Partial activity is seen with many substitutions of highly conserved active site residues in human Pseudouridine synthase 1

Abstract

Pseudouridine synthase 1 (Pus1p) is an enzyme that converts uridine to Pseudouridine (C) in tRNA and other RNAs in eukaryotes. The active site of Pus1p is composed of stretches of amino acids that are highly conserved and it is hypothesized that mutation of select residues would impair the enzyme's ability to catalyze the formation of C. However, most mutagenesis studies have been confined to substitution of the catalytic aspartate, which invariably results in an inactive enzyme in all C synthases tested. To determine the requirements for particular amino acids at certain absolutely conserved positions in Pus1p, three residues (R116, Y173, R267) that correspond to amino acids known to compose the active site of TruA, a bacterial C synthase that is homologous to Pus1p, were mutated in human Pus1p (hPus1p). The effects of those mutations were determined with three different in vitro assays of pseudouridylation and several tRNA substrates. Surprisingly, it was found that each of these components of the hPus1p active site could tolerate certain amino acid substitutions and in fact most mutants exhibited some activity. The most active mutants retained near wild-type activity at positions 27 or 28 in the substrate tRNA, but activity was greatly reduced or absent at other positions in tRNA readily modified by wild-type hPus1p.

In Human Pseudouridine Synthase 1 (hPus1), a C-Terminal Helical Insert Blocks tRNA from Binding in the Same Orientation as in the Pus1 Bacterial Homologue TruA, Consistent with Their Different Target Selectivities

“…It acts on mature and intron-containing tRNAs, the U2 snRNA and the steroid receptor RNA activator (SRA). 9 Pseudouridylation is critical for SRAs function as a co-activator of the nuclear receptor estrogen receptor α. 10; 11 …”

Pseudouridine synthase 1: a site-specific synthase without strict sequence recognition requirements

“…In addition, yeast Pus1p has been shown to modify positions 65 and 67 in vivo (34) and mouse (mPus1p) and human Pus1p (hPus1p) modify position 30 in tRNAs, whereas yeast Pus1p does not (29,33). The presence of the intron in yeast pre-tRNA Ile (UAU) is required for the formation of Ψ at positions 34 and 36 in the anticodon by Pus1p in vitro (26,34).…”

Formation of a stalled early intermediate of pseudouridine synthesis monitored by real-time FRET

“…Pseudouridine synthase 1 (Pus1), a member of the TruA family, is a multisite and multisubstrate specific enzyme, which catalyzes pseudouridine formation at several positions of mammalian cytosolic and mitochondrial tRNAs, as well as in snRNA from various species (Arluison et al 1998;Motorin et al 1998;Arluison et al 1999;Chen and Patton 1999;Massenet et al 1999;Chen and Patton 2000;Hellmuth et al 2000;Patton and Padgett 2005;BehmAnsmant et al 2006;Sibert et al 2008). These include positions in the anticodon stem of several tRNAs at positions 27 and 28.…”