Different mechanisms for pseudouridine formation in yeast 5S and 5.8S rRNAs

The selection of sites for pseudouridylation in eukaryotic cytoplasmic rRNA occurs by the base pairing of the rRNA with specific guide sequences within the RNA components of box H/ACA small nucleolar ribonucleoproteins (snoRNPs). Forty-four of the 46 pseudouridines (Psis) in the cytoplasmic rRNA of Saccharomyces cerevisiae have been assigned to guide snoRNAs. Here, we examine the mechanism of Psi formation in 5S and 5.8S rRNA in which the unassigned Psis occur. We show that while the formation of the Psi in 5.8S rRNA is associated with snoRNP activity, the pseudouridylation of 5S rRNA is not. The position of the Psi in 5.8S rRNA is guided by snoRNA snR43 by using conserved sequence elements that also function to guide pseudouridylation elsewhere in the large-subunit rRNA; an internal stem-loop that is not part of typical yeast snoRNAs also is conserved in snR43. The multisubstrate synthase Pus7 catalyzes the formation of the Psi in 5S rRNA at a site that conforms to the 7-nucleotide consensus sequence present in other substrates of Pus7. The different mechanisms involved in 5S and 5.8S rRNA pseudouridylation, as well as the multiple specificities of the individual trans factors concerned, suggest possible roles in linking ribosome production to other processes, such as splicing and tRNA synthesis.     

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.     

Elements essential for accumulation and function of small nucleolar RNAs directing site-specific pseudouridylation of ribosomal RNAs.

During site-specific pseudouridylation of eukaryotic rRNAs, selection of correct substrate uridines for isomerization into pseudouridine is directed by small nucleolar RNAs (snoRNAs). The pseudouridylation guide snoRNAs share a common 'hairpin-hinge- hairpin-tail' secondary structure and two conserved sequence motifs, the H and ACA boxes, located in the single-stranded hinge and tail regions, respectively. In the 5'- and/or 3'-terminal hairpin, an internal loop structure, the pseudouridylation pocket, selects the target uridine through formation of base-pairing interactions with rRNAs. Here, essential elements for accumulation and function of rRNA pseudouridylation guide snoRNAs have been analysed by expressing various mutant yeast snR5, snR36 and human U65 snoRNAs in yeast cells. We demonstrate that the H and ACA boxes that are required for formation of the correct 5' and 3' ends of the snoRNA, respectively, are also essential for the pseudouridylation reaction directed by both the 5'- and 3'-terminal pseudouridylation pockets. Similarly, RNA helices flanking the two pseudouridylation pockets are equally essential for pseudouridylation reactions mediated by either the 5' or 3' hairpin structure, indicating that the two hairpin domains function in a highly co-operative manner. Finally, we demonstrate that by manipulating the rRNA recognition motifs of pseudouridylation guide snoRNAs, novel pseudouridylation sites can be generated in yeast rRNAs.     

Pseudouridylation of yeast U2 snRNA is catalyzed by either an RNA-guided or RNA-independent mechanism

Yeast U2 small nuclear RNA (snRNA) contains three pseudouridines (Psi35, Psi42, and Psi44). Pus7p and Pus1p catalyze the formation of Psi35 and Psi44, respectively, but the mechanism of Psi42 formation remains unclear. Using a U2 substrate containing a single (32)P radiolabel at position 42, we screened a GST-ORF library for pseudouridylase activity. Surprisingly, we found a Psi42-specific pseudouridylase activity that coincided with Nhp2p, a protein component of a Box H/ACA sno/scaRNP (small nucleolar/Cajal body-specific ribonucleoprotein). When isolated by tandem affinity purification (TAP), the other protein components of the H/ACA sno/scaRNP also copurified with the pseudouridylase activity. Micrococcal nuclease-treated TAP preparations were devoid of pseudouridylase activity; however, activity was restored upon addition of RNAs from TAP preparations. Pseudouridylation reconstitution using RNAs from a Box H/ACA RNA library identified snR81, a snoRNA known to guide rRNA pseudouridylation, as the Psi42-specific guide RNA. Using the snR81-deletion strain, Nhp2p- or Cbf5p-conditional depletion strain, and a cbf5 mutation strain, we further demonstrated that the pseudouridylase activity is dependent on snR81 snoRNP in vivo. Our data indicate that snRNA pseudouridylation can be catalyzed by both RNA-dependent and RNA-independent mechanisms.     

Immunopurified small nucleolar ribonucleoprotein particles pseudouridylate rRNA independently of their association with phosphorylated Nopp140

The isomerization of up to 100 uridines to pseudouridines (Psis) in eukaryotic rRNA is guided by a similar number of box H/ACA small nucleolar RNAs (snoRNAs), each forming a unique small nucleolar ribonucleoprotein particle (snoRNP) with the same four core proteins, NAP57 (also known as dyskerin or Cbf5p), GAR1, NHP2, and NOP10. Additionally, the nucleolar and Cajal body protein Nopp140 (Srp40p) associates with the snoRNPs. To understand the role of these factors in pseudouridylation, we established an in vitro assay system. Short site-specifically (32)P-labeled rRNA substrates were incubated with subcellular fractions, and the conversion of uridine to Psi was monitored by thin-layer chromatography after digestion to single nucleotides. Immunopurified box H/ACA core particles were sufficient for the reaction. SnoRNPs associated quantitatively and reversibly with Nopp140. However, pseudouridylation activity was independent of Nopp140, consistent with a chaperoning role for this highly phosphorylated protein. Although up to 14 bp between the snoRNA and rRNA were required for the in vitro reaction, rRNA pseudouridylation and release occurred in the absence of ATP and magnesium. These data suggest that substrate release takes place without RNA helicase activity but may be aided by the snoRNP core proteins.     

