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The Complete Set of Trna Species in Nanoarchaeum Equitans

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The Complete Set of Trna Species in Nanoarchaeum Equitans FEBS 29588 FEBS Letters 579 (2005) 2945–2947 The complete set of tRNA species in Nanoarchaeum equitans Lennart Randaua, Michael Pearsonb, Dieter So¨lla,c,* a Department of Molecular Biophysics and Biochemistry, Yale University, 266 Whitney Avenue, New Haven, CT 06520-8114, USA b Department of Molecular, Cellular and Developmental Biology, Sinsheimer Laboratories, UCSC, Santa Cruz, CA 95064, USA c Department of Chemistry, Yale University, 266 Whitney Avenue, New Haven, CT 06520-8114, USA Received 22 April 2005; accepted 25 April 2005 Available online 3 May 2005 Edited by Lev Kisselev led to the reannotation of three tRNA genes and provide N. Abstract The archaeal parasite Nanoarchaeum equitans was found to generate five tRNA species via a unique process requir- equitans with a complete set of tRNAs. ing the assembly of seperate 50 and 30 tRNA halves [Randau, L., Mu¨nch, R., Hohn, M.J., Jahn, D. and So¨ll, D. (2005) Nanoar- chaeum equitans creates functional tRNAs from separate genes 2. Materials and methods for their 50- and 30-halves. Nature 433, 537–541]. Biochemical evidence was missing for one of the computationally-predicted, 2.1. Cell cultivation and tRNA isolation joined tRNAs designated as tRNATrp. Our RT-PCR and N. equitans cells were grown in a 300 l fermenter in a simultaneous sequencing results identify this tRNA as tRNALys (CUU) joined culture with Ignicoccus sp. and purified by gradient centrifugation as at the alternative position between bases 30 and 31. We show described [3]. Total tRNA was prepared by SDS-lysis of the cell pellet, that the intron-containing tRNATrp was misidentified in the ini- phenol/chloroform extraction and MonoQ anion-exchange chroma- tial Nanoarchaeum equitans genome annotation [E. Waters tography as described [1]. et al. (2003) The genome of Nanoarchaeum equitans: insights into early archaeal evolution and derived parasitism. Proc. Natl. 2.2. Reverse transcription and sequencing Acad. Sci. USA 100, 12984–12988]. Along with a previously Total tRNA from N. equitans was reverse transcribed with Thermo- unidentified joined tRNAGln (UUG), Nanoarchaeum equitans script reverse transcriptase and the resulting cDNA was PCR amplified with Platinum Taq DNA polymerase (Invitrogen) according to exhibits 44 tRNAs and is enabled to read all 61 sense codons. the manufacturerÕs directions. The tRNA template and primers Features unique to this set of tRNA molecules are discussed. were denatured at 100 °C for 5 min and cooled on ice for 5 min to Ó 2005 Federation of European Biochemical Societies. Published facilitate specific annealing. PCR products were cloned with the by Elsevier B.V. All rights reserved. pCR-2.1-TOPO cloning kit (Invitrogen). Plasmids were sequenced at the W.M. Keck Facility. The following oligonucleotides were used Keywords: tRNA splicing; tRNA maturation; Intron for PCR amplification of the indicated full-length tRNAs: (tRNALys) 50-GGGCCGGTAGCTCAGCCTGG-30 and 50-CGGGCCGGCG- GGGATTCGAACC-30, (tRNAGln)50-AGCCCCGTGGTGTAGC- GGC-30 and 50-TAGCCCCGCCCGGATTCGAACC-30, (tRNATrp) 50-GGGGCCGTAGCTCAGCCAGGCAG-30 and 50-TGGGGCC- GGGGGGATTCGAACC-30 (tRNAMet)50-GCCGCCGTAGCT- 0 0 0 1. Introduction CAGCGG-3 and 5 -TGCCGCCGGCGGGATTCGAACC-3 . The sequencing of the small genome of the hyperthermo- philic parasite Nanoarchaeum equitans raised some questions 3. Results and discussion since the genes for four essential transfer RNA species ap- peared to be missing [2]. Recently, this mystery was solved We reverse transcribed total tRNA from N.equitans with by the finding that N. equitans is the only known organism primers complementary to the 30 ends of the interrupted to assemble full-length tRNAs from separate genes encoding tRNAs. The resulting cDNAs were amplified by PCR with their 50- and 30-halves [1]. The assembly of four tRNA species an isotype-specific set of oligos described in Section 2 (Fig. Glu Glu His Met (tRNA (CUG), tRNA (UUG), tRNA and tRNAi ) 1). The sequencing results for the joined tRNA previously des- from nine tRNA halves was shown biochemically, but no se- ignated as tRNATrp stimulated our interest. This tRNA was quence information was obtained for four putative tRNA found to be tRNALys (CUU) with the two tRNA halves joined halves. It was predicted that the mature N. equitans tRNAs at an alternative position between nucleotides 30 and 31 (Fig. are joined one base after the anticodon-adjacent position 37, 2). This finding challenges the view that tRNA halves are spe- the location of most tRNA introns which led to identification cifically assembled at position 37 and led to the reinvestigation of a ‘‘missing’’ tRNATrp. We reinvestigated possible tRNAs of two additional tRNA halves identified by the long reverse joined at alternative solutions. Our new sequencing results complementary intervening sequence characteristic for