Published online 18 August 2006 Nucleic Acids Research, 2006, Vol. 34, No. 15 4293–4301 doi:10.1093/nar/gkl530 Formation of the conserved pseudouridine at position 55 in archaeal tRNA Martine Roovers, Caryn Hale1,2, Catherine Tricot, Michael P. Terns1,2, Rebecca M. Terns1,2,*, Henri Grosjean3 and Louis Droogmans4,* Institut de Recherches Microbiologiques J.-M. Wiame, Avenue E. Gryson 1, B-1070 Bruxelles, Belgium, 1Department of Biochemistry and Molecular Biology, 2Department of Genetics, University of Georgia, Life Sciences Building, Athens, GA 30602, USA, 3Laboratoire d’Enzymologie et Biochimie Structurales, Centre National de la Recherche Scientifique, Avenue de la Terrasse 1, F-91198 Gif-sur-Yvette, France and 4Laboratoire de Microbiologie, Universite´ Libre de Bruxelles, Institut de Recherches Microbiologiques J.-M. Wiame, Avenue E. Gryson 1, B-1070 Bruxelles, Belgium Received June 6, 2006; Revised and Accepted July 10, 2006 ABSTRACT Pseudouridine (C) located at position 55 in tRNA is a nearly universally conserved RNA modification found in all three domains of life. This modification is catalyzed by TruB in bacteria and by Pus4 in eukaryotes, but so far the C55 synthase has not been identified in archaea. In this work, we report the ability of two distinct pseudouridine synthases from the hyperthermophilic archaeon Pyrococcus furiosus to specifically modify U55 in tRNA in vitro. These enzymes are pfuCbf5, a protein known to play a role in RNA-guided modification of rRNA, and pfuPsuX, a previously uncharacterized enzyme that is not a member of the TruB/Pus4/Cbf5 family of pseudouridine synthases. pfuPsuX is hereafter renamed pfuPus10. Both enzymes specifically mod- ify tRNA U55 in vitro but exhibit differences in substrate recognition. In addition, we find that in a heterologous in vivo system, pfuPus10 efficiently complements an Escherichia coli strain deficient in the bacterial C55 synthase TruB. These results indicate that it is probable that pfuCbf5 or pfuPus10 (or both) is responsible for the introduction of pseudouridine at U55 in tRNAs in archaea. While we cannot unequivocally assign the function from our results, both possibilities represent unexpected functions of these proteins as discussed herein. INTRODUCTION Pseudouridine (Y) is the most abundant modiﬁed nucleoside in RNA. It has been identiﬁed in tRNAs, rRNAs, snRNAs and/or snoRNAs of all organisms that have been analyzed to date (1–4). Y is formed post-transcriptionally by enzymatic isomerization of uridine by a class of enzymes known as pseudouridine synthases. Most Y synthases have the ability to recognize and catalyze isomerization of one (or a few) unique target uridines (5). For many years, there were thought to be only four families of Y synthases, corresponding to TruA, TruB, RluA and RsuA in bacteria (6). Members of these families were found in all three domains of life. Recently however, Y syn- thases that share little sequence homology to any of these families (such as Escherichia coli TruD) have been found, leading to the recognition of additional groups of Y syn- thases, as well as the idea that there may be many more Y synthases that are yet to be discovered (7). Y55 in tRNA is a quasi-universally conserved modiﬁcation found in all forms of life analyzed to date. The Y synthases responsible for this very highly conserved modiﬁcation have been identiﬁed in eukaryotes and bacteria, and are members of the TruB/Pus4/Cbf5 family [Cluster of Orthologous Groups of proteins (COG) 0130 (8)]. In the yeast Saccharo- myces cerevisiae, Pus4 was shown to be the enzyme respons- ible for this modiﬁcation (9), and in E.coli, TruB is the enzyme responsible for formation of Y55 (10). Although Y55 is known to exist in archaeal tRNAs and the activity has been identiﬁed in Pyrococcus furiosus cell extract (11), no archaeal Y55 synthase has been identiﬁed to date. The goal of the present work was to identify the enzyme respons- ible for isomerization of U55 to Y55 in P.furiosus, our model archaeal system. We have tested two candidate Y synthases, pfuCbf5 and pfuPus10 (previously known as PsuX). The only identiﬁed archaeal protein within the TruB/Pus4/ Cbf5 family was recently shown to function in RNA-guided pseudouridylation of rRNA (12,13). While most Y synthases are dedicated to modiﬁcation of a single target uridine, in *To whom correspondence should be addressed. Tel: +32 2 526 72 83; Fax: +32 2 526 72 73; Email: ldroogma@ulb.ac.be *Correspondence may also be addressed to Rebecca M. Terns. Tel: +1 706 542 1703; Fax: +1 706 542 1752; Email: rterns@bmb.uga.edu The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors 2006 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.