Abstract
Modified nucleosides in tRNAs are critical for protein translation. N1-methylguanosine-37 and N1-methylinosine-37 in tRNAs, both located at the 3’-adjacent to the anticodon, are formed by Trm5 and here we describe Arabidopsis thaliana AtTrm5 (At3g56120) as a Trm5 ortholog. We show that AtTrm5 complements the yeast trm5 mutant, and in vitro methylates tRNA guanosine-37 to produce N1-methylguanosine (m1G). We also show in vitro that AtTRM5 methylates tRNA inosine-37 to produce N1-methylinosine (m1I) and in Attrm5 mutant plants, we show a reduction of both N1-methylguanosine and N1-methylinosine. We also show that AtTRM5 is localized to the nucleus in plant cells. Attrm5 mutant plants have overall slower growth as observed by slower leaf initiation rate, delayed flowering and reduced primary root length. In Attrm5 mutants, mRNAs of flowering time genes are less abundant and correlated with delayed flowering. Finally, proteomics data show that photosynthetic protein abundance is affected in mutant plants. Our findings highlight the bifunctionality of AtTRM5 and the importance of the post-transcriptional tRNA modifications m1G and m1I at tRNA position 37 in general plant growth and development.
Introduction
RNA has over 100 different post-transcriptional modifications that have been identified in organisms across all three domains of life (1–5). While several RNA modifications have been recently identified on mRNAs in plants and animals, tRNAs are still thought to be the most extensively modified cellular RNAs (6–9). These tRNA modifications are introduced at the post-transcriptional level by specific enzymes. These enzymes recognize polynucleotide substrates and modify individual nucleotide residues at highly specific sites. Some tRNA modifications have been shown to have a clear biological and molecular function (10, 11). Several tRNA modifications around the anticodon have been demonstrated to have crucial functions in translation, for example, by enhancing decoding (12), influencing the propensity to ribosomal frameshifting or facilitating wobbling (13–15). Modifications distal to the tRNA anticodon loop can also directly influence the tRNA recognition and/or translation process (16) or can have roles in tRNA folding and stability (1, 17). However, the precise functions of many tRNA modifications still remain unknown despite often being conserved across species. Often, loss of a tRNA modification does not negatively impair cell growth or cell viability under standard laboratory growth conditions (18). However, under environmental stress, such mutants display a discernible phenotype (10).
The tRNA anticodon loop position 37 is important to maintain translational fidelity and efficiency (11, 19) and almost all tRNAs are modified at this site. In bacteria, N1-methylation of guanosine at position 37, m1G37, is performed by TrmD-type enzymes (20). In Archaea and Eukaryotes, the m1G37 modification is enzymatically performed by functionally and evolutionarily unrelated Trm5-type proteins (21). In yeast Saccharomyces cerevisiae, m1G37 methylation of cytoplasmic and mitochondrial tRNAs is performed by Trm5p and complete loss of function mutants are lethal (22, 23). In humans, TRMT5 (tRNA methyltransferase 5), catalyzes the formation of m1G37 in vivo, and mitochondrial tRNAPRO and tRNALEU have been found to also contain m1G37 (24, 25). Mutations in human TRMT5 cause patients to have multiple respiratory-chain deficiencies and a reduction in mitochondrial tRNA m1G37 (11). In plants, the enzymes catalysing m1G37 methylation have not been identified to date.
N1-methylguanosine has been described in eukaryotic tRNAs at two positions; at position 37 catalysed by Trm5, and the other at position 9 catalysed by Trm10 (26). Trm5 in humans, yeast, and Pyrococcus abyssi has been described as having multifunctionality (23, 24, 27). In contrast to TrmD which requires a guanosine at position 37, human Trm5 can also recognise and methylate inosine at position 37 with some limited activity (24). Similarly,Trm5p have also been shown to catalyse inosine to m1I modification in yeast in a two-step reaction, where the first adenosine-to-inosine modification was mediated by Tad1p (10, 18, 22, 28). As m1G is an intermediate during the modification of guanosine to wybotusine (yW), tRNAs from trm5 mutants were also devoid of yW (22). The yeast Trm5p protein has been shown to be localised to the cytoplasm and mitochondria (23) and it is thought that Trm5p protein present in the mitochondria is required to prevent unmodified tRNA affecting translational frameshifting (22). In the unicellular parasite Trypanosome brucei, Trm5 was located in both the nucleus and mitochondria and reducing Trm5 expression led to reduced mitochondria biogenesis and impaired growth (29). Interestingly, Trm5 and m1G37 were shown to be essential for mitochondrial protein synthesis but not cytosolic translation (29).
Little is known about tRNA modifying enzymes in plants. Here, we report the identification and functional analysis of AtTrm5 (At3g56120) from the model plant Arabidopsis thaliana. We demonstrate that Attrm5 mutants are slower growing, have reduced shoot and root biomass and display late flowering. Furthermore, we demonstrate that in vitro TRM5 is required for m1G37 and m1I37 methylation at the position 3’ to the anticodon and in vivo tRNAs enriched from Attrm5 plants have reduced m1G and m1I.
