Human TRMT1 and TRMT1L paralogs ensure the proper modification state, stability, and function of tRNAs

Abstract

The tRNA methyltransferase 1 (TRMT1) enzyme catalyzes the N2,N2-dimethylguanosine (m2,2G) modification in tRNAs. Intriguingly, vertebrates encode an additional tRNA methyltransferase 1-like (TRMT1L) paralog. Here, we use a comprehensive tRNA sequencing approach to decipher targets of human TRMT1 and TRMT1L. We find that TRMT1 methylates all known tRNAs containing guanosine at position 26, while TRMT1L represents the elusive enzyme catalyzing m2,2G at position 27 in tyrosine tRNAs. Surprisingly, TRMT1L is also necessary for maintaining 3-(3-amino-3-carboxypropyl)uridine (acp3U) modifications in a subset of tRNAs through a process that can be uncoupled from methyltransferase activity. We also demonstrate that tyrosine and serine tRNAs are dependent upon m2,2G modifications for their stability and function in translation. Notably, human patient cells with disease-associated TRMT1 variants exhibit reduced levels of tyrosine and serine tRNAs. These findings uncover unexpected roles for TRMT1 paralogs, decipher functions for m2,2G modifications, and pinpoint tRNAs dysregulated in human disorders caused by tRNA modification deficiency.

Keywords: CP: Molecular biology; RNA modification; TRMT1; TRMT1L; Trm1; intellectual disability; m2,2G; paralog; tRNA; translation.

Figure 1.. Mapping tRNA modifications dependent upon TRMT1 and TRMT1L

(A) TRMT1 and TRMT1L protein domains. (B) Immunoblot of TRMT1 and TRMT1L in lysates from the indicated human cell lines. (C) Mismatch incorporation frequency across the tRNA transcriptome in control wild-type (WT) human cells without or with (+) AlkB treatment. (D) Heatmap of the relative mismatch incorporation frequency across the tRNA transcriptome in the human cell lines. Predicted modifications are noted above the map.

Figure 2.. TRMT1 and TRMT1L catalyze m2,2G modification at positions 26 and 27 of tRNA-Tyr, respectively

(A) Misincorporation frequency for tRNA-Tyr isoacceptors from the indicated cell lines. (B) tRNA-Tyr with known modifications. (C) Immunoblot of TRMT1 and TRMT1L levels in the indicated cell lines. (D) Relative peak intensity values of the indicated modifications detected by LC-MS in purified tRNA-Tyr from the cell lines versus the control WT cell line. (E) Primer extension analysis of tRNA-Tyr from the indicated cell lines. An asterisk represents a non-specific band. (F) Immunoblot analysis of TRMT1L expression in the TRMT1L-KO cell lines. (G) Primer extension analysis of tRNA-Tyr from the indicated cell lines. (H) PHA northern blot analysis to detect m2,2G at position 26. (I) Quantification of northern blot signal in (H). Bars represent the standard deviation from the mean. Statistical analysis was performed using one-way ANOVA, and significance was calculated using Dunnett’s multiple comparison test. ****p ≤ 0.0001, ***p ≤ 0.001.

Figure 3.. In vitro reconstitution of TRMT1 and TRMT1L methyltransferase activity on tRNA substrates

(A) Sypro Ruby-stained gel of purified TRMT1 and TRMT1L. (B) Peak intensity areas of m2,2G or m2G measured by LC–MS in in vitro-transcribed tRNA-Tyr after pre-incubation with buffer control, TRMT1, or TRMT1L. (C) Primer extension assay of tRNA-Tyr. (D) Quantification of G26 and/or G27 stop in (C) and (E)–(G). (E–G) Primer extension analysis of (E) intron-containing pre-tRNA-Tyr, (F) tRNA-Tyr variants, and (G) tRNA-Ile variants. An asterisk denotes a non-specific band.

Figure 4.. TRMT1- and TRMT1L-deficient human cells exhibit a reduction in acp3U modification in a subset of tRNAs

(A) Change in mismatch incorporation percentage at position U20 for tRNA-Ala or tRNA-Cys in the TRMT1-KO or TRMT1L-KO cell line relative to the control-WT cell line. (B) Relative peak intensity values of acp3U modification detected by LC-MS in the indicated tRNAs purified from HEK293T cell lines versus control-WT. (C) tRNA-Cys with modifications in red. (D) Primer extension gel of tRNA-Cys from the indicated HEK293T cell lines. (E) Quantification of the stop signal relative to readthrough plus stop signal of (D). (F) Sequence alignment of TRMT1L homologs encompassing the P512 residue. (G) Relative peak intensity values of the indicated modifications detected by LC-MS in purified tRNAs from fibroblast cell lines. (H and I) Primer extension assay gels to monitor m2,2G or acp3U in tRNA-Tyr or tRNA-Cys, respectively, from the indicated cell lines with quantification of percent stop. Statistical analysis was performed using one-way ANOVA, and significance was calculated using Dunnett’s multiple comparison test for (B), (E), (G), and (H) or Tukey’s multiple-comparisons test for (I). ****p ≤ 0.0001, ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05.

Figure 5.. TRMT1-catalyzed modification is required for the accumulation of tyrosine and serine tRNAs in human cells

(A) Volcano plot of tRNA levels in TRMT1-KO versus control WT cell lines. Triangles represent tRNAs that are modified with m2,2G at position 26. Circles represent tRNAs without m2,2G. Red and blue denote tRNAs that are increased and decreased, respectively, in the TRMT1-KO cell lines. (B) Northern blot of tRNAs from the indicated HEK293T cell lines. (C) Quantification of northern blots in (B). (D) Schematic of TRMT1 variants. (E) Immunoblot analysis of TRMT1 variants in the TRMT1-KO cell line. (F) Primer extension analysis of mt-tRNA-Ile or tRNA-Met-CAU from cell lines containing stable integration of the indicated TRMT1 construct. (G) Northern blot of tRNAs from control WT and TRMT1-KO cell lines containing the indicated TRMT1 construct. (H) Quantification of northern blots in (G). Bars represent the standard deviation from the mean. Statistical analysis was performed using one-way ANOVA, and significance was calculated using Dunnett’s multiple-comparisons test. ****p ≤ 0.0001, ***p ≤ 0.001, **p ≤ 0.01.

Figure 6.. Human patient cells containing biallelic pathogenic TRMT1 variants exhibit a reduction in serine and tyrosine tRNA levels

(A) Patient and control cell lines used for analysis. Red indicates patients with decreased m2,2G. (B) Northern blot analysis of cell lines derived from patient and control individuals. (C–G) Quantification of the relative northern blot signal for the indicated tRNAs from patient cell lines versus the C1 control fibroblast cell line or C2 control lymphoblastoid cell line. Error bars indicate the standard deviation from the mean. Statistical analysis was performed using one-way ANOVA, and significance was calculated using Dunnett’s multiple comparison test. ****p ≤ 0.0001, ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05.

Figure 7.. Loss of TRMT1 and TRMT1L impairs protein expression from specific codon reporters

(A) Codon reporter constructs. (B) Quantification of protein expression from the tyrosine UAC and UAU codon reporters. (C) Quantification of translation from serine codon reporters. Error bars indicate the standard error from the mean. Statistical analysis was performed using one-way ANOVA, and significance was calculated using Dunnett’s multiple-comparisons test. **p ≤ 0.01, *p ≤ 0.05.