FEBS Letters 462 (1999) 94^100 FEBS 22957 CORE Metadata, citation and similar papers at core.ac.uk Provided byThe Elsevier - Publisher ¢rst Connector determination of pseudouridine residues in 23S ribosomal RNA from hyperthermophilic Archaea Sulfolobus acidocaldarius Se¨verine Massenet1, Isabelle Ansmant1, Yuri Motorin, Christiane Branlant* Laboratoire de Maturation des ARN et Enzymologie Mole¨culaire, UMR 7567 CNRS-UHP Nancy I, Faculte¨ des Sciences, P.O. Box 239, 54506 Vandoeuvre-les-Nancy Cedex, France Received 16 October 1999 sites present in LSU rRNAs are evolutionarily conserved. The Abstract We describe the first identification of pseudouridine (8) residues in ribosomal RNA (23S rRNA) of an hyperthermo- situation is quite di¡erent for the 8 residues present in the philic Archaea Sulfolobus acidocaldarius. In contrast to Eucarya rRNA of the small ribosomal subunit (SSU). In this rRNA, rRNA, only six 8 residues were detected, which is rather close to the 8 residues are mostly present at sites speci¢c for each the situation in Bacteria. However, three modified positions organism and no apparent clustering of these modi¢ed nucleo- (82479, 82535 and 82550) are unique for S. acidocaldarius. Two 8 tides in de¢ned areas of SSU rRNA was observed (for review residues at positions 2060 and 2594 are universally conserved, see [2]) while one other 8 (position 2066) is also common to Eucarya. Synthesis of 8 residues in RNA molecules is catalysed by a Taken together the results argue against the conservation of 8- large family of enzymes, the RNA:8-synthases. Recent prog- synthases between Archaea and Bacteria and provide a basis for ress in studies of 8 residue formation demonstrated that Bac- the search of snoRNA-like guides for 8 formation in Archaea. teria and Eucarya cells do not share the same process to z 1999 Federation of European Biochemical Societies. modify U residues in rRNAs. Indeed, in Bacteria, 8 forma- Key words: Pseudouridine; rRNA; SnoRNA; tion is ensured by site- or multisite-speci¢c rRNA:8-syn- Pseudouridine-synthase; Archaea; Sulfolobus acidocaldarius thases. Several such enzymes have recently been characterised in E. coli and Bacillus subtilis [12^18]. Two multisite-speci¢c E. coli enzymes, RluC and RluD, catalyse 8 formation at six distinct sites in 23S rRNA (positions 955, 2504 and 2580, and 1. Introduction 1911, 1915 and 1917, respectively) [14^16]. Three other 8- synthases, RluB in B. subtilis and RsuA and RluE in E. The rRNAs and tRNAs of all organisms and the Eucarya coli, each modify a unique U residue into a 8 residue UsnRNAs examined so far undergo extensive post-transcrip- [13,18,19]. The ¢rst discovered rRNA:8-synthase in E. coli, tional modi¢cation (for review see [1^4]). The most frequent RluA, displays a dual substrate speci¢city, since it is impli- chemical modi¢cations in rRNAs are methylations of the 2P- cated in the formation of 8746 in 23S rRNA and of 832 in hydroxyl of the ribose moieties (2P-O-methylation) and iso- tRNAs [12,17]. merisations of uracil residues into pseudouridines (8). The In Eucarya, pre-rRNA maturation involves many di¡erent 8 residues in rRNA of the large ribosomal subunit (LSU) small nucleolar RNAs (snoRNAs) of two di¡erent classes: the have been mapped for several of species from the Eucarya H/ACA and the C/D snoRNAs. Most of these H/ACA and C/ and Bacteria domains (Homo sapiens, Mus musculus, Saccha- D snoRNAs are implicated in selection of sites of 8 forma- romyces cerevisiae, Escherichia coli, etc.), for one Archaea tion and 2P-O-methylations, respectively, by site-speci¢c base (Halobacterium halobium), and also for mitochondria and pairing with the pre-rRNA (for review see [2,4]). Speci¢c H/ chloroplasts of several Eucarya cells [5^10]. The number of ACA snoRNA guides have been found for at least 23 pseu- 8 residues per rRNA molecule varies widely from Bacteria douridylation sites out of 30 in yeast 26S rRNAs [20] and, in and Archaea to Eucarya (from nine 8 residues in E. coli LSU most cases, the direct implication of the corresponding sno- rRNA to 55 8 residues in its human counterpart). However, RNA in modi¢cation at a given site was proved experimen- all detected 8 residues are concentrated in three domains of tally [21,22]. In yeast, this snoRNA-guided modi¢cation sys- the LSU rRNA (domains II, IV and V). These domains are tem comprises several protein components and a unique located at or are functionally linked to the peptidyl transferase 8-synthase, Cbf5p [23,24]. S. cerevisiae Cbf5p and the centre (PTC) ([5,6,11]; for review see [2]). Despite the cluster- tRNA:855-synthase Pus4p are both homologous to the E. ing of 8 residues in the functional site of the