Methods
Plant material and root growth experiments
Arabidopsis thaliana (Columbia accession) wild type and mutant plants were grown in Phoenix Biosystems growth under metal halide lights as previously described (30). For plate experiments, seeds were first surface sterilized, plated on ½ MS medium supplemented with 1% sucrose and sealed as previously described (30, 31). All plants were grown under either long-day photoperiod conditions of 16 h light and 8 h darkness or short-day photoperiods of 10 h light and 14 h darkness.
Characterization of the mutant alleles, trm5-1 (SALK_022617) and trm5-2 (SALK_032376) are as described previously (32). The tad1-2 mutant was used as described previously (10). Nucleotide sequence data for the following genes are available from The Arabidopsis Information Resource (TAIR) database under the following accession numbers: TRM5 (At3g56120), At4g27340, At4g04670, and TAD1 (At1g01760).
Analysis of root phenotypes was carried out on 11-day-old seedlings grown on ½ MS agar plates. A flatbed scanner (Epson) was used to non-destructively acquire images of seedling roots grown on the agar surface. Once captured, the images were analyzed by software package RootNav (33, 34).
Plasmid construction and generation of transgenic plants
For the 35SCaMV:TRM5 construct, the full-length genomic region of At3g56120 including the 5’UTR and 3’UTR was amplified from Col-0 genomic DNA template with primers provided in (Supplementary Table S1) and cloned into Gateway entry vector pENTRTM/SD/D-TOPO (Invitrogen). The insert was sequenced and then cloned into the binary destination vector pGWB5 by an LR recombination reaction, using the Gateway cloning system following the manufacturers protocol (Invitrogen), resulting in the 35S:TRM5 construct. For the TRM5Pro::TRM5 construct, the full-length genomic region of At3g56120 that included the promoter, 5’UTR and 3’UTR was amplified from Col-0 genomic DNA template with primers provided in (Supplementary Table S1) and cloned into Gateway entry vector PCR8 TOPO-TA (Invitrogen). The insert was sequenced and then cloned into the a modified (35S promoter removed) destination pMDC32 vector, using the Gateway cloning system (35) following the manufacturers protocol (Invitrogen), resulting in TRM5Pro::TRM5. The 35S:TRM5 construct was transformed into A. thaliana wild type Col-0 plants or trm5-1 mutant plants by Agrobacterium-mediated floral dip method respectively (36). The TRM5Pro::TRM5 construct was transformed into trm5-2 mutant plants by Agrobacterium-mediated floral dip method. Transgenic plants were selected on ½ MS media supplemented with 50 μg ml−1 kanamycin. TRM5 transcript abundance was assessed in at least five independent T1 plants using qRT-PCR and two lines showing the highest TRM5 transcript levels were carried through to homozygous T3 generation for phenotypic analysis.
Sub-cellular localization of TRM5
For analysis of subcellular localization of TRM5, the construct described above was introduced into A. tumefaciens strain GV3101 and transiently expressed in 5-week-old Nicotiana benthamiana leaves. Fluorescence was analyzed using a confocal laser-scanning microscope (Zeiss microscope, LSM700) and excited with 488-nm line of an argon ion laser. GFP fluorescence was detected via a 505- to 530-nm band-pass filter. The cut leaves were immersed in 10 μM 4′, 6-diamidino-2-phenylindole (DAPI) at room temperature for 45 min, and then washed with PBS for 3 times (5 min each). The blue fluorescence of DAPI was imaged using 404-nm line for excitation and a 435- to 485nm band pass filter for emission.
Shoot apical meristem sections
14, 18, or 22-day-old seedlings were fixed for 1 day in FAA containing 50% ethanol, 5% acetic acid and 3.7% formaldehyde. The samples were then dehydrated through an ethanol series of five one-hour steps (50%, 60%, 70%, 85%, 95% ethanol) ending in absolute ethanol (100%). The ethanol was gradually replaced with Histoclear containing safranine to stain the tissue, as following: incubated in a Histoclear series (75:25, 50:50, 25:75 Ethanol: Histoclear) for 30 min and followed by twice one-hour incubation in 100% Histoclear. The paraffin was polymerised by baking overnight at 60 °C, and the samples were embedded in paraffin. Sections were cut, attached to slides and dried on a slide warmer overnight at 42 °C to allow complete fixation. The shoot apical meristem was observed using light microscopy.
