Loss of both wobbleU34 modifications in mcm5s2U tRNAs impairs rRNA biosynthesis, growth, and development in Arabidopsis thaliana

In eukaryotic cells, the first anticodon uridine base of cytosolic tRNALys(UUU), tRNAGlu(UUC), and tRNAGln(UUG) (wobbleU₃₄) is post transcriptionally modified through adding a methoxycarbonylmethyl group and substituting a sulfur at the fifth and second carbons, respectively, to form 5-methoxycarbonylmethyl-2-thiouridine. The simultaneous deletion of these two wobbleU₃₄ modifications causes lethality in mice and flies. Here, we report that deletion of both wobbleU₃₄ modifications results in severe growth retardation and morphological abnormalities in Arabidopsis thaliana. The results of Ribo-seq and RNA-seq analyses indicate that the ribosome occupancy of many transcripts is substantially different in the Arabidopsis mutant lacking both wobbleU₃₄ modifications compared with the wild type. Gene Ontology analysis shows that genes with altered ribosome occupancy are categorized as having RNA-binding properties. Several pre-rRNA processing precursors accumulate in the mutant lacking both wobbleU₃₄ modifications. In the mutant, ribosomes likely pause when the cognate [A/G/C]AA codons of tRNALys(UUU), tRNAGlu(UUC), and tRNAGln(UUG) are positioned at the A site during translation of transcripts encoding proteins involved in pre-rRNA processing, such as DRH1 and ATRH7. These findings suggest that deleting both wobble U₃₄ modifications impairs rRNA maturation, leading to the accumulation of rRNA precursors adversely affecting growth and morphogenesis in plants.

1 Introduction

Posttranscriptional RNA modifications affect a variety of functions in gene transcription and translation processes to maintain cellular homeostasis (Delaunay and Frye, 2019). Many nucleoside modifications occur in various tRNAs, and modifications mostly found in the first anticodon nucleosides of specific tRNAs help maintain translation efficiency (Suzuki, 2021). The uridine at the first anticodon position of tRNALys(UUU), tRNAGlu(UUC), and tRNAGln(UUG) in the eukaryotic cytosol is modified to 5-methoxycarbonylmethyl-2-thiouridine (mcm⁵s²U) (the wobbleU₃₄ modification), which comprises a methoxycarbonylmethyl group added to the fifth carbon of the base (mcm⁵) via the Elongator complex (Jaciuk et al., 2023; Abbassi et al., 2024) and a sulfur atom on the second carbon of the base, attached by the intracellular sulfur transport relay system (Nakai et al., 2008; Sokołowski et al., 2024).

The wobbleU₃₄ modification in the plant Arabidopsis thaliana requires the Elongator complex (Mehlgarten et al., 2010) and the methyltransferase AtTRM9 (Leihne et al., 2011) to form the mcm⁵ group, and two Arabidopsis URM genes, URM11 and URM12, are required for the sulfur modification (Nakai et al., 2012, 2019). We previously found that the first pair of true leaves in the urm11–1 urm12–1 double mutant (referred to as urm in this article) or in the elo3–10 Elongator mutant (referred to as elo in this article) of A. thaliana were slightly delayed in entering the endoreduplication phase of the cell cycle. The arrangement of mesophyll cells was slightly disturbed compared with that of the wild-type plant (WT) (Nakai et al., 2019).

These two distinct modifications of wobbleU₃₄ independently contribute to adjusting the stacking rigidity and flexibility of the codon–anticodon pair structure that specifies Lys, Glu, or Gln (Yarian et al., 2000; Murphy et al., 2004; Duechler et al., 2016; Vendeix et al., 2012; Larsen et al., 2015), and lack of one of the two wobbleU₃₄ modifications may have a limited impact on plants (Nakai et al., 2019). The wobbleU₃₄ modification may affect translation of almost all gene transcripts, but the effect of the complete loss of both wobbleU₃₄ modifications on translation and on plant growth and development remains unknown. Here, we created a triple mutant tri (urm11–1 urm12–1 elo3-10) lacking both wobbleU₃₄ modifications, characterized the mutant phenotypes, and analyzed gene expression and translation using RNA-seq and Ribo-seq, respectively.

2 Materials and methods

2.1 Plant materials, growth condition, and treatments

Arabidopsis thaliana Columbia-0 (Col-0) was used as the WT plant. The urm (urm11–1 urm12-1) and elo mutants (elo3-10), both of which have a Col-0 background, were described previously (Nakai et al., 2019). The tri mutant (urm11–1 urm12–1 elo3-10) was obtained by crossing the urm and elo strains. The plants were initially cultured on half-concentration Murashige and Skoog (MS) agar medium (Murashige and Skoog, 1962) with 2% sucrose. Water-absorbed seeds were dormant in the dark at 4°C for 3 days, and then sown and cultured under illuminated conditions, with the first day of growth under illumination designated as 0 days after stratification (DAS). Seedlings were also grown under white fluorescent light (∼90-110 µmol/m²/s) with a 16-h light/8 h dark cycle at 22°C in a growth chamber BIOTRON LPH 200 (NK System). Seedlings were grown in sterile soil or Rockwool, as previously reported (Nakai et al., 2019).

