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.
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