The Effect of tRNA^[Ser]Sec Isopentenylation on Selenoprotein Expression

Transfer RNA^[Ser]Sec carries multiple post-transcriptional modifications. The A37G mutation in tRNA^[Ser]Sec abrogates isopentenylation of base 37 and has a profound effect on selenoprotein expression in mice. Patients with a homozygous pathogenic p.R323Q variant in tRNA-isopentenyl-transferase (TRIT1) show a severe neurological disorder, and hence we wondered whether selenoprotein expression was impaired. Patient fibroblasts with the homozygous p.R323Q variant did not show a general decrease in selenoprotein expression. However, recombinant human TRIT1^R323Q had significantly diminished activities towards several tRNA substrates in vitro. We thus engineered mice conditionally deficient in Trit1 in hepatocytes and neurons. Mass-spectrometry revealed that hypermodification of U₃₄ to mcm⁵Um occurs independently of isopentenylation of A₃₇ in tRNA^[Ser]Sec. Western blotting and ⁷⁵Se metabolic labeling showed only moderate effects on selenoprotein levels and ⁷⁵Se incorporation. A detailed analysis of Trit1-deficient liver using ribosomal profiling demonstrated that UGA/Sec re-coding was moderately affected in Selenop, Txnrd1, and Sephs2, but not in Gpx1. 2'O-methylation of U₃₄ in tRNA^[Ser]Sec depends on FTSJ1, but does not affect UGA/Sec re-coding in selenoprotein translation. Taken together, our results show that a lack of isopentenylation of tRNA^[Ser]Sec affects UGA/Sec read-through but differs from a A37G mutation.