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.     

Identification of 13 novel human modification guide RNAs

Members of the two expanding RNA subclasses termed C/D and H/ACA RNAs guide the 2'-O-methylations and pseudouridylations, respectively, of rRNA and spliceosomal RNAs (snRNAs). Here, we report on the identification of 13 novel human intron-encoded small RNAs (U94-U106) belonging to the two subclasses of modification guides. Seven of them are predicted to direct 2'-O-methylations in rRNA or snRNAs, while the remainder represent novel orphan RNA modification guides. From these, U100, which is exclusively detected in Cajal bodies (CBs), is predicted to direct modification of a U6 snRNA uridine, U(9), which to date has not been found to be pseudouridylated. Hence, within CBs, U100 might function in the folding pathway or other aspects of U6 snRNA metabolism rather than acting as a pseudouridylation guide. U106 C/D snoRNA might also possess an RNA chaperone activity only since its two conserved antisense elements match two rRNA sequences devoid of methylated nucleotides and located remarkably close to each other within the 18S rRNA secondary structure. Finally, we have identified a retrogene for U99 snoRNA located within an intron of the Siat5 gene, supporting the notion that retro-transposition events might have played a substantial role in the mobility and diversification of snoRNA genes during evolution.     

A small nucleolar guide RNA functions both in 2'-O-ribose methylation and pseudouridylation of the U5 spliceosomal RNA

In eukaryotes, two distinct classes of small nucleolar RNAs (snoRNAs), namely the fibrillarin-associated box C/D snoRNAs and the Gar1p-associated box H/ACA snoRNAs, direct the site-specific 2'-O-ribose methylation and pseudouridylation of ribosomal RNAs (rRNAs), respectively. We have identified a novel evolutionarily conserved snoRNA, called U85, which possesses the box elements of both classes of snoRNAs and associates with both fibrillarin and Gar1p. In vitro and in vivo pseudouridylation and 2'-O-methylation experiments provide evidence that the U85 snoRNA directs 2'-O-methylation of the C45 and pseudouridylation of the U46 residues in the invariant loop 1 of the human U5 spliceosomal RNA. The U85 is the first example of a snoRNA that directs modification of an RNA polymerase II-transcribed spliceosomal RNA and that functions both in RNA pseudouridylation and 2'-O-methylation.     

The snoRNA domain of vertebrate telomerase RNA functions to localize the RNA within the nucleus.

Telomerase RNA is an essential component of the ribonucleoprotein enzyme involved in telomere length maintenance, a process implicated in cellular senescence and cancer. Vertebrate telomerase RNAs contain a box H/ACA snoRNA motif that is not required for telomerase activity in vitro but is essential in vivo. Using the Xenopus oocyte system, we have found that the box H/ACA motif functions in the subcellular localization of telomerase RNA. We have characterized the transport and biogenesis of telomerase RNA by injecting labeled wild-type and variant RNAs into Xenopus oocytes and assaying nucleocytoplasmic distribution, intranuclear localization, modification, and protein binding. Although yeast telomerase RNA shares characteristics of spliceosomal snRNAs, we show that human telomerase RNA is not associated with Sm proteins or efficiently imported into the nucleus. In contrast, the transport properties of vertebrate telomerase RNA resemble those of snoRNAs; telomerase RNA is retained in the nucleus and targeted to nucleoli. Furthermore, both nuclear retention and nucleolar localization depend on the box H/ACA motif. Our findings suggest that the H/ACA motif confers functional localization of vertebrate telomerase RNAs to the nucleus, the compartment where telomeres are synthesized. We have also found that telomerase RNA localizes to Cajal bodies, intranuclear structures where it is thought that assembly of various cellular RNPs takes place. Our results identify the Cajal body as a potential site of telomerase RNP biogenesis.     

Plant U13 orthologues and orphan snoRNAs identified by RNomics of RNA from Arabidopsis nucleoli

Small nucleolar RNAs (snoRNAs) and small Cajal body-specific RNAs (scaRNAs) are non-coding RNAs whose main function in eukaryotes is to guide the modification of nucleotides in ribosomal and spliceosomal small nuclear RNAs, respectively. Full-length sequences of Arabidopsis snoRNAs and scaRNAs have been obtained from cDNA libraries of capped and uncapped small RNAs using RNA from isolated nucleoli from Arabidopsis cell cultures. We have identified 31 novel snoRNA genes (9 box C/D and 22 box H/ACA) and 15 new variants of previously described snoRNAs. Three related capped snoRNAs with a distinct gene organization and structure were identified as orthologues of animal U13snoRNAs. In addition, eight of the novel genes had no complementarity to rRNAs or snRNAs and are therefore putative orphan snoRNAs potentially reflecting wider functions for these RNAs. The nucleolar localization of a number of the snoRNAs and the localization to nuclear bodies of two putative scaRNAs was confirmed by in situ hybridization. The majority of the novel snoRNA genes were found in new gene clusters or as part of previously described clusters. These results expand the repertoire of Arabidopsis snoRNAs to 188 snoRNA genes with 294 gene variants.     

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.     

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.     

