Figure 3

Loss of Tarsl2 exerts no effect on the steady-state or charging of tRNA^Thrs in vivo.A, schematic showing Tarsl2-mutant mouse construction by CRISPR/Cas9 gene editing technology. Two sgRNAs were designed around exon 3 to delete a sequence fragment. Primers 1 and 2 and Primers 3 and 4 were used to detect DNA mutations. B, PCR detection of the Tarsl2^+/+, Tarsl2^+/−, and Tarsl2^−/− mouse genotypes using Primers 1 and 2 and Primers 3 and 4. C, sequencing results of Tarsl2^+/+ and Tarsl2^−/− mice. In the mutant mice, Tarsl2 mRNA translation was prematurely terminated at nt 342. D, Tarsl2 mRNA levels in the muscle of the 6-week-old Tarsl2^+/+ and Tarsl2^−/− mice, as determined by RT‒PCR with two pairs of primers. E, protein abundance of Tarsl2 in the 6-week-old Tarsl2^+/+ and Tarsl2^−/− mouse muscle, as detected by Western blotting. Gapdh was used as the loading control. F and G, levels of tRNA^Thr(UGU), tRNA^Thr(CGU), and tRNA^Thr(AGU) in the muscle tissues of the 6-week-old Tarsl2^+/+ and Tarsl2^−/− mice. tRNA^Gly(CCC) was included as the loading control. H and I Aminoacylation levels of tRNA^Thr(UGU), tRNA^Thr(CGU), and tRNA^Thr(AGU) in the muscle tissues of the 6-week-old Tarsl2^+/+ and Tarsl2^−/− mice. tRNA^Gly(CCC) was included as the control. De: tRNA deaminoacylation control. ∗∗∗∗p < 0.0001. Error bars in (G and I) represent the mean ± SD with the p values indicated (two-tailed Student’s t test).