Box C/D RNA guides for the ribose methylation of archaeal tRNAs. The tRNATrp intron guides the formation of two ribose-methylated nucleosides in the mature tRNATrp

Abstract

Following a search of the Pyrococcus genomes for homologs of eukaryotic methylation guide small nucleolar RNAs, we have experimentally identified in Pyrococcus abyssi four novel box C/D small RNAs predicted to direct 2'-O-ribose methylations onto the first position of the anticodon in tRNALeu(CAA), tRNALeu(UAA), elongator tRNAMet and tRNATrp, respectively. Remarkably, one of them corresponds to the intron of its presumptive target, pre-tRNATrp. This intron is predicted to direct in cis two distinct ribose methylations within the unspliced tRNA precursor, not only onto the first position of the anticodon in the 5' exon but also onto position 39 (universal tRNA numbering) in the 3' exon. The two intramolecular RNA duplexes expected to direct methylation, which both span an exon-intron junction in pre-tRNATrp, are phylogenetically conserved in euryarchaeotes. We have experimentally confirmed the predicted guide function of the box C/D intron in halophile Haloferax volcanii by mutagenesis analysis, using an in vitro splicing/RNA modification assay in which the two cognate ribose methylations of pre-tRNATrp are faithfully reproduced. Euryarchaeal pre-tRNATrp should provide a unique system to further investigate the molecular mechanisms of RNA-guided ribose methylation and gain new insights into the origin and evolution of the complex family of archaeal and eukaryotic box C/D small RNAs.

Figure 1

Novel Pyrococcus box C/D small RNAs with tRNA antisense elements. (A) RNA coding sequences aligned by reference to the C, D’, C’ and D box motifs (in bold). Small RNAs predicted from genomic search were detected by northern hybridization of P.abyssi total cellular RNA with oligonucleotide probes and their 5′ ends mapped by primer extension. Small RNA 3′ ends were inferred from size determination (by northern) and 5′ end mapping, using oligonucleotides S, T, U and V. Sequence coordinates in the P.abyssi genome (database maintained at the Genoscope, Centre National de Séquençage, Evry, France, http://www.cns.fr/cgi-bin/Pab.cgi) are given into brackets with indication of the DNA strand. Nucleotide sequences of the homologous RNAs in P.horikoshii and P.furiosus, respectively, are shown below each P.abyssi sequence. Nucleotides immediately upstream from box C and downstream from box D, which form a 5′–3′ terminal stem–box structure typical of eukaryotic box C/D antisense snoRNAs (1–3) are denoted by horizontal arrows in opposite orientation. Potential tRNA antisense elements are in red. (B) Structure of potential guide RNA duplexes involving one of the novel sRNAs and a P.abyssi mature or precursor tRNA. The tRNA nucleotide paired to the fifth nucleotide upstream from box D or D’ is denoted by an arrow, with indication of its position in the P.abyssi mature tRNA sequence (into brackets: position in the universal numbering system for tRNAs). The anticodon is overlined. For pre-tRNAMet and pre-tRNATrp, intron nucleotides are in blue. For duplexes involving sR47 and sR49, nucleotide differences in homologous RNAs of A.fulgidus and M.jannaschii, respectively, are denoted. In M.thermoautrotrophicum, the duplex between pre-tRNAMet and the likely sR49 homolog (accession number AE000852, positions 4049–4100 for complement to the box C/D interval) shows a sequence identical to the Pyrococcus.

Figure 2

Sequence of the box C/D-containing intron of the euryarchaeal tRNATrp gene. (A) General alignment of the box C/D intron (and flanking exonic) sequences in GenBank. Intron nucleotides are in lower case. The 5′- and 3′-AE are denoted by a filled bar and their cognate complementary sequences by an open bar (with indication of the corresponding antisense element into brackets). In the tRNA exons the two nucleotide positions targeted for ribose methylation by the pair of antisense elements in the intron are denoted by a filled circle. Nucleotides involved in the formation of the BHB motif cleaved by the tRNA-splicing endonuclease are underlined. Nucleotide differences, which are compensatory within presumptive methylation guide RNA duplexes, are in red. (B) Further intragroup comparisons based on the determination of additional euryarchaeal sequences. Stars denote novel sequences determined after amplification of genomic DNA by PCR with primers matching conserved exonic segments (Tb, T.barophilus; Ap, A.profundus; Hso, H.sodomense; Hsa, H.salinarium; Hh, H.hispanica; GenBank accession numbers AY047490, AY047491, AY047489, AY047492 and AY047493, respectively). For halophiles, an additional copy of the 5′-AE (5′-AE-2) and its cognate box D” motif are denoted, and the two RNA duplexes which could both direct formation of Cm39 in tRNATrp are depicted below the alignment. The nucleotides potentially involved in the formation of a 5′–3′ stem, adjacent to boxes C and D, are overlined by arrows in opposite orientation.