Quantitative RT-PCR
For the transcription profiling of flowering-related genes and circadian clock-related genes, 17-day-old seedlings were sampled from Zeitgeber time (ZT) 1 and collected every 3h during the day and night cycles, respectively. Total RNA was extracted the leaf samples using Trizol reagent (Invitrogen). The relative expression levels of AtTRM5 were determined using quantitative real-time PCR with gene-specific primers (Supplementary Table S1). Real-time PCR was performed using the StepOnePlus real-time PCR system (Applied Biosystems) using Absolute SYBR Green ROX mix (Applied Biosystems) for quantitation. Three biological replicates were carried out for each sample set. The relative expression was corrected using a reference gene EF1alpha (At5g60390) and calculated using the 2−ΔΔCt method as described previously (10).
mRNA-sequencing
Total RNA, 1 ug, was extracted from 20-day-old Arabidopsis leaf samples using Trizol reagent (Invitrogen) and purified using the RNAeasy Mini RNA kit (Qiagen). One hundred nanograms of RNA were used for RNA-seq library construction according the manufacturer’s recommendations (Illumina). First-strand cDNA was synthesized using SuperScript II Reverse Transcriptase (Invitrogen). After second strand cDNA synthesis and adaptor ligation, cDNA fragments were enriched, purified and then sequenced on the Illumina Hiseq X Ten. Three biological replicates were used for RNA-seq experiments.
tRNA purification and tRNA-sequencing
Total RNA was isolated from wild type and trm5 10-day-old Arabidopsis seedlings using the Spectrum Plant total RNA kit (SIGMA-ALDRICH) and contaminating DNA removed using DNase I (SIGMA-ALDRICH). To enrich for tRNAs, 10μg of total RNA was separated on a 10% polyacrylamide gel, the region containing 65-85 nts was removed and RNA was purified as previously described (31). Purified tRNAs were used for library construction using NEB Ultradirectional RNA library kit. Given the short sequences of tRNAs, the fragmentation step of the library preparation was omitted, and samples were quickly processed for first-strand cDNA synthesis after the addition of the fragmentation buffer. The remaining steps of library construction were performed as per the manufacturer’s instructions. Illumina sequencing was performed on a MiSeq platform at The Australian Cancer Research Foundation (ACRF) Cancer Genomics Facility, Adelaide.
Yeast complementation
AtTrm5 (At3g56120) and ScTrm5 (YHR070W) was PCR amplified from cDNA and cloned into pYE19 using a Gibson assembly reaction (NEB). Mutant AtTrm5 (R166D) was generated by synthesising gene blocks (IDT) with nucleotides that mutated the translated proteins at R166 and the gene block was cloned into pYE19 using a Gibson assembly reaction (NEB). Yeast Δtrm5 (Mat a, hisD1, leu2D1, met15D0, trm5:KanMX) was previously described (37). Recombinant plasmids were transformed into Δtrm5 mutant strain and the resulting strain was analysed for growth phenotypes and m1G nucleoside levels.
AtTrm5, AtTAD1, ScTrm5 protein expression and purification and tRNA methylation
Full length AtTrm5, mutant AtTrm5 (R166D) and AtTAD1 (At1g01760) cDNAs were cloned into pGEX resulting in GST-AtTrm5, GST-AtTRM5-mutant, GST-TAD1, respectively. Mutant AtTAD1 (E76S) was generated by synthesising a gene block (IDT) with mutated nucleotides and the gene block was cloned into pGEX using a Gibson assembly reaction (NEB) resulting in GST-TAD1 mutant. IPTG (0.5 μM) was used to induce expression of the proteins and the recombinant proteins were purified on a GST resin column (ThermoFisher Scientific). tRNA-Asp-GUC or tRNA-Ala-AGC were transcribed in vitro with T7 polymerase (Promega). Methylation reactions were performed in 100 mM Tris-HCl, 5mM MgCl2, 100 mM KCl, 2mM DTT, 50mM EDTA, 0.03 mg/mL BSA and 25 μM AdoMet. Substrate tRNA was provided in a final concentration of 1-5 μM, AtTrm5 or AtTrm5 mutant proteins from 6.0 to 12 μM and AtTAD1 or AtTAD1 mutant proteins at 5.0 μM.
Bioinformatics analysis of mRNA-seq and tRNA-seq
Global Mapping
Reads were first adapter trimmed using Trim galore v0.4.2 (http://www.bioinformatics.babraham.ac.uk/projects/trimgalore/) with default parameter. The quality of the trimmed reads were checked using Fastqc (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and ngsReports (38). Reads were then globally mapped to Arabidopsis reference genomes (TAIR10) (39) using STAR v2.5.3 (40). Mapped reads were counted using featureCounts (41). The raw read counts were normalized using sample size factor for sequence depth and differential expression analysis between wild type and trm5-1 mutant samples was performed using DESeq2 v1.18.1 (42). The tRNA-enriched reads were mapped to tDNA reference acquired from GtRNAdb (generated from tRNA-Scan SE v2.0) of Arabidopsis thaliana derived from TAIR10 reference genome (39, 43–45) using segemehl v0.2.0 (45). The mapped reads were then processed for variant calling using GATK v3.7 (46). Haplotype Caller in GATK v3.7 was implied to call for variants, with the variant filtered using hard filtering as recommended.
Proportion estimation
The common SNPs identified in wild type and trm5-1 samples were cross compared. SNPs at position 37 were extracted and analysed using vcftools (47). Changes in base pair modifications were indicated by base substitution due to the property of next generation sequencing as mentioned in (11, 48). The ratio of the expected A-to-T conversion in wild type samples and both the ratio of A-to-T (indicating no change in comparison to wild type) and A-to-G in trm5-1 were analyzed as an indication of m1I depletion. Identification of other base changes was attempted to identify putative m1I modification.
