Nuclear receptors (NRs) induce transcription through association with coactivator complexes. We identified a pseudouridine synthase (PUS), mPus1p, as a coactivator for retinoic acid receptor (mRAR)gamma and other NR-dependent transactivation. mPus1p is a member of the truA subfamily of PUSs, a class of enzymes that isomerize uridine to pseudouridine in noncoding RNAs, such as tRNA, to ensure proper folding and function. mPus1p binds the first zinc finger of mRARgamma and also associates with other NRs. Interestingly, mPus1p pseudouridylates coactivator Steroid Receptor RNA Activator (SRA), and when coexpressed, mPus1p and SRA cooperatively enhance mRARgamma-mediated transcription. mPus1p, mRARgamma, and SRA exist in a retinoid-independent, promoter bound complex in the nucleus although mPus1p is also expressed in the nucleolus, where it likely modifies tRNA. Finally, we show that mPus1p-coactivator function required SRA, mPus1p-associated mRARgamma binding, and PUS activities. mPus1p-dependent pseudouridylation of SRA represents an additional type of posttranscriptional modification of a NR-coactivator complex that is important for NR signaling.

Although the Ul small nuclear ribonucleoprotein particle (snRNP) was the first mRNA-splicing cofactor to be identified, the manner in which it functions in splicing is not precisely understood. Among the information required to understand how Ul snRNP participates in splicing, it will be necessary to know its structure. Here we describe the in vitro reconstitution of a particle that possesses the properties of native Ul snRNP. 32P-labeled Ul RNA was transcribed from an SP6 promoter-human Ul gene clone and incubated in a HeLa S100 fraction. A Ul particle formed which displayed the same sedimentation coefficient (-1OS) and buoyant density (1.40 g/cm3) as native Ul snRNP. The latter value reflects the ability to withstand isopycnic banding in Cs2SO4 without prior fixation, a property shared by native Ul snRNP. The reconstituted Ul particle reacted with both the Sm and RNP monoclonal antibodies, showing that these two classes of snRNP proteins were present.Moreover, the reconstituted Ul snRNP particle was found to display the characteristic Mg2+ switch of nuclease sensitivity previously described for native Ul snRNP: an open, nuclease-sensitive conformation at a low Mg2+ concentration (3 mM) and a more compact, nuclease-resistant organization at a higher concentration (15 mM). The majority of the Ul RNA in the reconstituted particle did not contain hypermethylated caps, pseudouridine, or ribose 2-0-methylation, showing that these enigmatic posttranscriptional modifications are not essential for reconstitution of the Ul snRNP particle. The extreme 3' end (18 nucleotides) of Ul RNA was required for reconstitution, but loop II (nucleotides 64 to 77) was not. Interestingly, the 5' end (15 nucleotides) of Ul RNA that recognizes pre-mRNA 5' splice sites was not required for Ul snRNP reconstitution.Ul RNA is one of several small nuclear RNAs that participate in mRNA splicing (21). Ul RNA is associated with at least nine proteins in a complex known as the Ul small nuclear RNP particle (snRNP) (3, and references cited therein). Several of these proteins are common to snRNPs that contain U2, U5, or U4/U6 RNAs, but three are specific to Ul snRNP (2,3,10,11,30,36).The detailed structure of the Ul snRNP has not been determined. Nuclease digestion studies of human Ul, U2, U4, and U5 snRNPs have revealed a major protected region containing the sequence A(U),G, where n is 2 3, flanked by stem-loop structures (17). This is the binding site for snRNP proteins reactive with Sm antibodies and is therefore known as the Sm binding site or Sm domain (14,17,23). The structure of the Ul snRNP is Mg2> dependent: at high Mg2> concentrations (>7 mM), the particle undergoes a switch to a more compact conformation that renders additional regions of the Ul RNA nuclease resistant (18,32).In this paper we report that under appropriate conditions, it is possible to assemble a particle that possesses several properties of native Ul snRNP, including the characteristic Mg2+-dependent conformational switch. We also define some of the RNA sequences requi...