18S rRNA processing requires base pairings of snR30 H/ACA snoRNA to eukaryote‐specific 18S sequences

The H/ACA RNAs represent an abundant, evolutionarily conserved and functionally diverse class of non‐coding RNAs. Many H/ACA RNAs direct pseudouridylation of rRNAs and snRNAs, while members of the rapidly growing group of ‘orphan’ H/ACA RNAs participate in pre‐rRNA processing, telomere synthesis and probably, in other nuclear processes. The yeast snR30 ‘orphan’ H/ACA snoRNA has long been known to function in the nucleolytic processing of 18S rRNA, but its molecular role remained unknown. Here, we provide biochemical and genetic evidence demonstrating that during pre‐rRNA processing, two evolutionarily conserved sequence elements in the 3′‐hairpin of snR30 base‐pair with short pre‐rRNA sequences located in the eukaryote‐specific internal region of 18S rRNA. The newly discovered snR30‐18S base‐pairing interactions are essential for 18S rRNA production and they constitute a complex snoRNA target RNA transient structure that is novel to H/ACA RNAs. We also demonstrate that besides the 18S recognition motifs, the distal part of the 3′‐hairpin of snR30 contains an additional snoRNA element that is essential for 18S rRNA processing and that functions most likely as a snoRNP protein‐binding site.     

U2 snRNA is inducibly pseudouridylated at novel sites by Pus7p and snR81 RNP

All pseudouridines identified in RNA are considered constitutive modifications. Here, we demonstrate that pseudouridylation of Saccharomyces cerevisiae U2 snRNA can be conditionally induced. While only Ψ35, Ψ42 and Ψ44 are detected in U2 under normal conditions, nutrient deprivation leads to additional pseudouridylation at positions 56 and 93. Pseudouridylation at position 56 can also be induced by heat shock. Detailed analyses have shown that Pus7p, a single polypeptide pseudouridylase known to modify U2 at position 35 and tRNA at position 13, catalyses Ψ56 formation, and that snR81 RNP, a box H/ACA RNP known to modify U2 snRNA at position 42 and 25S rRNA at position 1051, catalyses Ψ93 formation. Using mutagenesis, we have demonstrated that the inducibility can be attributed to the imperfect substrate sequences. By introducing Ψ93 into log-phase cells, we further show that Ψ93 has a role in pre-mRNA splicing. Our results thus demonstrate for the first time that pseudouridylation of RNA can be induced at sites of imperfect sequences, and that Pus7p and snR81 RNP can catalyse both constitutive and inducible pseudouridylation.     

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.     

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.     

Multiple functional interactions between components of the Lsm2-Lsm8 complex, U6 snRNA, and the yeast La protein

The U6 small nuclear ribonucleoprotein is a critical component of the eukaryotic spliceosome. The first protein that binds the U6 snRNA is the La protein, an abundant phosphoprotein that binds the 3' end of many nascent small RNAs. A complex of seven Sm-like proteins, Lsm2-Lsm8, also binds the 3' end of U6 snRNA. A mutation within the Sm motif of Lsm8p causes Saccharomyces cerevisiae cells to require the La protein Lhp1p to stabilize nascent U6 snRNA. Here we describe functional interactions between Lhp1p, the Lsm proteins, and U6 snRNA. LSM2 and LSM4, but not other LSM genes, act as allele-specific, low-copy suppressors of mutations in Lsm8p. Overexpression of LSM2 in the lsm8 mutant strain increases the levels of both Lsm8p and U6 snRNPs. In the presence of extra U6 snRNA genes, LSM8 becomes dispensable for growth, suggesting that the only essential function of LSM8 is in U6 RNA biogenesis or function. Furthermore, deletions of LSM5, LSM6, or LSM7 cause LHP1 to become required for growth. Our experiments are consistent with a model in which Lsm2p and Lsm4p contact Lsm8p in the Lsm2-Lsm8 ring and suggest that Lhp1p acts redundantly with the entire Lsm2-Lsm8 complex to stabilize nascent U6 snRNA.     

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.     

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.     

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 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.     

Identification of novel proteins associated with yeast snR30 small nucleolar RNA

H/ACA small nucleolar RNPs (snoRNPs) that guide pseudouridylation reactions are comprised of one small nucleolar RNA (snoRNA) and four common proteins (Cbf5, Gar1, Nhp2 and Nop10). Unlike other H/ACA snoRNPs, snR30 is essential for the early processing reactions that lead to the production of 18S ribosomal RNA in the yeast Saccharomyces cerevisiae. To determine whether snR30 RNP contains specific proteins that contribute to its unique functional properties, we devised an affinity purification strategy using TAP-tagged Gar1 and an RNA aptamer inserted in snR30 snoRNA to selectively purify the RNP. Northern blotting and pCp labeling experiments showed that S1-tagged snR30 snoRNA can be selectively purified with streptavidin beads. Protein analysis revealed that aptamer-tagged snR30 RNA was associated with the four H/ACA proteins and a number of additional proteins: Nop6, ribosomal proteins S9 and S18 and histones H2B and H4. Using antibodies raised against Nop6 we show that endogenous Nop6 localizes to the nucleolus and that it cosediments with snR30 snoRNA in sucrose density gradients. We demonstrate through primer extension experiments that snR30 snoRNA is required for cleavages at site A0, A1 and A2, and that the absence of Nop6 decreases the efficiency of cleavage at site A2. Finally, electron microscopy analyses of chromatin spreads from cells depleted of snR30 snoRNA show that it is required for SSU processome assembly.     

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.     

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.