Sequence analysis of tRNA editing
tRNA purification and tRNA editing analysis were performed as described previously (10). Cytosolic tRNA-Ala (AGC) were amplified by reverse transcriptase (RT)-PCR with specific primers (tRNA-Ala-f and tRNA-Ala-r, Supplementary Table S1), and purified PCR products were directly Sanger sequenced.
TMT-based proteome determination and data analysis
Total protein was extracted from 20-day-old Arabidopsis leaf samples and purified according to a method described by (49). Protein digestion was performed according to FASP procedure described previously (50), and 100 ųg peptide mixture of each sample was labelled using TMT reagent according to the manufacturer’s instructions (Thermo Fisher Scientific). LC-MS/MS analysis was performed on a Q Exactive mass spectrometer (Thermo Fisher Scientific) that was coupled to Easy nLC (Thermo Fisher Scientific). MS data was acquired using a data-dependent top20 method dynamically choosing the most abundant precursor ions from the survey scan (200-1800 m/z) for HCD fragmentation (Shanghai Applied Protein Technology Co., Ltd). Determination of the target value is based on predictive Automatic Gain Control (pAGC). SEQUEST HT search engine configured with Proteome Discoverer 1.4 workflow (Thermo Fischer Scientific) was used for mass spectrometer data analyses. The latest Arabidopsis protein databases (2018) was downloaded from http://www.arabidopsis.org/download/index-auto.jsp?dir=/downloadfiles/Proteins and configured with SEQUEST HT for searching the datasets. The following screening criteria was deemed as differentially expression: fold-changes >1.2 or <0.83 (up- or down-regulation) and P < 0.05 were considered significant.
tRNA nucleoside analysis
tRNAs were purified as previously described (31). Twenty-five μg of tRNAs were digested with P1 nuclease (Sigma-Aldrich) and 1.75 units of calf intestine alkaline phosphatase in 20 mM Hepes–KOH (pH 7.0) at 37 °C for 3 h. API 4000 Q-Trap mass spectrometer coupled with LC-20A HPLC system was used for nucleoside separation and detection, with a diode array UV detector (190–400 nm) set for positive ion mode.
RESULTS
Identification of At3g56120 as a TRM5 homolog
In yeast (Saccharomyces cerevisiae), m1G37 nucleoside modification is catalysed by ScTrm5 (22). We searched for Arabidopsis thaliana homologs by using blastp and HMMER and identified a high confidence candidate, At3g56120, with 49% similarity to ScTrm5 (Supplementary Figure S1). Alignment of At3g56120 with yeast, human, Drosophilia, Pyrocococcus, and Methanococcus Trm5 homologs identified three conserved motifs and catalytically required amino acids (R166, D192, E206) present in At3g56120 (Supplementary Figure S1). We subsequently will refer to At3g56120 as AtTrm5. In the Arabidopsis genome, AtTrm5 has homology to At4g27340 and At4g04670 and both genes were recently been named as Trm5B and Trm5C respectively (Supplementary Figure S1 and (51)). We have also identified Trm5 homologs in algae, bryophytes and vascular plants (Figure 1A).
To functionally characterize AtTrm5 we isolated two T-DNA insertions, SALK_022617 and SALK_032376, and identified homozygous mutant plants for each insertion (Figure 1B). SALK_022617 and SALK_032376 were named trm5-1 and trm5-2, respectively. Next, we measured AtTrm5 mRNA abundance in both mutants and detected almost no transcripts in both mutants (Figure 1C). We generated a genomic construct of AtTrm5 that contained the endogenous promoter, coding region and UTRs, transformed the construct into trm5-2 and demonstrated that the AtTrm5 mRNA levels were similar in two complemented lines when compared to wild type plants. Subsequently, the extracted tRNAs from wild type and the trm5 mutants were purified, digested and modified nucleosides measured by mass spectrometry (Figure 1D). In both trm5 mutant alleles, nucleoside m1G levels were reduced to about 30% of the wild type and m1G levels were restored to wild type levels in both complemented lines (Figure 1D). Nucleoside m1G is present at tRNA positions 9 and 37 (22), therefore the residual m1G levels in both trm5 mutants may be the result of tRNA m1G at position 9.
In yeast, Trm5 has also been reported to also catalyse m1I on tRNAs (10, 22, 51). We therefore measured m1I levels in purified tRNAs from both Arabidopsis trm5 mutants and wild type control plants. In both trm5-1 and trm5-2 mutant alleles, nucleoside m1I levels were reduced to about 10% of wild type levels and were restored to wild type levels in plants of both complemented lines (Figure 1D). In summary, in Arabidopsis thaliana we identified At3g56120 as a Trm5 homolog in Arabidopsis thaliana, identified two AtTrm5 mutant alleles, trm5-1 and trm5-2, and both mutants showed a significant reduction in m1G and m1I.
