Deficiency of the tRNA:)35-synthase aPus7 in Archaea of the Sulfolobales order might be rescued by the H/ACA sRNA-guided machinery

Up to now, ) formation in tRNAs was found to be catalysed by stand-alone enzymes. By computational analysis of archaeal genomes we detected putative H/ACA sRNAs, in four Sulfolobales species and in Aeropyrum pernix, that might guide )35 formation in pre-tRNA(GUA). This modification is achieved by Pus7p in eukarya. The validity of the computational predictions was verified by in vitro reconstitution of H/ACA sRNPs using the identified Sulfolobus solfataricus H/ACA sRNA. Comparison of Pus7-like enzymes encoded by archaeal genomes revealed amino acid substitutions in motifs IIIa and II in Sulfolobales and A. pernix Pus7-like enzymes. These conserved RNA:)-synthasemotifs are essential for catalysis. As expected, the recombinant Pyrococcus abyssi aPus7 was fully active and acted at positions 35 and 13 and other positions in tRNAs, while the recombinant S. solfataricus aPus7 was only found to have a poor activity at position 13. We showed that the presence of an A residue 3’ to the target U residue is required for P. abyssi aPus7 activity, and that this is not the case for the reconstituted S. solfataricus H/ACA sRNP. In agreement with the possible formation of )35 in tRNA(GUA) by aPus7 in P. abyssi and by an H/ACA sRNP in S. solfataricus, the A36G mutation in the P. abyssi tRNA(GUA) abolished )35 formation when using P. abyssi extract, whereas the A36G substitution in the S. solfataricus pre-tRNA did not affect )35 formation in this RNA when using an S. solfataricus extract. INTRODUCTION In all domains of life, pseudouridine residues ( ) are the most frequent post-transcriptionally modified residues in RNAs. They are universally found in ribosomal RNA (rRNAs) and in tRNAs (1,2). U to conversions are catalysed either by stand-alone enzymes [specific RNA: -synthases, (3)] or by small ribonucleoprotein particles [H/ACA snoRNPs or H/ACA scaRNPs in eukarya, and H/ACA sRNPs in Archaea, (4)]. H/ACA RNPs contain a small RNA that defines the targeted U residue by basepair interaction with the RNA substrate (5–8). Archaeal and eukaryal H/ACA RNPs contain 4 proteins: Nop10, Gar1, L7ae/Nhp2p and Cbf5/Dyskerin (9). CBF5 belongs to the TruB family of RNA: -synthases. Whereas aCBF5 alone has no activity on rRNAs (5–8), recent data showed an in vitro activity of aCBF5 at position 55 in tRNAs. This activity does not require the presence of a guide RNA (10–12). The additional free N1-H of the nucleobase allows the formation of an additional hydrogen bond, either in cis, within the RNA molecule, or in trans, with another RNA molecule or a protein. For instance, residue 35 in the eukaryal cytoplasmic tRNA(GUA) allows the formation of an hydrogen bond with the O20 of residue U33, which stabilizes the anticodon loop structure (13,14). Furthermore, substitution of the C-N bond between the ribose and the nucleobase by a C-C bond limits the flexibility of the ribose-phosphate backbone and favours RNA-RNA base-pair interactions (13–15). In all organisms, pseudouridylations were found to occur at numerous positions in tRNAs (seven in Escherichia coli, at least 15 in Saccharomyces cerevisiae and even more in higher eukaryotes) (1). Formation of residue 55 in the T C loop is the most frequent *To whom correspondence should be addressed. Tel: +33 3 83 68 43 03; Fax: +33 3 83 68 43 07; Email: [email protected]