Mass spectrometry analysis of tRNA modification

Total RNA was isolated from the liver of WT and Ftsj1 KO mice at 8 weeks or human lymphoblast cells using TRIzol (Invitrogen) according to the manufacturer’s instructions. Individual mouse tRNA and human tRNA were purified from total RNA using a 5′-amino–modified DNA oligo probe by the reciprocal circulating chromatography (RCC) method (49). For analysis of tRNA fragments, purified tRNA was digested with RNase T1 or RNase A and subjected to mass spectrometry analysis as described (50). For analysis of nucleosides, purified tRNA was digested with 2.5 U of nuclease P1 (Wako) and 0.2 U alkaline phosphatase (TaKaRa, 2250A) in 5 mM ammonium acetate (pH 5.3) and 20 mM Hepes-KOH (pH 7.0) for 3 hours at 37°C. Samples were subject to mass spectrometry analysis as described (50). The sequences of DNA oligo probes for isolation of tRNAs are as follows: tRNA^Phe, 5′-GAACCAGGGACCTTTAGATCTTCAGTCTAACGCTC; tRNA^Trp, 5′-TCTGATCTGGAGTCAGACGCGCTACCGTTGCGCCA; tRNA^Sec, 5′-TTGAAGCCTGCACCCCAGACCACTGAGGATCA; tRNA^Leu(UAA), 5′-GATCTTAAGTCCAACGCCTTAACCACTCGGCCATCCTGGT; tRNA^Leu(CAA), 5′-TGGTGTCAGAAGTGGGATTCGAACCCACGCCTCCATCCGG; *tRNA^Leu(AAG), 5′-TGGTGGCAGCGGTGGGATTCGAACCCACGCCCCCGAAGAG; tRNA^Leu(CAG), 5′-AGACTGCGACCTGAACGCAGCGCCTTAGACCGCTCGGCCA; tRNA^Val(AAC), 5′-ACCTTTCGCGTGTTAGGCGAACGTGATAACCACTACACTA; tRNA^Gln(CUG), 5′-GTCGCTGGATTCAGAGTCCAGAGTGCTAACCATTACACCAT; tRNA^Arg(ACG), 5′-AATCTTCTGATCCGTAGTCAGACGCGTTATCCATTGCGCCAC. Asterisk (*) indicates that probe for tRNA^Leu(AAG) was used to isolate tRNA^Leu(UAG).