AtTRM5 m1G methyltransferase activity
To test AtTrm5 m1G methyltransferase activity in vivo, a yeast, a Δtrm5 mutant strain in yeast (Saccharomyces cerevisiae, Sc) that is defective for the tRNA m1G37 modification was used for genetic complementation. The mutant not only has defective tRNA m1G37 but also a slow growth phenotype when compared to wild type or a congenic strain (Figure 2A). Full-length ScTrm5 and AtTrm5 were cloned into yeast expression vectors. From the AtTrm5 expression vector, a catalytically inactive mutant Attrm5 R166D was generated by site-directed mutagenesis. After the three vectors had been transformed into the yeast Δtrm5 mutant, cell growth and m1G nucleoside levels were observed (Figure 2). Not only were the slow growth and nucleoside levels rescued when expressing ScTrm5 but they were also rescued when expressing AtTrm5 (Figure 2A and B). However, the catalytically inactive Attrm5 R166D did not rescue either the slow growth or m1G nucleoside levels (Figure 2A and B).
To test the m1G methyltransferase activity of AtTRM5 in vitro, we incubated purified recombinant proteins with tRNA substrates and measured the m1G levels. We expressed AtTRM5 as a GST fusion protein and purified the recombinant GST-AtTRM5 protein. We also generated a catalytically inactive GST-AtTRM5 recombinant protein by using site directed mutagenesis, expressed and purified the GST-AtTRM5 recombinant fusion protein. In yeast, ScTrm5 methylates tRNA-His-GUG, tRNA-Leu-UAA, tRNA-Asp-GUC and other tRNA isoacceptors (10, 22, 51). Yeast tRNA-Asp-GUC RNA transcripts were generated in vitro by using T7 polymerase, the tRNA transcripts incubated with the recombinant fusion proteins in the presence of the methyl donor S-adenosyl-methionine (AdoMet) and m1G nucleoside levels were measured (Figure 2D). m1G was detected only when AtTRM5 was provided (Figure 2D) and in a dosage dependent manner. No m1G was detected when the catalytically inactive mutant AtTRM5 was provided (Figure 2D). To test the specificity of the methyltransferase activity on tRNA-Asp guanine at position 37, the guanine nucleotide was mutated to an adenine nucleotide (tRNA-Asp-A37) and the m1G methyltransferase activity was measured. No m1G was detected after incubation with the fusion proteins (Figure 2D). The overall results of the yeast complementation experiments suggest that guanosine methylation occurred at position 37 of tRNA.
AtTRM5 tRNA m1I methyltransferase activity
Previously in plants, TAD1 was demonstrated to oxidatively deaminate adenosine at position 37 of tRNA-Ala-(UGC) to inosine, and subsequently methylated by an unknown enzyme to N1-methylinosine (m1I; Figure 3A). Human TRM5 has been reported to methylate tRNA I37 but with limited activity (24). Given our observation that Attrm5 mutant plants had reduced m1I (Figure 1D), we asked the question whether AtTRM5 has methyltransferase activity on tRNA I37. We developed a two-step approach, whereby purified AtTAD1 was first incubated with the substrate tRNA-Ala-A37 to produce tRNA-Ala-I37 and then the inosine methyltransferase activity of AtTRM5 was measured by incubating AtTRM5 with the tRNA-Ala-I37 substrate. Previously, in yeast ScTAD1 was demonstrated to deaminate tRNA-Ala-A37 to tRNA-Ala-I37 in vitro (22) and Arabidopsis thaliana tad1 mutants were reported to have reduced tRNA-Ala-I37 (10). We expressed AtTAD1 as a GST fusion protein and purified the recombinant GST-AtTAD1 protein. We also generated a catalytically inactive GST-AtTAD1 mutant recombinant protein by using site-directed mutagenesis, and expressed and purified the GST-AtTAD1 mutant fusion protein. In a two-step assay, yeast tRNA-Ala-UGC RNA transcripts were generated by in vitro transcription using T7 RNA polymerase, the tRNA transcripts were then incubated with the recombinant fusion proteins in the presence of Mg2+ and methyl donor S-adenosyl-methionine (AdoMet), and the m1I nucleoside levels measured (Figure 3B). m1I was detected only when GST-AtTAD1 and GST-AtTRM5 were provided, and its production occurred in a dosage-dependent manner (Figure 3B). No m1I was detected when the catalytically inactive mutants AtTAD1 E76S or AtTRM5 R166D were provided (Figure 3B). To test the specificity of the methyltransferase activity on tRNA-Ala alanine at position 37, the alanine nucleotide was mutated to a cytosine nucleotide (tRNA-Ala-C37) and the m1I methyltransferase activity was measured. No m1I was detected after incubation with the fusion proteins (Figure 3B). Collectively, these findings interestingly suggest that inosine methylation also occurrs at position 37, in addition to guanosine methylation.
