Cloning and characterization of a mammalian pseudouridine synthase

Abstract

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.

Mitochondrial Myopathy and Sideroblastic Anemia (MLASA)

“…Labeled tRNA Ser (UGA) was transcribed in vitro by T7 RNA polymerase in the presence of [ 32 P]GTP, using the plasmid pHtS (20) digested with BstNI as a template (21). This labeled substrate (43,000 cpm, 0.41 ng) was incubated with each of the extracts (30 g of total protein in 20 mM Hepes, 20% glycerol, 0.1 M KCl, 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, and 0.5 mM dithiothreitol), recombinant mouse Pus1p (mPus1p) (0.7 g), or Lac protein (0.7 g) for 2 h at 37°C as described (2,22). The RNA was isolated from the reactions and gel-purified on a 10% polyacrylamide/8.3 M urea gel (23).…”

“…4 for relative values). Each RNA sample was then digested with RNaseT 2 and chromatographed on TLC plates using isobutyric acid:ammonium hydroxide (25%):water (50:1.1: 28.9; v/v/v) in the first dimension and isopropanol:concentrated HCl: water (70:15:15; v/v/v) in the second dimension (2,24). The TLCs were then exposed to a phosphorimaging screen for 48 h and scanned using a Bio-Rad Personal Molecular Imager FX TM system.…”

“…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). 1 Pus1p has been characterized from yeast (1), mouse (2), and Caenorhabditis elegans (3), and homologues have been identified in many eukaryotes, including humans (2). Pus1p has been shown to modify positions 27, 28, 34, and 36 in tRNAs in vitro and in vivo (1,2).…”

“…1 Pus1p has been characterized from yeast (1), mouse (2), and Caenorhabditis elegans (3), and homologues have been identified in many eukaryotes, including humans (2). Pus1p has been shown to modify positions 27, 28, 34, and 36 in tRNAs in vitro and in vivo (1,2). Additional sites in tRNAs have been identified for yeast Pus1p in vivo (1).…”

“…Genetic analysis has implicated a missense mutation in the PUS1 gene as the cause of mitochondrial myopathy and sideroblastic anemia (MLASA (MIM 600462)), 2 a rare autosomal recessive disorder of oxidative phosphorylation and iron metabolism (9,10). This mutation, a replacement of an arginine with a tryptophan at position 116, affects an amino acid in Pus1p that is highly conserved throughout evolution.…”