Mining small RNA sequencing data: a new approach to identify small nucleolar RNAs in Arabidopsis

Small nucleolar RNAs (snoRNAs) are noncoding RNAs that direct 2′-O-methylation or pseudouridylation on ribosomal RNAs or spliceosomal small nuclear RNAs. These modifications are needed to modulate the activity of ribosomes and spliceosomes. A comprehensive repertoire of snoRNAs is needed to expand the knowledge of these modifications. The sequences corresponding to snoRNAs in 18–26-nt small RNA sequencing data have been rarely explored and remain as a hidden treasure for snoRNA annotation. Here, we showed the enrichment of small RNAs at Arabidopsis snoRNA termini and developed a computational approach to identify snoRNAs on the basis of this characteristic. The approach successfully uncovered the full-length sequences of 144 known Arabidopsis snoRNA genes, including some snoRNAs with improved 5′- or 3′-end annotation. In addition, we identified 27 and 17 candidates for novel box C/D and box H/ACA snoRNAs, respectively. Northern blot analysis and sequencing data from parallel analysis of RNA ends confirmed the expression and the termini of the newly predicted snoRNAs. Our study especially expanded on the current knowledge of box H/ACA snoRNAs and snoRNA species targeting snRNAs. In this study, we demonstrated that the use of small RNA sequencing data can increase the complexity and the accuracy of snoRNA annotation.     

snoRNA的结构与功能

核仁小分子RNA（snoRNA）是一类广泛分布于真核生物细胞核仁的小分子非编码RNA,具有保守的结构元件,并以此划分为3大类：boxC／DsnoRNA、boxH／ACAsnoRNA和MRPRNA。其中boxC／D和boxH／ACA是已知snoRNA的主要类型,以碱基配对的方式分别指导着核糖体RNA的甲基化和假尿嘧啶化修饰。研究发现,snoRNA除了在核糖体RNA的生物合成中发挥作用之外,还能够指导snRNA、tRNA和mRNA的转录后修饰。此外,还有相当数量的snoRNA功能不明,被称为孤儿sn0RNA（orphansnoRNA）。在哺乳动物的孤儿snoRNA中,印迹snoRNA（imprintedsnoRNA）是最为特殊的一群,由基因组印迹区编码,具有明显的组织表达特异性。原核生物古细菌中类snoRNA的鉴定表明这些非编码RNA家族成员的古老起源;而哺乳动物中大量的snoRNA反转座子的存在更为人们探索snoRNA在基因组中扩增和功能进化提供了新的思路。     

Biogenesis of small nucleolar ribonucleoproteins

Eukaryotic cells contain a very complex population of small nucleolar RNAs. They function, as small nucleolar ribonucleoproteins, in pre-ribosomal RNA processing reactions, and also guide methylation and pseudouridylation of ribosomal RNA, spliceosomal small nuclear RNAs, and possibly other cellular RNAs. Synthesis of small nucleolar RNAs frequently follows unusual strategies. Some newly discovered brain-specific small nucleolar RNAs of unknown function are encoded in introns of tandemly repeated units, expression of which is paternally imprinted. Recent studies of the protein components and factors participating in small nucleolar ribonucleoprotein assembly have revealed interesting connections with other classes of cellular ribonucleoproteins such as spliceosomal small nuclear ribonucleoproteins and telomerase. Cajal bodies emerge as nuclear structures important for the biogenesis and function of small nucleolar ribonucleoproteins.     

Solution structure of an rRNA substrate bound to the pseudouridylation pocket of a box H/ACA snoRNA

Base pairing between the RNA components of box H/ACA small nucleolar ribonucleoproteins (snoRNPs) and sequences in other eukaryotic RNAs target specific uridines for pseudouridylation. An RNA called HJ1 has been developed that interacts with the rRNA sequence targeted by the 5' pseudouridylation pocket of human U65 snoRNA the same way as intact U65 snoRNA. Sequences on both strands of the analog of the U65 snoRNP pseudouridylation pocket in HJ1 pair with its substrate sequence, and the resulting complex, called HJ3, is strongly stabilized by Mg(2+). The solution structure of HJ3 reveals an Omega-shaped RNA interaction motif that has not previously been described, which is likely to be common to all box H/ACA snoRNP-substrate complexes. The topology of the complex explains why the access of substrate sequences to snoRNPs is facile and how uridine selection may occur when these complexes form.     

Box H/ACA snoRNAs are preferred substrates for the trimethylguanosine synthase in the divergent unicellular eukaryote Trichomonas vaginalis

The 2,2,7-trimethylguanosine caps of eukaryal snRNAs and snoRNA are formed by the enzyme Tgs1, which catalyzes sequential guanine-N2 methylations of m(7)G caps. Atypically, in the divergent unicellular eukaryote Trichomonas vaginalis, spliceosomal snRNAs lack a guanosine cap and the recombinant T. vaginalis trimethylguanosine synthase (TvTgs) produces only m(2,7)G in vitro. Here, we show by direct metabolic labeling that endogenous T. vaginalis RNAs contain m(7)G, m(2,7)G, and m(2,2,7)G caps. Immunodepletion of TvTgs from cell extracts and TvTgs add-back experiments demonstrate that TvTgs produces m(2,7)G and m(2,2,7)G caps. Expression of TvTgs in yeast tgs1Δ cells leads to the formation of m(2,7)G and m(2,2,7)G caps and complementation of the lethality of a tgs1Δ mud2Δ strain. Whereas TvTgs is present in the nucleus and cytosol of T. vaginalis cells, TMG-containing RNAs are localized primarily in the nucleolus. Molecular cloning of anti-TMG affinity-purified T. vaginalis RNAs identified 16 box H/ACA snoRNAs, which are implicated in guiding RNA pseudouridylation. The ensemble of new T. vaginalis H/ACA snoRNAs allowed us to predict and partially validate an extensive map of pseudouridines in T. vaginalis rRNA.     

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.