To test the AtTRM5 inosine methyltransferase activity in vivo, we measured tRNA position modifications by cDNA sequencing from either mutant or wild type plants (Figure 3C and D). In the sequencing assay, modification events at position 37 of tRNAs can be directly detected by sequencing of amplified cDNA obtained by reverse transcription and comparison to the DNA reference sequence as inosine is read as 1 guanine (G) and m1I is read as thymine (T) by the reverse transcriptase (11, 48). As expected, we detected substitutions of A in the reference to T at position 37 for tRNA-Ala-TGC and tRNA-Ala-CGC by using Illumina sequencing in wild-type plants which 11 is consistent with the presence of m1I (Figure 3C). m1I modifications have previously been described in tRNA-Ala at position 37 in eukaryotes (10, 28, 52). In the trm5-1 mutant, no T’s were detected at position 37 (Figure 3C) which is consistent with AtTRM5 acting as a tRNA m1I methyltransferase at position 37. Based on our in vitro assay results, AtTAD1 first deaminates tRNA-Ala-A37 to tRNA-Ala-I37, and then AtTRM5 methylates tRNA-Ala-I37 to tRNA-Ala-m1I37. We confirmed this pathway in tad1 and trm5 mutant plants by Sanger sequencing of tRNA-Ala-AGC (Figure 3D). As expected, at position 37, A was substituted to T in the wild type, whereas in trm5 mutants a G and in tad1 mutants an A were observed (Figure 3D). These sequencing results are consistent with AtTAD1 first deaminating A37 to I37 as previously reported by (10), and AtTRM5 then methylating I37 to m1I. We also attempted to detect the putative loss of m1G in the tRNA-sequencing data, as it has been reported that m1G is prone to be called as a T in sequencing (48). However, this was not observed in our datasets. Together, our in vitro and in vivo data provide support for AtTRM5 possessing tRNA m1I methyltransferase activity.
AtTRM5 is localized to the nucleus
In yeast, ScTRM5 is localized to both the nucleus and mitochondria (23, 53). Localisation to mitochondria is thought to be important as yeast strains with only nuclear-localized ScTRM5 exhibited a significantly lower rate of oxygen consumption (23). In order to determine to which subcellular compartment(s) AtTRM5 is localized in Nicotiana benthamiana, we fused TRM5 to the Green Fluorescent Protein (GFP) reporter, transiently infiltrated the construct into leaves and performed laser-scanning confocal microscopy to detect GFP fluorescence. To unambiguously identify the nucleus, we stained the cells with DAPI. When we imaged the cells (n=100), we observed distinct DAPI fluorescence in a single large circular structure per cell, as expected for the nucleus (Figure 4). Next, we imaged the same cells for GFP fluorescence (Figure 4C) and overlayed the DAPI and GFP fluorescence. The two fluorescence signals showed perfect overlap (Figure 4D). We then searched for nuclear localisation signals (NLS) using the LOCALIZER (http://localizer.csiro.au/), LocSigDB (http://genome.unmc.edu/LocSigDB/), and cNLS mapper (http://nls-mapper.iab.keio.ac.jp/cgi-bin/NLS_Mapper_form.cgi) programs (54–56). While LOCALIZER and LocSigDB did not predict any canonical or bipartite NLS, cNLS mapper predicted with high confidence a 29 amino acid importin α-dependent NLS, QKGCFVYANDLNPDSVRYLKINAKFNKVD, that starts at amino acid 236. Previous finding in yeast suggested that no common canonical or bipartite NLS was detected in ScTrm5 (22), which explains the outcome from LOCALIZER and LocSigDB. In summary, we conclude that, unlike in yeast, AtTRM5 in Arabidopsis is only localized to the nucleus and may be imported from the cytoplasm into the nucleus by the importin α-dependent pathway.
Proteins involved in photosynthesis are affected in trm5 mutant plants
Next, we performed a proteomic analysis to identify proteins that differentially accumulate in trm5-1 plants when compared to the wild type by using the Tandem Mass Tag (TMT) method (Figure 5). A total of 61571 peptide-spectrum match (PSM) were recorded, corresponding to 29011 peptides and 23055 unique peptides, respectively. 5242 protein groups were identified by blastp searches against the TAIR_pep database. Proteins with fold changes ≥1.20 or ≤0.83 and a significance level of P ≤0.05 were considered to be differentially expressed. In this way, a total of 263 proteins were identified (Supplementary Table S2). 102 proteins were upregulated, and 151 proteins were downregulated in trm5 (Figure 5A). GO annotation of these differentially accumulating proteins revealed enrichment of the GO terms thylakoid, chloroplast, and photosystem I (Figure 5B). KEGG annotation revealed enrichment of proteins involved in photosynthesis, and photosynthetic proteins (Figure 5C). Taken together, the GO and KEGG analysis demonstrated that most differentially accumulating proteins are involved in processes related to photosynthesis.