This report describes the cloning and characterization of a pseudouridine (psi) synthase from mouse that we have named mouse pseudouridine synthase 1 (mpus1p). The cDNA is approximately 1.5 kb and when used as a probe on a Northern blot of mouse RNA from tissues and cultured cells, several bands were detected. The open reading frame is 393 amino acids and has 35% identity over its length with yeast psi synthase 1 (pus1p). The recombinant protein was expressed in Escherichia coli and the purified protein converted specific uridines to psi in a number of tRNA substrates. The positions modified in stoichiometric amounts in vitro were 27/28 in the anticodon stem and also positions 34 and 36 in the anticodon of an intron containing tRNA. A human cDNA was also cloned and the smaller open reading frame (348 amino acids) was 92% identical over its length with mpus1p but is shorter by 45 amino acids at the amino terminus. The expressed recombinant human protein has no activity on any of the tRNA substrates, most probably the result of the truncated open reading frame.

It was previously shown that mouse Pus1p (mPus1p), a pseudouridine synthase (PUS) known to modify certain transfer RNAs (tRNAs), can also bind with nuclear receptors (NRs) and function as a coactivator through pseudouridylation and likely activation of an RNA coactivator called steroid receptor RNA activator (SRA). Use of cell extract devoid of human Pus1p activity derived from patients with mitochondrial myopathy and sideroblastic anemia, however, still showed SRA-modifying activity suggesting that other PUS(s) can also target this coactivator. Here, we show that related mPus3p, which has a different tRNA specificity than mPus1p, also serves as a NR coactivator. However, in contrast to mPus1p, it does not stimulate sex steroid receptor activity, which is likely due to lack of binding to this class of NRs. As expected from their tRNA activities, in vitro pseudouridylation assays show that mPus3p and mPus1p modify different positions in SRA, although some may be commonly targeted. Interestingly, the order in which these enzymes modify SRA determines the total number of pseudouridines. mPus3p and SRA are mainly cytoplasmic; however, mPus3p and SRA are also localized in distinct nuclear subcompartments. Finally, we identified an in vivo modified position in SRA, U206, which is likely a common target for both mPus1p and mPus3p. When U206 is mutated to A, SRA becomes hyperpseudouridylated in vitro, and it acquires dominant-negative activity in vivo. Thus, Pus1p- and Pus3p-dependent pseudouridylation of SRA is a highly complex posttranscriptional mechanism that controls a coactivator-corepressor switch in SRA with major consequences for NR signaling.

Incubation of a SP6-transcribed human U2 RNA precursor molecule in a HeLa cell S100 fraction resulted in the formation of ribonucleoprotein complexes. In the presence of ATP, the particles that assembled had several properties of native U2 snRNP, including resistance to dissociation in Cs2SO4 gradients, their buoyant density, and pattern of digestion by micrococcal nuclease. These particles also reacted with Sm monoclonal antibody and a human autoantibody with specificity for the U2 snRNP-specific proteins A' and B", but not with antibodies for U1 snRNP-specific proteins. In contrast, the particles that formed in the absence of ATP did not have these properties. ATP analogs with non-hydrolyzable beta-gamma bonds did not substitute for ATP in U2 snRNP assembly. Additional experiments with a mutant U2 RNA confirmed that nucleotides 154-167 of U2 RNA are required for binding of the U2 snRNP-specific proteins but not of the "Sm" core proteins. Pseudouridine formation, a major post-transcriptional modification of U2 RNA, was enhanced under assembly permissive conditions.

Mouse pseudouridine synthase 1 (mPus1p) was the first vertebrate RNA:pseudouridine synthase that was cloned and characterized biochemically. The mPus1p was previously found to catalyze C formation at positions 27, 28, 34, and 36 in in vitro produced yeast and human tRNAs. On the other hand, the homologous Saccharomyces cerevisiae scPus1p protein was shown to modify seven uridine residues in tRNAs (26, 27, 28, 34, 36, 65, and 67) and U44 in U2 snRNA. In this work, we expressed mPus1p in yeast cells lacking scPus1p and studied modification of U2 snRNA and several yeast tRNAs. Our data showed that, in these in vivo conditions, the mouse enzyme efficiently modifies yeast U2 snRNA at position 44 and tRNAs at positions 27, 28, 34, and 36. However, a tRNA:C26-synthase activity of mPus1p was not observed. Furthermore, we found that both scPus1p and mPus1p, in vivo and in vitro, have a previously unidentified activity at position 1 in cytoplasmic tRNA Arg (ACG). This modification can take place in mature tRNA, as well as in pre-tRNAs with 59 and/or 39 extensions. Thus, we identified the protein carrying one of the last missing yeast tRNA:C synthase activities. In addition, our results reveal an additional activity of mPus1p at position 30 in tRNA that scPus1p does not possess.