all split tRNAs. We determined that these tRNA halves are joined at position 32 and a previously unidentified tRNAGln (UUG) is * Corresponding author. Fax: +1 203 432 6202. formed (Fig. 2). This isoacceptor features the same recognition E-mail address: [email protected] (D. So¨ll). elements for glutaminyl-tRNA synthetase present in the unin- Gln Abbreviations: BHB, bulge helix bulge; RT-PCR, reverse transcriptase terrupted isoacceptor tRNA (CUG) which includes the un- PCR ique A1-U72 basepair [4]. The two split tRNAGlu isoacceptors 0014-5793/$30.00 Ó 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2005.04.051 2946 L. Randau et al. / FEBS Letters 579 (2005) 2945–2947 motifs [1] usually found in the distance of 26 nucleotides to- wards the tRNA gene suggests the possibility that the accep- tor stem might be extended by one base pair which would allow the presence of a consensus A–U pair at its end. Thus, Met we propose that the N. equitans tRNAi has an 8 bp accep- tor stem. Fig. 1. RT-PCR results. The RT-PCR products obtained for tRNAGln (UUG) (Gln), tRNALys (CUU) (Lys) and tRNATrp (Trp) were We initially observed tRNA splicing of N. equitans elongator Met separated on a 3% ethidium bromide-stained agarose gel. The tRNA when reverse transcriptase PCR (RT-PCR) experi- fragments of a low molecular weight DNA ladder are indicated for ments amplified the mature, processed tRNA as well as the pri- Met size comparison. RT-PCR of elongator tRNA (eMet) yielded two mary transcript containing the 66 bp long intron. In order to amplified fragments. The individual bands were gel-excised, cloned and sequenced. The upper band was shown to be the intron-containing pre- investigate the mechanism of tRNA splicing in N. equitans tRNAMet and the lower band the spliced mature tRNAMet annotated we took a closer look at the sequences around the tRNA split in [2]. sites. Interestingly, we noted the possible formation of relaxed Bulge-Helix-Bulge (BHB) motifs for the cis-spliced tRNAs (Fig. 3A) as well as for the joined tRNA halves (Fig. 3C). This and both tRNAGln isoacceptors exhibit a deletion of base 47 correlation suggests the possibility of a trans-splicing mecha- (Fig. 3A). The bona fide tRNATrp from N.equitans conse- nism in the assembly of the tRNA halves. In this case a splicing quently is not assembled from separate tRNA halves but endonuclease would be required to recognize the proposed re- rather is the product of an intron-containing tRNA gene which laxed BHB-motifs. Possible evidence for biological significance was previously misidentified as tRNASer during the initial gen- of these relaxed motifs is the observation that a large number ome annotation using tRNAScan-SE [5]. The position of the of tRNA introns in Crenarchaeota can only be folded into intron was proposed to be between bases 37 and 38, thus pre- non-standard BHB motifs, termed hBH or HBh0 [8]. Here, dicting a second cis-spliced tRNASer (CGA) isoacceptor. How- only the central 4-bp helix H, one bulge B and a flanking helix ever, this tRNASer does not contain the signature elements on either side (h or h0) can be identified. Experimental investi- required for recognition by seryl-tRNA synthetase, namely gations will be needed to define the biochemical mechanism of the extended variable loop [6]. We found that the 13 nucleotide tRNA trans-splicing. The overall similarity of the proposed long intron is removed between the base 30 and 31 and that the splicing motifs point out an appealing connection between resulting mature tRNATrp features the anticodon CCA and the the mechanism of assembly of tRNA halves and the presence discriminator base A73 recognized by tryptophanyl-tRNA of introns in tRNAs. synthetase [7] (Fig. 2). The new annotations describe six split A function for divided tRNA genes is not immediately obvi- tRNAs and four cis-spliced tRNAs, finally providing N. equi- ous. However, the observation that site-specific integration by tans with a set of 44 non-reduntant tRNAs able to read all 61 archaeal viruses and conjugative plasmids may occur exclu- sense codons (Fig. 3A). sively at tRNA genes [9] leads us to consider that an adaptive Two features unique to the tRNA set of N. equitans value may lie in providing resistance to the integration of mo- should be noted. Firstly, N. equitans is the only known bile DNA elements. As N. equitans has virtually no metabolic archaeon displaying a tRNAIle (UAU) anticodon which or biosynthetic genes [2], the added burden of simply replicat- might correlate with the absence of a protein coding gene re- ing the DNA of an integrated element would be expected to quired to modify the CAU anticodon. Secondly, the initiator slow growth considerably. While a typical tRNA intron might tRNA exhibits a C1–G72 base pair whereas a A1–U72 base also function to prevent integration, extreme genome reduc- pair is the consensus element in all other archaeal and tion would favor its deletion when the selection pressure of eukaryotic initiator tRNAs.
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