention of particles containing these elements inside nucleoplasmic Cajal bodies. Two highly conserved elements, the m1 and m2 boxes, have been identified in the 3' stem of the atypical H/ACA snR30/U17 RNAs. These conserved sequence elements are necessary for early pre-rRNA cleavage events and consequently for mature 18S rRNA production. Finally, convincing evidence has been provided that the conserved C and D sequence motifs of C/D-type small RNAs fold into a helix-bulge-helix structure, called a kink-turn, that provides a platform for assembly of C/D-type ribonucleoprotein particles.     

Small nucleolar RNA

The review considers small nucleolar RNAs (snoRNAs), an abundant group of non-protein-coding RNAs. In association with proteins, snoRNAs determine the two most common nucleotide modifications in rRNA and some other cell RNAs: 2′-O-methylation of ribose and pseudouridylation. In addition, snoRNAs are involved in pre-mRNA cleavage and the telomerase function. Almost all snoRNAs fall into two families, C/D and H/ACA, distinguished by conserved sequence boxes. Although the proteins of C/D and H/ACA snoRNPs have homologous regions, these snoRNPs are assembled differently. The RNA components of RNases P and MRP are also classed with snoRNAs. Another problem considered is the structure and function of small RNAs from Cajal bodies (small organelles associated with the nucleoli), which are similar to snoRNAs.