ribosome, the coli tRNA:855-synthase TruB [23,25]. Cbf5p counterparts exact importance of these 8 residues in ribosomal assembly have been previously reported for a variety of Eucarya, such and peptidyl transferase reaction remains unknown. Several 8 as Kluyveromyces lactis [26], Drosophila melanogaster (Nop60p) [27], rat (NAP 57) [28] and human (DKC1) [29]. Although the Archaea and Bacteria appear rather similar in terms of general genome organisation, some of the Archaea *Corresponding author. Fax: (33) 3.83.91.20.93. E-mail: [email protected] genes show greater sequence similarity to Eucarya than to Bacteria counterparts (for review see [30]). Up to now, it is 1 These authors contributed equally to the work. not known whether the Archaea domain also possesses RNA- guided modi¢cation systems. Several observations argue for Abbreviations: 8, pseudouridine; nt, nucleotides; RT, reverse tran- the existence of only C/D-type RNPs, but not for H/ACA- scriptase; CMCT, N-cyclohexyl-NP-(2-morpholinoethyl)-carbodiimide metho-p-toluolsulfonate; PTC, peptidyl transfer centre; LSU, large type RNPs in Archaea [31]. Indeed, the S. cerevisiae C/D ribosomal subunit; SSU, small ribosomal subunit snoRNAs are associated with proteins Nop1p (¢brillarin in 0014-5793 / 99 / $20.00 ß 1999 Federation of European Biochemical Societies. All rights reserved. PII: S0014-5793(99)01524-0 FEBS 22957 18-11-99 S. Massenet et al./FEBS Letters 462 (1999) 94^100 95 human), Nop56p and Nop58p [32^35]. A gene homologous to which is slightly higher than the 3.7 þ 0.1 mol/mol of 8 resi- the yeast NOP1 gene was detected in three methanogenic dues detected by chromatography/mass spectrometry in the S. Archaea [36,37], and a homologue of the yeast NOP58 gene sulfolobus LSU rRNA [40] and the four 8 residues localised in was found in the Archaea Archaeoglobus fulgidus, Methano- the H. halobium LSU rRNA. Only a few modi¢cation posi- bacterium thermoautotrophicum and Methanococcus jannaschii tions are conserved between Bacteria and Archaea, while the [31,37^39]. The estimated number of 2P-O-methylations in others seem to be speci¢c for Archaea or common to Archaea Sulfolobus solfataricus LSU rRNA (about 44 sites) [40] is and Eucarya. These observations will be discussed in terms of much higher than that found in Bacteria (four sites in E. the evolution of 8-synthase activities. coli LSU rRNA), and quite similar to that observed in Eucar- ya (55 sites in yeast LSU rRNA, for review see [4]). This huge 2. Materials and methods number of 2P-O-methylation sites in Archaea rRNAs suggests that this type of post-transcriptional modi¢cation in Archaea 2.1. RNA preparation may be RNA-guided like in Eucarya. As far as rRNA pseu- S. acidocaldarius cells were a generous gift of P. Forterre (Orsay, douridylation is concerned, the situation with Archaea seems France). The method of RNA preparation was adapted from Aiba et to be di¡erent. H/ACA snoRNA guides directing pseudouri- al. [44], 0.3^0.6 g of cells were washed with 10 ml of 50 mM glucose, dylation of rRNA in S. cerevisiae are found to be associated 10 mM EDTA, 25 mM Tris-HCl pH 8.2 bu¡er and centrifuged for 10 min at 4000 rpm. Cells were resuspended in 300 Wl of 1 mM EDTA, with the Gar1p protein [24,41^43] and no homologous protein 0.5% SDS, 20 mM sodium acetate pH 5.5 bu¡er, treated with 300 Wl has been reported up to now in Archaea [31]. In fact, the only of phenol, incubated for 10 min at 65³C and centrifuged. The extrac- observation in favour of an RNA-guided pseudouridylation tion was done twice, recovered RNAs were treated with equal vol- system in Archaea is the ¢nding of an open reading frame, umes of chloroform/isoamyl alcohol (24:1) and ethanol precipitated in the presence of 0.3 M NaCl. conserved in all studied Archaea genomes, that shows 62% sequence similarity with Cbf5p and only 29% with TruB 2.2. Mapping of 8 residues in the S. acidocaldarius LSU rRNA [24]. The authors concluded that the Archaea protein is The N-cyclohexyl-NP-(2-morpholinoethyl)-carbodiimide metho-p- most likely a Cbf5p and not a TruB homologue. toluolsulfonate (CMCT) modi¢cation protocol was adapted from Ba- Thus, the question remains open about the existence of a kin and Ofengand [8], with the modi¢cation described previously [45]. RNA-guided system for 8 synthesis in Archaea rRNAs. To Modi¢cation by CMCT was performed with 10 WgofS. acidocaldar- search for the presence of genes encoding H/ACA-like guide ius total RNA. Positions of CMCT modi¢cations were identi¢ed by primer exten- RNAs in the Archaea genomes, one has ¢rst to identify the sion analysis, using the AMV RT (Life Science, USA), in the condi- positions of 8 residues in rRNAs from these organisms.