Figure 3

In vitro processing of the H.volcanii pre-tRNATrp. An in vitro synthesized [α-³²P]CTP-labeled pre-tRNATrp transcript was incubated at 37°C with a H.volcanii S100 extract as described (27), except for the addition of 0.02 mM S-adenosyl methionine prior to incubation. Aliquots taken after increasing times of incubation, as indicated above the lanes, were analyzed by electrophoresis on a 7 M urea–6% acrylamide gel (lane 0, no incubation; lane M, DNA size marker). The different processing products (indicated on the right) have been assigned by northern hybridizations and primer extensions with appropriate oligonucleotides (data not shown). The previously unreported form of the intron is denoted by an asterisk (see Results).

Figure 4

Formation of Cm34 and Um39 in a pre-tRNATrp transcript incubated with a H.volcanii S100 extract. The in vitro synthesized H.volcanii pre-tRNATrp transcript, labeled by incorporation of [α-³²P]NTP, was incubated at 37°C for 2 h in the presence of a H.volcanii S100 extract supplemented with S-adenosyl methionine. Processing products were separated by electrophoresis on 7 M urea–8% acrylamide gel, purified and digested by nuclease P1 or RNase T2. (A) 2D-TLC in system C of nuclease P1 digests of the 5′ and 3′ exons obtained from an [α-³²P]CTP- or [α-³²P]UTP-labeled pre-tRNATrp transcript, respectively (digests of a pre-tRNATrp transcript not incubated with the S100 extract serve as controls). Open circles, position of unlabeled nucleoside 5′-monophosphate markers. (B) 2D-TLC in system B of RNase T2 digests of the 5′ exon obtained from an [α-³²P]CTP- or [α-³²P]ATP-labeled pre-tRNATrp transcript (left and middle, respectively) and of the RNase T1 7-nt long oligonucleotide spanning C34, which was purified from an [α-³²P]ATP-labeled 5′ exon (right: the oligonucleotide sequence is shown, with sites of cleavage by RNase T1 and T2 denoted by long and short arrows, respectively, and labeled phosphates by stars). (C) 2D-TLC in system B of RNase T2 digests of the 3′ exon obtained from an [α-³²P]CTP- or [α-³²P]ATP-labeled pre-tRNATrp transcript. (D) 2D-TLC in system B of RNase T2 digests of the [α-³²P]CTP-labeled pre-tRNATrp transcript in the absence of, or following, a 2 h incubation with the S100 extract. (E) Structure of mature H.volcanii tRNATrp. Previously reported, naturally occurring nucleotide modifications (35) are indicated, using the universal numbering system for tRNAs. Modifications analyzed upon in vitro incubation of the pre-tRNATrp transcript are boxed. The modification in parenthesis, Cm32, did not form in our assay. The location of the intron is denoted.

Figure 5

Effects of alterations of the pre-tRNATrp intron structure on in vitro formation of Cm34 and Um39. (A) Structure of the two large intronic deletion mutants, Del1 and Del2. The intact H.volcanii pre-tRNATrp structure is schematized (top line), with indication of sequence coordinates. (B) Various box D, D’ and D” deletions. Structure of the H.volcanii pre-tRNATrp transcript corresponds to its splicing-competent folding. The exon–intron junctions (arrows), the BHB motif (boxed), the 5′- and 3′-AE (filled overline) and matching exonic tracts (open overline), and the 2 nt (filled circles) targeted for ribose methylation by the intronic guide sequences are denoted. The 5 nt in lower case at the 3′ end of the in vitro transcript are not present in cellular pre-tRNATrp. The 5′ and 3′ boundaries of mutant ExDel, which harbors major exonic deletions leaving intact the splice-site proximal exonic nucleotides participating in the formation of the BHB, are denoted. (C) Yields of cleavage and formation of modified nucleotides for the various pre-tRNATrp transcripts upon incubation with the H.volcanii S100 extract. For mutants denoted by an asterisk modified nucleotides were assessed on the uncleaved pre-tRNA transcript. The experimental error was ±0.1 mol/mol tRNA for Um39 and ±0.05 mol/mol tRNA for Cm34 and Ψ55.

Figure 6

Alternative foldings of H. volcanii pre-tRNATrp involved in ribose methylation and splicing, respectively. The two mutually exclusive structures competent for guided-ribose methylation and splicing are shown on the left and right, respectively. (Left) The two pairs of sequence tracts involved in the formation of methylation guide duplexes directing formation of Cm34 and Um39 are denoted by broken lines. (Right) The canonical BHB splicing motif is depicted in a dotted box. Two elementary stems (termed 5′–3′ stem and apical stem, respectively) shared by both folding patterns and conserved in all halophilic tRNATrp introns are indicated.