Defects in tRNA m1G methylation can be expected to affect mRNA translation, particularly of proteins that have high numbers of affected codons. Therefore, we were interested in identifying genes that showed reduced expression at the protein level in our proteomics analysis of trm5 plants, but no detectable reduction in mRNA abundance. To identify such mRNAs, we performed RNA-seq on wild type and trm5 plants. We identified 1186 transcripts that were reduced in abundance in trm5 and 580 transcripts that were increased in abundance by at least 2 fold and hierarchically clustered these transcripts (Supplementary Figure S4 and Supplementary Table S4). Comparison of the RNA-seq and proteomics datasets identified 133 proteins with reduced abundance, but with no detectable reduction in mRNA abundance (Supplementary Figure S4). We further inspected the data by selecting four candidate proteins with the highest fold change reported in the proteomics data (Table 1). From the selected candidate proteins, we discovered that three of the differentially expressed proteins reported in the proteomics data were not differentially expressed in the RNA-seq data, indicating that there is no change in transcripts levels leading to the fluctuation of their corresponding proteins. Only one protein candidate (AT2G45180.1) showed decreased fold change of mRNA, which correlates with the decrease of its corresponding protein reported in the proteomics data.
Finally, we performed a codon usage frequency analysis for this class of genes and detected no codon bias towards triplets read by both m1I and m1G modified tRNAs (Supplementary Figure S4).
AtTRM5 is involved in leaf and root development and flowering time regulation
Before undertaking growth measurements, we grew wild-type Columbia, trm5-1, two complemented lines, and two overexpression lines together under long-day conditions, harvested and dried the seeds to minimise any maternal or environmental effects. To observe the early growth stages of seedlings, we grew the six lines (wild type, trm5-1, two complemented lines and two overexpression lines) on plates for 10 days (Figure 6A). The trm5-1 seedlings were noticeably smaller than the wild type. In contrast, no clear differences were evident between wild type, the complemented and overexpression lines. To rule out the possibility that the reduced growth in trm5-1 seedlings was due to slower germination, we measured the germination of trm5-1 and wild type and no difference was observed (Supplementary Figure S2). Reduced growth of trm5-1 roots was also evident on plate-grown plants (Figure 6B). Interestingly, trm5-1 primary, lateral and total (primary + lateral) root lengths were reduced in trm5-1 when compared to wild-type plants (Supplementary Figure S3). We also measured the lateral root number and found that trm5-1 plants had reduced numbers when compared to the wild type (Supplementary Figure S3). In contrast, no differences in the root growth were evident upon comparison of the wild type and the complemented lines. In TRM5 overexpression lines, primary and lateral root lengths were slightly longer than in the wild type (Figure 6B and Supplementary Figure S3).
At inflorescence emergence in wild-type plants grown under long days, we observed reduced rosette leaf numbers, smaller leaves and reduced fresh weight in trm5-1 plants (Figure 6C and D; Supplementary Figure S2). Sectioning of the shoot apical meristems of wild type and trm5-1 plants at wild-type floral transition, confirmed that trm5 plants were later flowering (Supplementary Figure S2). We measured the flowering time of wild type, trm5-1, complemented, and overexpression lines under both short and long days and observed that mutants produced more rosette leaves and flowered later than the wild type (Figure 7A; Supplementary Figure S2). Plants overexpressing TRM5 flowered slightly earlier than wild type under both long and short-day conditions (Figure 6D; Supplementary Figure S2).
AtTRM5 is involved in circadian clock and flowering time gene expression
To explore the molecular basis of delayed flowering in trm5-1, we measured the mRNA abundance of circadian clock and flowering time related genes by quantitative RT-PCR over a 24-hour period (Figure 7). In trm5-1 plants, lower abundance of the clock genes TIMING OF CAB EXPRESSION 1 (TOC1), and Pseudo Response Regulators 7 (PPR7), and the flowering time regulator genes GIGANTEA (GI), CONSTANS (CO) and FLOWERING LOCUS T (FT) were observed at ZT10 to ZT22. The reduced abundance of the flowering time regulators GI, CO, and FT in trm5-1 mutants correlates with delayed flowering. As expected, the downstream floral meristem identity gene LEAFY (LFY) had lower abundance at almost all tested time points in the trm5 mutant when compared to wild type (Figure 7B). Together these results support a role for TRM5 in plant growth, development, and flowering time regulation.
Discussion
The discovery of m1G and m1I at position 37 in tRNAs of a wide range of eukaryotic and prokaryotic organisms underscores its importance as a key regulator of tRNA function and, presumably, translation (11, 20, 23). Previous studies in bacteria have shown that m1G37 is required for translational fidelity (20, 57, 58) and that mutations in the enzymes catalyzing m1G37 severely impact growth or cause lethal phenotypes (11, 23, 29). In eukaryotes, m1G37 modification requires the methyltransferase TRM5. Here we report that in plants, trm5 mutants have a 60% reduction in m1G and m1I levels, display severely reduced growth and delayed transition to flowering. This is somewhat similar to human patients that are heterozygous for one mutant and one functional allele of Trm5. They show childhood failure to thrive and exercise intolerance symptoms (11).
With the 60% reduction of m1G in trm5 plants, it is reasonable to anticipate a significant impact on protein translation due to the reduced stacking effect of m1G37 on base-pairing between position 36 and the first nucleoside in translated mRNA codons. We observed a reduction in abundance of proteins involved in ribosome biogenesis and photosynthesis, and this is likely to account for the observed reduced growth of the mutant plants. In the future, it would be interesting to test if translational errors, such as ribosome stalling or frame shifting, occur in trm5 plants which would be similar to what has been previously reported in bacteria with reduced tRNA m1G (22).
