The Arabidopsis TRM61/TRM6 complex is a bona fide tRNA N1-methyladenosine methyltransferase

Abstract

tRNA molecules, which contain the most abundant post-transcriptional modifications, are crucial for proper gene expression and protein biosynthesis. Methylation at N1 of adenosine 58 (A58) is critical for maintaining the stability of initiator methionyl-tRNA (tRNAiMet) in bacterial, archaeal, and eukaryotic tRNAs. However, although research has been conducted in yeast and mammals, it remains unclear how A58 in plant tRNAs is modified and involved in development. In this study, we identify the nucleus-localized complex AtTRM61/AtTRM6 in Arabidopsis as tRNA m1A58 methyltransferase. Deficiency or a lack of either AtTRM61 or AtTRM6 leads to embryo arrest and seed abortion. The tRNA m1A level decreases in conditionally complemented Attrm61/LEC1pro::AtTRM61 plants and this is accompanied by reduced levels of tRNAiMet, indicating the importance of the tRNA m1A modification for tRNAiMet stability. Taken together, our results demonstrate that tRNA m1A58 modification is necessary for tRNAiMet stability and is required for embryo development in Arabidopsis.

Keywords: Arabidopsis; AtTRM6; AtTRM61; N1-methyladenosine; embryo; tRNA.

Fig. 1.

Identification of putative genes for tRNA m¹A58 modification. (A) Structure of the RNA m¹A modification. (B) Immuno-northern blotting analysis of the tRNA m¹A modification in Arabidopsis. Total RNA was isolated from different organs and examined by staining with Midori Green and immuno-Northern blotting using an anti-m¹A antibody. Rt, roots; St, stems; CL, cauline leaves; RL, rosette leaves; IF, inflorescences; OF, open flowers; Sil, siliques. (C) Unrooted Neighbor-joining tree of TRM61 spanning three Domains, including archaeobacteria (Thermus thermophilus, Pyrococcus abyssi), eubacterium (Mycobacterium tuberculosis), protists (Paramecium tetraurelia), fungi (Saccharomyces cerevisiae), plants (A. thaliana, Oryza sativa, Zea mays), and animals (Caenorhabditis elegans, Drosophila melanogaster, Danio rerio, Mus musculus, Homo sapiens). Numbers are values of bootstrap analysis, was performed with 1000 iterations. (D) Phylogenetic tree of TRM6 in Eukarya.

Fig. 2.

Yeast complementation assays using AtTRM61 and AtTRM6. (A) BY4741 and trm61-4 cells were transformed with the control vector pESC-Leu, and trm61-4 cells were also transformed with pESC-Leu carrying TRM61, AtTRM61, or AtTRM61/AtTRM6. The transformants were incubated on SD/–Leu plates and then serial dilutions were dotted onto selective SCgal/raf plates at a permissive (28 ℃) or non-permissive (37℃) temperature for 3 d. Each column is a 10-fold dilution. (B) Detection of tRNA m¹A levels by immuno-northern blotting in the trm61-4 complement strains. (C) BY4741 and trm6-506 cells transformed with the control vector pESC-Leu, and trm6-506 cells complemented with TRM6, AtTRM6, or AtTRM61/AtTRM6. (D) Detection of tRNA m¹A levels by immuno-Northern blotting in the trm6-506 complement strains.

Fig. 3.

AtTRM61 and AtTRM6 form a complex and show tRNA m¹A methyltransferase activity in vitro. (A) AtTRM61 interacts with AtTRM6 in yeast two-hybrid assays. SD/2, SD/–Leu–Trp; SD/4, SD/–Trp–Leu–His–Ade. (B) AtTRM61 interacts with AtTRM6 in pull-down assays. (C) Co-immunoprecipitation assays indicate the interaction between AtTRM61 and AtTRM6 in Arabidopsis protoplasts. (D) SDS-PAGE of the purified AtTRM61-His/AtTRM6-GST complex. (E) tRNA of trm6-506 was used as the substrate in m¹A methyltransferase activity assays. AdoMet, S-adenosyl-L-methionine. (F) In vitro transcribed tRNAi^Met of Arabidopsis was used as the substrate in m¹A methyltransferase activity assays.

Fig. 4.

AtTRM61 and AtTRM6 are essential for embryo development in Arabidopsis. (A) T-DNA insertion site of Attrm61 and (B) Attrm6. A single-nucleotide polymorphism between Col-0 and Ts-1 in (C) AtTRM61 and AtTRM6(D). (E) Sequencing of AtTRM61 RT-PCR products from F₁ plants of the cross Attrm61×Ts-1. The arrow indicates transcripts of the Col-0 version. (F) Comparison of the ratio of Col-0 transcripts in F₁ plants with or without the T-DNA insertion. (G) Sequencing of AtTRM6 RT-PCR products from F₁ plants of the cross Attrm6×Ts-1. The arrow indicates transcripts of the Col-0 version. (H) Silique phenotype of T-DNA mutants, complemented plants, and CRISPR-Cas9-based knockout plant. Scale bars are 1 mm. Arrows indicate arrested seeds. Normal and arrested embryos in the same siliques of (I) Attrm61 and (J) AtTRM61CR at 4 d after pollination (DAP), and of (K) Attrm6 and (L) AtTRM6CR at 3 DAP. Scale bars in (I–L) are 20 μm.

Fig. 5.

AtTRM61 and AtTRM6 are nuclear proteins. Subcellular localization of AtTRM61 and AtTRM6 in the root cells of 6-d-old seedlings as determined by DAPI staining and green fluorescent protein (GFP) analysis of transformed plants.

Fig. 6.

AtTRM61 and AtTRM6 are highly expressed in fast-growing tissues of Arabidopsis. Relative expression of (A) AtTRM61 and (B) AtTRM6 in different tissues as determined by qRT-PCR. Rt, roots; St, stems; CL, cauline leaves; RL, rosette leaves; IF, inflorescences; OF, open flowers; Sil, siliques. Expression is relative to that of the ACTIN2 gene. Data are means (±SE) of three replicates. (C) GUS staining of AtTRM61pro::GUS plants. Arrows indicate regions where AtTRM61 was actively expressed. Scale bars are 1 mm in the top row of images and 20 μm in the bottom row.

Fig. 7.

Decreased expression of AtTRM61 results in reduced tRNA m¹A modification and decreased levels of tRNAi^Met. (A) Phenotypes of Attrm61/LEC1pro::AtTRM61 and the wild-type (WT). Arrows indicate siliques. (B, C) Close-up images of the short siliques of Attrm61/LEC1pro::AtTRM61. (D) Seed numbers in Attrm61/LEC1pro::AtTRM61 and the WT. Data are means (±SE), n=30. The significant difference was determined using Student’s t-test: **P<0.01. (E) Relative expression of AtTRM61 in different tissues of the WT and Attrm61/LEC1pro::AtTRM61, as determined by qRT-PCR. Expression is relative to that of the ACTIN2 gene. Data are means (±SE) of three replicates. (F) tRNA m¹A modification in different tissues as determined by immuno-northern blotting and northern blotting of tRNAi^Met. Lanes 1, 3, 5 are the WT; Lanes 2, 4, 6 are Attrm61/ LEC1pro::AtTRM61.