2.2 Ribo-seq and RNA-seq

The Ribo-seq and RNA-seq datasets were acquired as previously described (Kurihara et al., 2018). In brief, the cell extracts prepared from the plant sample 11 DAS were treated with DNase I (Thermo Fisher Scientific) and used for total RNA and ribosome footprint preparations. The total RNA was extracted using TRIzol LS reagent (Thermo Fisher Scientific) and a Direct-zol RNA kit (ZYMO RESEARCH), and libraries for RNA-seq were constructed using a TruSeq Stranded Total RNA Library Prep Kit (Illumina). Purified ribosome footprints were used to construct Ribo-seq libraries as previously described (Kurihara et al., 2018). RNA sequence data were obtained from the RNA-seq and Ribo-seq libraries using HiSeq X (Illumina). RNA-seq reads were mapped to the Arabidopsis TAIR11 genome using STAR (Dobin et al., 2013) after rRNA/tRNA removal. The Ribo-seq reads were mapped to the TAIR11 genome using TopHat version 2.1.1. The mapping data from WT and mutant plants were merged into a single dataset, and open reading frames (ORFs) were predicted using RiboTaper v1.2 (Calviello et al., 2016) with 27–29-nt ribosome footprint reads. Read counts for each predicted ORF were normalized using DESeq (Trapnell et al., 2012). All samples were analyzed with two biological replicates.

2.3 Generating P site plot using Ribo-seq data and analyzing ribosome footprints

The Ribo-seq data records corresponding to 28- and 29-nt, 100% matched ribosome-protected sequences (ribosome footprints) for each of particular transcript were extracted using local BLAST. The extracted 28- or 29-nt sequences were aligned across the transcript to generate a P site plot with a 12- or 13-nt offset, respectively. Their 3-nt periodicities were confirmed throughout their coding sequences from the first ATG codon to the last codon adjacent to the stop codon. The ribosome footprint counts from the WT and the mutants were normalized based on their total Ribo-seq read counts and compared in association with the particular codons positioned at the P, A, or E site, as well as the −6- or +6-nt site relative to the P site. The ribosome footprint counts for the cognate [A/G/C]AA and [A/G/C]AG codons corresponding to the wobbleU₃₄-containing tRNAs positioned at these six positions on the translating ribosomes were separately compared between the WT and the tri, urm, or elo mutants. Their differences were plotted. Unrelated [A/G/C]GA and [A/G/C]GG codons were analyzed and plotted as controls.

2.4 Comparing gene expression profiles between mutants and WT

We calculated the fold-change log₂ values between the normalized mRNA read counts of each mutant and those of the WT (mRNA-FClog₂) as well as the fold-change log₂ values between the normalized ribosome-occupied RNA read counts of each mutant and those of the WT (ribo-FClog₂). The translational efficiency (TE) of each transcript was defined as the ribosome occupancy per transcript. The TE value was calculated by dividing the Ribo-seq read counts by the RNA-seq read counts using DESeq (Calviello et al., 2016). The significant differences in the TE between each mutant and the WT (log₂ fold-change of the TE, TE_FClog₂) with a threshold-adjusted p-value <0.05 were analyzed using the Benjamini–Hochberg (BH) method. The TE_FClog₂ value was plotted against the log₂ value of the normalized mRNA read count (mRNA-NormClog₂).

2.5 Gene Ontology enrichment analysis

Gene Ontology (GO) enrichment was analyzed using DAVID (https://davidbioinformatics.nih.gov/home.jsp) and PANTHER (https://pantherdb.org) against the Arabidopsis gene annotation using TAIR11. The false discovery rate for GO items was determined using the BH method, and only items with an adjusted p-value < 0.05 were selected. The results of the GO enrichment analysis were visualized using the GO plot (https://wencke.github.io).

2.6 Extracting total RNA for northern analysis of A. thaliana

Total RNA was prepared from whole plants 11 DAS using an ISOSPIN Plant RNA kit (Nippon-Gene). Approximately 10–20 µg of total RNA was used for northern hybridization analysis, performed as previously described (Kojima et al., 2018). Briefly, the RNA samples were separated on a 1%–1.2% denaturing agarose gel, blotted onto the nylon membrane (Hybond-N⁺, Cytiva), and hybridized with digoxigenin (DIG; Roche Diagnostics GmbH)-labeled DNA probes to detect specific rRNA precursor regions. The primers used to amplify the DIG-labeled DNA probes are shown in Supplementary Table S4. All samples were analyzed in triplicate.