We localized Arabidopsis TRM5 to the nucleus which is in contrast to the cytoplasmic and mitochondrial localisation in other eukaryotes: Trypanosoma brucei, Homo sapiens (HeLa cells) and Saccharomyces cerevisiae (11, 23, 29), although in one study S. cerevisiae Trm5 has also been localized exclusively to the nucleus (53). Interestingly, in yeast, Trm5 acts in the nucleus to form m1G on retrograde imported tRNAPhe after initial export from the nucleus and subsequent splicing at the mitochondrial outer membrane. As retrograde tRNA import is conserved from yeast to vertebrates (59, 60), it is tempting to speculate that TRM5-mediated m1G formation occurs in retrograde imported tRNAs in Arabidopsis.
Direct comparison of RNA-seq and proteomics data has been challenging as variation in protein abundance in proteomics datasets can be confounded by multiple factors. Several factors can cause fluctuation in protein levels with no change of mRNA abundance: mRNA transcript abundance, translation rate, and translation resources availability (as reviewed in (as reviewed in (61)). This may explain the results of the initial comparison between our RNA-seq and proteomics data, and the seeming discrepancies in the derived sets of differentially expressed genes (Supplementary Figure S4). In our RNA-seq data, microtubule-related genes appeared to be significantly downregulated (AT1G21810, AT1G52410, AT2G28620, AT2G44190, AT3G23670, AT3G60840, AT3G63670, AT5G67270, AT4G14150, AT5G27000). However, we could not detect any differential expression of these proteins in our proteomics dataset. This is not unexpected, as it has been reported that protein and transcript abundances only rarely correlate in data analysis (61). However, it has been shown that transcript abundance can still be used to infer protein abundance (62). Among the selected four candidate proteins (Table 1), we could observe only one event of protein downregulation due to decreased transcript level: the lipid transfer protein (AT2G45180) which is involved in lipid transport in chloroplasts. Lipid transport is crucial in the formation of photosynthetic membranes in plants (63). As several lipids are important functional components of thylakoidal protein complexes involved in photosynthesis, the impacted lipid transport and lipid synthesis revealed by the GO term analysis may contribute to the observed dysregulation of photosynthesis-related genes. The remaining three candidate genes displayed no changes in transcript levels, but one candidate gene showed upregulation and the other two downregulation at the protein level. The exact function of pentatricopeptide repeat (PPR-like) superfamily protein (AT1G03540.1) is unknown, but the protein was suggested to play a significant role in post-transcriptional modification of tRNAs (64). The P-glycoprotein 6 (AT2G39480) belongs to the large P-glycoprotein family and is known to play a role in mediating auxin transport, and disturbed auxin transport is known to affect plant growth (65). Our proteomics data suggested that the auxin transport is affected (AT5G35735, AT3G07390, AT4G12980, AT5G35735) in the trm5 mutant. Ferric reduction oxidase 8 (AT5G50160/AtFRO8) has been shown to participate in iron reduction and was implicated in leaf vein transport (66). We proposed that disturbed photosynthesis in trm5 mutant plants is a secondary effect of dysregulated transport processes.
The role of post-transcriptional RNA modifications in tRNA and mRNA metabolism and their impact on plant growth and development in plants are only beginning to be elucidated. Here, we have described the TRM5-mediated m1G methylation in tRNAs and identified crucial links between this modification, photosynthesis, and plant growth. It appears likely that the many other tRNA modifications in plant tRNAs also play important roles in translation and/or translational regulation which remain to be discovered.
Data Access
The data sets supporting the results of this article are available in NCBI’s GEO database repository and are accessible through GEO Series accession number GSE114898. Proteomics raw data has been deposited on iProX, with under accession number IPX0001222000.
Author’s contributions
Experiments were designed by I.S. and W.Z. Experiments were performed by R.D., PQ.N., I.S., Q.G., S.S., D.F., H.W., M.Z., T.D. and W.Z. Data analysis was performed by I.S., PQ.N., J.L., W.Z., and R.B. The manuscript was prepared and edited by PQ.N., Q.G., W.Z., I.S., and R.B. All authors read and approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Acknowledgements
We thank the staff at The Australian Cancer Research Foundation (ACRF) Cancer Genomics Facility, Adelaide. This research was partially supported by ARC grant FT130100525, partially funded an Australia-China Science and Research Fund grant ACSRF48187 awarded to I.S. and an APA awarded to PQ.N and J.L. This research was also partially supported by the National Natural Science Foundation (31570234) awarded to W.Z., W.Z. was supported by the Innovation Program of Chinese Academy of Agricultural Sciences and the Elite Youth Program of the Chinese Academy of Agricultural Science.
Footnotes
↵† The authors wish it to be known that, in their opinion, the first 2 authors should be regarded as joint first authors.