Taurine-containing uridine modifications in tRNA anticodons are required to decipher non-universal genetic codes in ascidian mitochondria

Abstract

Variations in the genetic code are found frequently in mitochondrial decoding systems. Four non-universal genetic codes are employed in ascidian mitochondria: AUA for Met, UGA for Trp, and AGA/AGG(AGR) for Gly. To clarify the decoding mechanism for the non-universal genetic codes, we isolated and analyzed mitochondrial tRNAs for Trp, Met, and Gly from an ascidian, Halocynthia roretzi. Mass spectrometric analysis identified 5-taurinomethyluridine (τm(5)U) at the anticodon wobble positions of tRNA(Met)(AUR), tRNA(Trp)(UGR), and tRNA(Gly)(AGR), suggesting that τm(5)U plays a critical role in the accurate deciphering of all four non-universal codes by preventing the misreading of pyrimidine-ending near-cognate codons (NNY) in their respective family boxes. Acquisition of the wobble modification appears to be a prerequisite for the genetic code alteration.

FIGURE 1.

Mass spectrometric analysis of H. roretzi mt tRNAs. A, base peak chromatograms (BPC) of RNase T₁-digested fragments are shown for tRNA^Gly(AGR) (top), tRNA^Met(AUR) (middle), and tRNA^Trp(UGR) (bottom). Mass chromatograms for the anticodon-containing fragments of each tRNA are also indicated. Sequences and molecular masses of the RNA fragments numbered on base peak chromatograms are listed in supplemental Table 1. The anticodons are underlined. B, collision-induced dissociation spectrum of the anticodon-containing fragment derived from tRNA^Gly(AGR). The triply charged negative ion (m/z 1200.8) was employed as a precursor ion for collision-induced dissociation. Product ions of the c- and y-series (21) are indicated on the spectrum and assigned to the sequence.

FIGURE 2.

Clover leaf structures including modifications of H. roretzi mt tRNAs. Secondary structures of H. roretzi mt tRNAs responsible for the non-universal (A) and universal (B) genetic codes. Abbreviations for modified nucleosides are as follows: m¹G, 1-methylguanosine; m²G, N²-methylguanosine; m¹A, 1-methyladenosine; i⁶A, N⁶-isopentenyladenosine; ms²i⁶A, 2-methylthio-N⁶-isopentenyladenosine; Ψ, pseudouridine; τm⁵U, 5-taurinomethyluridine; and s²U, 2-thiouridine. The numbering system of tRNAs is based on the tRNA database (tRNAdb 2009) (47). Pseudouridines in tRNAs^Gly and tRNA^Met(AUR) were determined previously (14, 16, 22). Variations of the wobble bases are indicated in parenthesis (A). Other partial modifications were also found: N²-methylguanosine 10 (7%), ms²i⁶A37 (56%) and i⁶A37 (44%) in tRNA^Trp(UGR); m¹A9 (88%) in tRNA^Gly(AGR); and 1-methylguanosine 37 (97%) in tRNA^Met(AUG). At position 63 in tRNA^Gly(GGN), 24% of A63 is replaced with G63, probably due to heteroplasmy or a polymorphism in the mtDNA. The unpaired top bases and consecutive GCs are boxed in tRNA^Met(AUG).

FIGURE 3.

Decoding system of the non-universal codons in H. roretzi mitochondria. Non-universal AGR and universal GGN codons are decoded by tRNA^Gly with the τm⁵UCU and UCC anticodons, respectively. AUR codons for Met in elongation are decoded by tRNA^Met with the τm⁵(s²)UAU anticodon. The AUG codon for initiation can be recognized by tRNA^Met with the CAU anticodon. UGR codons for Trp are recognized by tRNA^Trp with the τm⁵UCA anticodon. Non-universal codons are boxed.