2.7 cRT-PCR and sequencing of plant RNA

cRT-PCR was performed as previously described (Maekawa et al., 2018). Briefly, total RNA was treated with DNase I, and a circularized library was formed using RNA ligase (TaKaRa Bio). First-strand cDNA was synthesized via reverse transcription using a primer bound to the target 25S rRNA region. PCR was performed using different primer sets. The length of the amplified DNA was confirmed by agarose gel electrophoresis, and the sequences encompassing the junction points formed by RNA circularization were determined by DNA cloning with the Mighty Cloning Kit (TaKaRa Bio) followed by DNA sequencing. The primers and probes used are shown in Supplementary Table S4. All samples were analyzed in triplicate.

2.8 Histological observations

The entire leaf and subepidermal parenchyma cells were observed as previously described (Nakai et al., 2019). The seeds in the silique were visualized using a clearing agent, ClearSeeTM (Fujifilm WAKO) following the manufacturer’s instructions. Plant tissues were observed under a stereomicroscope All-in-One BZ X-700 (KEYENCE) using the related products. The entire leaf area and leaf cell size were measured, and cells were counted using ImageJ. More than 20 mesophyll cells in the lower epidermal layer, from the midrib of the leaf vein at the center of the leaf blade to the leaf margin, were analyzed for each leaf. The transverse section of the leaf and the upper epidermal tissue were observed using scanning electron microscopy (LSM800, Zeiss), and the number of cells were measured as previously described (Nakai et al., 2019).

2.9 Germination assay

The WT and the tri seeds were grown on agar medium supplemented with or without gibberellin (GA) (GA3, Fujifilm WAKO). After sowing and stratification, seeds were germinated under normal growth conditions. GA was added to the medium before sowing the seeds at final concentrations of 1 or 10 µM.

3 Results

3.1 Lack of both wobbleU₃₄ modifications in Arabidopsis causes severe growth retardation and various morphological changes

By crossing two distinct wobbleU₃₄ modification mutants (urm and elo), we first obtained a mutant line carrying the heterozygous ELO3/elo3–10 alleles with the homozygous urm11–1 urm12–1 mutations. Then, from the progenies of this line, we successfully obtained the homozygous triple gene mutant (urm11–1 urm12–1 elo3-10) and named it the tri mutant (Supplementary Figure S1A). The growth of the tri mutant, lacking the both wobbleU₃₄ modifications, was substantially slower from the early stages of germination than that of the urm mutant lacking sulfur modification, the elo mutant lacking the mcm⁵ modification, and the WT plant (Figure 1A; Supplementary Figure S1A). The leaf development of the tri mutant was even slower than that of the elo mutant, with only eight to nine true leaves 21 DAS (Figure 1B), and the whole size of the tri mutant plant was smaller than that of the other strains (Figure 1C). Segregation analysis using seeds obtained from the abovementioned parental line (urm11–1 urm12–1 ELO3/elo3-10) confirmed that such slower growth was only observed for the urm11–1 urm12–1 elo3–10 homozygous triple mutant progenies but not for the urm11–1 urm12–1 ELO3/ELO3 or the urm11–1 urm12–1 ELO3/elo3–10 progenies (Supplementary Figure S1A).

Figure 1

Figure 1. Growth and morphological phenotypes of Arabidopsis woblbeU₃₄ mutants. (A) Growth of WT, urm, elo, and tri plants was compared at 5, 10, 14, 17, and 24 DAS. Scale bar, 1 cm. (B) Cotyledons and true leaves of WT and mutants of 21 DAS were aligned. Scale bar, 1 cm. (C) Shoots of WT and the wobbleU₃₄ modification mutants grown for 26 days. Scale bar, 1 cm. (D) First leaves of indicated genotypes. Scale bar, 5 mm. (E) Palisade cells in the adaxial subepidermal layer. Air space is indicated in green. Scale bar, 100 μm. (F) Adaxial epidermal tissues observed by a scanning electron microscope. Scale bar, 100 μm. (G–L) show the quantitative characteristics of the leaf phenotype of the wild type and the wobbleU₃₄ modification mutant strains: leaf blade area (G), palisade cell number (H), palisade cell area (I), pavement cell number (J), pavement cell area (K), and proportion of air space (L) in the first leaves of indicated genotypes. Numbers of palisade cells in adaxial subepidermal layer (H) and of adaxial pavement cells (J) were estimated by multiplying the leaf blade area by the corresponding cell density in each leaf. The number of leaves examined was 12 to 18 in (G, H, I, L), and 10 to16 in (J, K), respectively. The adaxial subepidermal layer was examined to measure projected areas of palisade cells and air space. A total of 20 palisade cells were measured per leaf. The number of pavement cells examined ranged from 379 to 1,093 in (K). Means ± s.d. are shown in (G, H, I, J, L). In (K), data are shown using violin plots and box plots. The 25th and 75th percentiles are indicated by lower and upper box edges, respectively, the median by a thick horizontal bar, and maximum and minimum values by whiskers. Different letters in each graph indicate statistically significant differences among genotypes (p < 0.05). p-values in (G, H, I, J, L) were determined using the Tukey honest significant difference test, and those in (K) were determined using the Kruskal–Wallis one-way analysis of variance test followed by Dunn’s test with Bonferroni correction. (M) Transverse sections of leaves were observed after staining cell walls with Calcofluor White. Magenta indicates chloroplast autofluorescence. Scale bar, 50 µm. (N) Whole sheath shape of WT and mutants. Scale bar, 5 mm. (O) Representative example of seed attachment in WT and tri dishes with transparent sheaths. (P) Number of ovaries per silique (top), number of ripe seeds per silique (middle), and fertility rate per silique (bottom) for WT and tri. Fertility is expressed as the number of ripe seeds per total number of ovaries per sheath.

