Taurine-containing Uridine Modifications in tRNA Anticodons Are Required to Decipher Non-universal Genetic Codes in Ascidian Mitochondria

Abstract

Non-universal codons are known to be used in the mitochondrial (mt) 3 genetic code (1-6). DNA sequencing of proteincoding genes in various metazoan mtDNAs followed by comparison of the deduced amino acid sequences with those of other species revealed amino acid assignment deviations for the codons UGA, AUA, AGR, and AAA. The termination codon UGA in the universal genetic code specifies Trp in the mitochondria of all species investigated so far (1, 2). The AUA codon for Ile changes to specify Met in most metazoa except echinoderms, planaria, and coelenterates (2-4). AGR codons are used for Ser in most invertebrates, for Gly in urochordates, and for termination in vertebrates (7-10). The AAA codon specifies Asn in echinoderms (7) and platyhelminths (11).Our group previously reported that the mitochondrial decoding system of ascidian Halocynthia roretzi employs four non-universal genetic codes: UGA for Trp, AUA for Met and AGR for Gly (Table 1) (9, 12). Sequence analysis of H. roretzi mtDNA identified three tDNAs responsible for these non-universal codons. Each of these tDNAs contains thymidine (T) at the first (wobble) position of their anticodons (Table 1). Because an unmodified U at the wobble position can decode any of the four bases at the third letter of the codon, according to the mitochondrial four-way wobble rule (5, 13), the uridines of these tRNAs at the wobble position must be modified in order to decipher their respective non-universal genetic codes accurately. To elucidate the molecular mechanism of the decoding system, it is necessary to determine the chemical structure of the anticodon wobble base in H. roretzi mt tRNAs. We previously reported 5-carboxymethylaminomethyluridine at the wobble position of mt tRNA Met (AUR) from H. roretzi (14). However, the nucleotide identification was inaccurate because it was analyzed by conventional two-dimensional TLC, and the modification was determined based on a comparison of its rate of flow value with that of known modified nucleotides (15). In addition, we also reported that H. roretzi mt tRNA Gly -(AGR) has an unidentified modified U at the wobble position (16). We describe here the chemical structures of the wobble bases in mt tRNAs for non-universal genetic codes in the H. roretzi mitochondrial decoding system and discuss evolutionary insights into the role of wobble modification in genetic code alteration. EXPERIMENTAL PROCEDURESIsolation of mt tRNAs-Two hundred grams of the commercially available edible muscle of an ascidian, H. roretzi, was minced, and the tRNA fraction was obtained by phenol extraction as described previously (17). Two thousand A 260 units of crude tRNA were separated by the DEAE-cellulose column * This work was supported by grants-in-aid for scientific research (C) from The Japan Society for the Promotion of Science (to K. W.) and by grants-in-aid for scientific research on priority areas from the Ministry of Education, Science, Sports, and Culture of Japan (to Takeo Suzuki and Tsutomu Suzuki). □ S The on-line version of this article...

Distribution and frequencies of post-transcriptional modifications in tRNAs

“…positions 2, 3 and 6). In particular mitochondria-specific base modifications tm 5 U and tm 5 s 2 U were discovered, 33 and linked with 2 major classes of mitochondrial diseases, MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes) and MERRF (myoclonus epilepsy associated with ragged red fibers). 39 Taken together, these observations suggest that the general patterns of tRNA modifications as depicted above are not likely to change greatly as more tRNA sequences become available.…”

“…Other positions in tRNA are much less frequently modified and the functions of these modifications are still largely unknown. Only a few positions have never been found modified in any tRNA sequenced so far (these are positions 5,11,23,24,33,42,43,45,53,59, 62, 63, 73-76 and the majority of positions from the variable loop: 13e, 15-17e, 1e, 3-5e, 21-27e).…”

“…Taurine-containing residues in the first position of the anticodon are found only in mitochondria. 33 …”

How the Early Genetic Code Was Established?: Inference from the Analysis of Extant Animal Mitochondrial Decoding Systems

“…In the last case, no structural study on the corresponding tRNA was performed, and no release factor (RF) relevant to the UAA codon was identified, so discussion on the codonanticodon relationship was not possible in this instance. There are 42 different anticodons and a total of 48 tRNAs with different body sequences in E. coli (Näsvall et al 2007) Modified nucleosides in the tRNA anticodons are associated with mitochondrial codon changes in all cases (Watanabe and Yokobori 2011): (1) The anticodon wobble position (the 34th) of tRNA Trp changes to 5-carboxymethyluridine (cmnm 5 U) in nematode mitochondria (Sakurai et al 2005) and to 5-taurinomethyluridine (τm 5 U) in ascidian (Suzuki et al 2011b), molluscan (Ohira et al 2013), and vertebrate (Suzuki et al 2002(Suzuki et al , 2011a mitochondria; (2) The anticodon wobble position of tRNA Met changes to 5-formylcytidine (f 5 C) in vertebrate (Moriya et al 1994), arthropod (Tomita et al 1999a), and nematode mitochondria (Watanabe et al 1994) and to τm 5 U in ascidian mitochondria (Suzuki et al 2011b); (3) The anticodon wobble nucleoside of tRNA Gly for AGR codons becomes τm 5 U in ascidian mitochondria (Suzuki et al 2011b). The anticodon wobble nucleoside of tRNA Ser becomes m 7 G in most invertebrate mitochondria (Matsuyama et al 1998;Tomita et al 1998) and unmodified U in nematode mitochondria (Watanabe et al 1994); (4) The starfish mitochondrial tRNA Asn that deciphers AAA as Asn possesses the anticodon GΨU (Ψ, pseudouridine) (Tomita et al 1999b); this is unusual in that it is a change at the second anticodon position.…”

The central role of tRNA in genetic code expansion

“…Thus, by introducing an aberrant decoder (here M. mobile tRNA Ile UAU ), direct AUA decoding is possible, and if accompanied by a fully orthogonal isoleucyl-tRNA synthetase (IleRS), AUA codons may be reassigned to a ncAA [104]. In nature, reassignment of Ile AUA codons to Met exists in metazoan and S. cerevisiae mitochondria where a 5-formyl-cytidine (f 5 C34) containing Met-tRNA Met reads both AUG and AUA [105, 106], as well as in ascidian mitochondria where the reassignment is mediated by a 5-taurine-2-thio-methyl-2-thiouridine on position 34 (Tm 5 s 2 U34) and an unmodified U37 in Met-tRNA Met [107]. …”