Natural human tRNA^Ala anticodon variants mistranslate the genetic code

tRNA sequencing quantifies tRNA^Ala anticodon variant levels in cells. We used hydro-tRNA-seq (Gogakos et al. 2017) to sequence the complete tRNAome of murine N2a cells expressing wild-type tRNA^Ala_AGC or a Gly-decoding G35C variant (tRNA^Ala_ACC(Gly)) that we showed previously causes misincorporation of Ala at Gly codons in N2a cells (Hasan et al. 2023). (A) The volcano plot shows the differential expression of endogenous tRNA genes as the significance of the difference (−log₁₀ of the P-value) as a function of the log₂ of fold change. Significant changes above 1.5-fold are annotated. Only one tRNA gene (mt-Arg-TCG) showed a significant change of more than twofold. (B) Levels of the indicated human tRNA^Ala_AGC (wild-type or G35C variant) and mouse tRNA^Ala and tRNA^Gly species expressed as log₁₀ of the normalized read count. The level of the tRNA^Ala G35C variant is plotted as (C) a proportion of the level of human tRNA^AlaAGC 6-1 G36C variant detected in cells relative to the level of the homologs mouse tRNA^Ala (Ala-AGC-1-1), as (D) a proportion of the tRNA^Ala_AGC isodecoder pool, as (E) a proportion of the total tRNA^Ala isoacceptor pool, and as (F) a proportion of the pool of competing tRNA^Gly_ACC and tRNA^Gly_GCC isodecoders. Independent sample t-tests were computed (*** P < 0.001, * P < 0.05) based on at least n = 3 biological replicates.