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. 2021 Oct 23;22(21):11454.
doi: 10.3390/ijms222111454.

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

Noelia Fradejas-Villar  1 , Simon Bohleber  1 , Wenchao Zhao  1 , Uschi Reuter  1 , Annika Kotter  2 , Mark Helm  2 , Rainer Knoll  1 , Robert McFarland  3 , Robert W Taylor  3 , Yufeng Mo  4 , Kenjyo Miyauchi  4 , Yuriko Sakaguchi  4 , Tsutomu Suzuki  4 , Ulrich Schweizer  1
Affiliations

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

Noelia Fradejas-Villar et al. Int J Mol Sci. .

Abstract

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 TRIT1R323Q 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.

Keywords: Trit1; isopentenylation; selenoproteins; tRNA[Ser]Sec.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Modifications and mutations in tRNA[Ser]Sec. Sequence of murine tRNA[Ser]Sec with post-transcriptional modifications indicated [20]. Where proposed, we mentioned the respective enzymes responsible for the modifications (black). Labelled in red are mutations in the primary sequence of tRNA[Ser]Sec in transgenic mouse models or observed in a human patient. The U34A mutant tRNA[Ser]Sec is further deaminated in vivo to inosine (I).
Figure 2
Figure 2
Selenoprotein expression in patient fibroblasts carrying a pathogenic homozygous TRIT1R323Q variant. (A) Western blot comparing selenoprotein expression in TRIT1 patient fibroblasts with two control fibroblast lines. The signal corresponding to TRIT1 protein is reduced almost to the detection limit in the TRIT1 patient cells, while the unspecific (lower) band suggests equal loading. β-Actin served as control. (B) Metabolic 75Se-labeling of cultured fibroblasts reveals normal 75Se incorporation in selenoproteins. Coomassie brilliant blue stained gel shows equal protein loading. Asterisks represent wells loaded with un-labelled protein to avoid diffusion (C) RT-PCR to determined ms2i6A in tRNAs. The two steps, reverse transcription of tRNA and qPCR of cDNA, are depicted. Primers are represented as half arrows (R1 and R2 are reverse primers and Fw is the forward primer) Arrowhead shows the position of the ms2i6A. (D) Determination of tRNA modification index based on RT-PCR. Traces from mt-tRNATrp analysis. (E) Modification index of several mt-tRNAs normally containing ms2i6A37 modifications depends on functional TRIT1. (F) Heatmap of significantly regulated genes from human fibroblasts focused on selenoprotein and NRF2 target genes. Up-regulated and down-regulated genes in the patient fibroblasts are depicted in red and blue, respectively.
Figure 3
Figure 3
Activity assays using recombinant TRIT1. (A) In vitro assay using wild type and p.R323Q variant TRIT1 recombinant proteins and in vitro transcribed tRNA[Ser]Sec. Isopentenylated tRNA was also detected in a urea-acrylamide gel. (B) Representative results of kinetic analyses of TRIT1 with ACF substrates corresponding to mt-tRNASer(UGA) and tRNA[Ser]Sec. (C) Specific activities determined for eight substrates using TRIT1 (Ctl) and p.R323Q. N = 3. * p < 0.05, Student’s t-test. The ACF oligonucleotide concentration in the endpoint assay corresponded to the KM of the oligonucleotide with the wild-type enzyme (Table 1).
Figure 4
Figure 4
Knockout of Trit1 in liver abrogates formation of i6A in tRNA and tRNA[Ser]Sec isopentenylation. (A) Western blot on liver extract using an antibody against TRIT1. (B) Levels of i6A in the tRNA fraction isolated from liver are significantly reduced in Trit1 KO. N = 3. *** p < 0.001, Student’s t-test. (C) Northern blot against tRNA[Ser]Sec and 5S rRNA as control. (D) Mass spectrometric analysis of the tRNA[Ser]Sec isolated from Ctl (left panels) and Trit1 KO (right panels) livers. Each panel from top to bottom shows an extracted-ion chromatogram for the RNase T1-digested anticodon-containing fragments with different modification status at positions 34 and 37. Modification status, sequence of the fragment, m/z value, and charge state (z) are shown on the right for each panel. Relative abundance of each fragment is denoted in each panel. Non-specific peaks are marked with asterisks.
Figure 5
Figure 5
Selenoprotein expression in Trit1-knockout (KO) mice. (A) Western blot against a panel of 8 selenoproteins in mouse liver. N = 6–7 individual mice. Liver protein, 50 µg, separated on SDS-PAGE. (B) Densitometric analysis of the western blot in (A). Ponceau was used for normalization. Results are expressed as mean ± SD of the percentage relative to the control (Ctl). GPX4 and SEPHS2 showed significant differences according two-tailed t-test. * p < 0.05. p-values of GPX4 and SEPHS2 were 4 × 10−6 and 2.25 × 10−4, respectively. (C) Metabolic labeling with 75Se-selenite of isolated primary hepatocytes. Coomassie brilliant blue-stained gel for loading control (left) and autoradiogram (right). N = 3 individual cultures. (D) Selenoprotein western blot from cortices of neuron-specific Trit1 KO mice. (E) 75Se-labeling of Trit1 KO and Ctl neuron cultures (a representative experiment). Coomassie showed equal loading.
Figure 6
Figure 6
Selenoprotein RiboSeq analysis of Trit1 knockout (KO) liver. (A) RPFs with the UGA/Sec in the A-site expressed as reads per million mapped reads (RPM) over all selenoproteins. (B) UGA recoding efficiency (URE, 3′RPF/5′RPF) calculated for selenoproteins with UGA/Sec far from the termination codon. ΔURE is calculated as URE(KO)/URE(Ctl). (C–G) RPF coverage of selected selenoprotein mRNAs in Trit1 KO mouse liver. The mean values of the groups were plotted. Start and stop positions are marked as green and red circles. Reads are plotted in blue for control (Ctl) and in orange for Trit1 KO livers. The position of the UGA/Sec codon is indicated by a black “x” mark. In the case of Selenop, following UGA codons after the first are displayed as black vertical lines. Cumulative sums of RPF are shown below the corresponding profiles. RPM: reads per million mapped reads.
Figure 7
Figure 7
Re-analysis of Fstj1 knockout (KO) brain RiboSeq data focussed on selenoproteins. (A) RPFs with the UGA/Sec in the A-site expressed as reads per million mapped reads (RPM) over all selenoproteins. (B) UGA recoding efficiency (URE, 3′RPF/5′RPF) calculated for selenoproteins with UGA/Sec far from the termination codon. ΔURE is calculated as URE(KO)/URE(Ctl). (C,D) RPF coverage of selected selenoprotein mRNAs in Ftsj1 KO mouse brain. The mean values of the groups were plotted. Start and stop positions are marked as green and red circles. Reads are plotted in blue for control (Ctl) and in orange for Ftsj1 KO brains. The position of the UGA/Sec codon is indicated by a black “x” mark. Cumulative sums of RPF are shown below the corresponding profiles. RPM: reads per million mapped reads.
Figure 8
Figure 8
Ribosomal profiling of Gpx1 in Elp3 knockout (KO) developing brain. (A) Ribosomal coverage plot. The position of the UGA/Sec codon is indicated by a black “x” mark. Note the decreased ribosomal coverage in Elp3 KO 3′ from the UGA/Sec codon. (B) Cumulative sum plot. The net translation of Gpx1 in Elp3 KO is apparently adjusted by increased translation/initiation 5′ of the UGA/Sec. Data from [62] were re-analyzed with the methods presented here.
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