A missense mutation in the PUS1 gene affecting a highly conserved amino acid has been associated with mitochondrial myopathy and sideroblastic anemia (MLASA), a rare autosomal recessive oxidative phosphorylation disorder. The PUS1 gene encodes the enzyme pseudouridine synthase 1 (Pus1p) that is known to pseudouridylate tRNAs in other species. Total RNA was isolated from lymphoblastoid cell lines established from patients, parents, unaffected siblings, and unrelated controls, and the tRNAs were assayed for the presence of pseudouridine (⌿) at the expected positions. Mitochondrial and cytoplasmic tRNAs from MLASA patients are lacking modification at sites normally modified by Pus1p, whereas tRNAs from controls, unaffected siblings, or parents all have ⌿ at these positions. In addition, there was no Pus1p activity in an extract made from a cell line derived from a patient with MLASA. Immunohistochemical staining of Pus1p in cell lines showed nuclear, cytoplasmic, and mitochondrial distribution of the protein, and there is no difference in staining between patients and unaffected family members. MLASA is thus associated with absent or greatly reduced tRNA pseudouridylation at specific sites, implicating this pathway in its molecular pathogenesis.Following transcription, all stable RNAs are processed, and nucleotides are modified. The most abundant modification is pseudouridine (⌿), formed by the action of pseudouridine synthases. A number of these modification enzymes have been characterized from several species, and one that has been particularly well studied is pseudouridine synthase 1 (Pus1p).

The formation of pseudouridine (I) in U5 RNA during ribonucleoprotein (RNP) assembly was investigated by using HeLa cell extracts. In vitro transcribed, unmodified U5 RNA assembled into an RNP particle with the same buoyant density and sedimentation velocity as did U5 small nuclear RNP from extracts. The greatest amount of W modification was detected when a combination of S100 and nuclear extracts was used for assembly. ' With the recent development of in vitro systems for the splicing of pre-mRNA, there has been a rapid elucidation of the mechanism of pre-mRNA splicing (reviewed in references 10 and 29). Several small nuclear ribonucleoprotein particles (snRNPs) are cofactors in this process (reviewed in references 12 and 35). These particles, Ul, U2, US, and U4/U6 snRNPs, are abundant, stable, and located in the nucleus. The U RNA sequences and secondary structures are highly conserved, and the RNAs contain a number of modified nucleotides (33). These snRNPs form a complex during splicing, called the spliceosome, that has been extensively studied (12). In addition, U5 and U4/U6 snRNPs interact in a presplicing complex that is dependent on the presence of ATP (2).The spliceosomal snRNAs bind a core of seven polypeptides, several of which are recognized by autoimmune patients anti-Sm antibodies (20,22). The Sm proteins bind to a conserved sequence A(U).G, found in Ul, U2, U4, and U5 RNAs (21). These proteins range in molecular weight from 9,000 to 29,000. In addition to these core proteins, a number of snRNP-specific proteins have been identified (22). Recently, Bach et al. (1) revealed that the protein composition of the 20S U5 snRNP is complex and includes six U5-specific proteins in addition to the common Sm proteins. U5 particles isolated by a different procedure had sedimentation values of 8S to 10S on glycerol gradients and did not contain these proteins (1).The RNAs contained in these snRNPs are highly modified (33). These modifications include, but are not limited to, a trimethylguanosine (TMG) cap of the 5' end and pseudouridine (P), a modified form of uridine. The TMG cap appears to be necessary for the nuclear targeting of newly assembled snRNPs (8, 13). A function for T in snRNAs has not yet been determined, but in tRNAs, T is necessary for the interaction of tRNA with the ribosome (6, 28). hisT mutants of Salmonella typhimurium lack T at positions 38, 39, and 40 in tRNA (4,5,26). This mutation causes a derepression of the histidine operon because the undermodified tRNAH1S produces inefficient reading of the histidine codons present in the histidine leader peptide mRNA (14).This study combines the use of extracts and in vitrotranscribed, unmodified U5 RNA to study the formation of T under various conditions and with mutant substrates. The results show that the modifications are specific, that in vitro, RNP formation precedes T formation, and that there is a requirement for Sm protein binding to the RNA for T formation. MATERIALS AND METHODSSynthesis of a human U5a gene. Six overlapping oligodeoxynuc...