A previously unidentified activity of yeast and mouse RNA:pseudouridine synthases 1 (Pus1p) on tRNAs

Abstract

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.

RNA methylation by Dnmt2 protects transfer RNAs against stress-induced cleavage

“…The recent development of RNA bisulfite sequencing (Schaefer et al 2009), and the use of methods for the purification and site-specific methylation analysis of defined tRNAs (Hengesbach et al 2008) allowed us to identify additional tRNAs that were methylated by Dnmt2 in Drosophila, thus providing evidence for multisubstrate methyltransferase activity of Dnmt2. Multisubstrate specificity has been described for other RNA modification enzymes, including pseudouridine synthase 1 (Behm-Ansmant et al 2006), and, significantly, TRM4, the only other characterized cytosine-5 tRNA methyltransferase (Motorin and Grosjean 1999). Considering that TRM4 methylates minisubstrates as small as 40 nucleotides, it remains a possibility that other unidentified substrate RNAs (e.g., small regulatory RNAs) might be methylated by RNA methyltransferases such as Dnmt2.…”

Novel small Cajal-body-specific RNAs identified in Drosophila: probing guide RNA function

“…Another interpretation is that a separate pseudouridine synthase exists for position 44 and that its activity is influenced by the presence or absence of pugU2-35/45. Such a synthase (Pus1p) has been identified in yeast (Massenet et al 1999), and its orthologs are presumably responsible for the corresponding modification in higher eukaryotes (Behm-Ansmant et al 2006).…”

The Saccharomyces cerevisiae Pus2 protein encoded by YGL063w ORF is a mitochondrial tRNA:Ψ27/28-synthase

“…To this end, the tRNA:C72-synthase activity of cell-free extracts prepared from different strains was tested on a transcript of the mitochondrial tRNA Met i(CAU) labeled by [a-32 P]ATP incorporation. As evidenced by thin layer chromatography (TLC) analysis of the labeled RNAse T2 digestion products, none of the gene disruptions affected the activity of the extracts (Table 1, see also Behm- Ansmant et al 2006). Thus, Pus1p and Pus2p are probably not involved in C formation at position 72.…”

“…We found that two distinct enzymes, Rib2p/Pus8p and Pus9p, are required for C32 formation in tRNAs, depending on their cytoplasmic or mitochondrial localization, and cytoplasmic Rib2p/Pus8p contains both a tRNA:C32-synthase domain and a DRAP-deaminase domain (Behm- Ansmant et al 2004). Pus1p is an RNA:C-synthase with a multisite specificity (tRNA positions 1, 26, 27, 28, 34, 36, 65, and 67) Behm-Ansmant et al 2006). In addition, it also modifies U2 snRNA at position 44 (Massenet et al 1999).…”