We performed microscopic observations of leaves to compare their quantitative characteristics using the first pair of true leaves 21 DAS from WT and the mutants lacking the wobbleU₃₄ modifications (Figures 1D–L). The leaf area of the tri mutant was much smaller than that of the WT, whereas those of the urm and the elo mutants were slightly larger (Figures 1D, G). By observing the palisade cells in the adaxial subepidermal layer (Figure 1E), cell densities (cell numbers per unit area) were calculated. Total palisade cell numbers per leaf (Figure 1H) were estimated by multiplying the cell density by the total leaf area (Figure 1G). As a result, the total palisade cell numbers in the urm and elo mutants were slightly higher than that of the WT, whereas that in the tri mutant was substantially lower than the others (Figure 1H). In addition, the individual palisade cell areas were slightly reduced in the urm and elo mutants but were further reduced in the tri mutant (Figures 1E, I). The pavement cell density of the tri and elo mutants appeared to be higher than that of the others (Figure 1F) because of their smaller individual pavement cell areas (Figures 1F, K). Depending on the differences in leaf areas (Figures 1D, G), the total pavement cell number per leaf in the tri mutant was substantially smaller than that in the others, whereas that of the elo mutant was much larger than that of the WT or the urm mutant (Figure 1J). As a consequence, in the tri mutant, both palisade and pavement cells were smaller and fewer per leaf than in the others. These results are consistent with the macroscopic observation that leaves were smaller in the tri mutant (Figures 1B, D).

The proportion of air space in the tri mutant was much larger than that of the WT or the urm mutant, and even larger than that of the elo mutant (Figures 1E, L). The palisade mesophyll cells in the first layer under the epidermis in the tri mutant were not arranged in parallel. The overall cell arrangement was sparse compared with that of the other strains (Figure 1M), indicating that the mesophyll cells in the leaf tissue of the tri mutant were spatially disordered compared with those of the WT and other mutants. Taken together, these results suggest that the deletion of both wobbleU₃₄ modifications affects leaf cell proliferation and differentiation, disrupting the balance between area and number of mesophyll and epidermal cells, altering cellular spatial arrangement, and leading to dwarf leaves.

Abnormal morphogenesis was also seen in other organs in the tri mutant. Although the siliques shapes of the urm and elo strains were similar to those of WT (Figure 1N), the tri mutant pistils had an abnormal shape characterized by shorter valves and a longer gynophore (Figure 1N), which is known as the short-valve trait (Nishimura et al., 2005). The tri mutant produced mature seeds; however, it produced considerably fewer seeds per silique than the WT (Figures 1O, P). Less than half the ovules per silique and mature seeds were produced by the tri mutant compared with the WT (Figure 1P), and many gametophytes were stunted in the tri mutant. In addition, based on the results of segregation analysis for the progenies derived from the heterozygous ELO3/elo3–10 parental line under the homozygous urm11–1 urm12–1 background as mentioned above, the tri seedlings appeared at below the expected ratio of 0.25, suggesting that the tri seeds were apt to lose the ability to germinate (Supplementary Figure S1B).

To examine the possibility that the observed slow growth phenotype of the tri mutant shown in Figure 1A results from delayed germination, we performed germination analysis with and without the addition of the gibberellin (GA) in the culture media (Supplementary Figure S2A). In the absence of exogenous GA, the tri mutant seeds began to germinate at around 48 h after stratification, whereas the WT seeds began to germinate at approximately 24 h. Increasing GA concentrations enhanced germination rates, and this effect was observed similarly in WT and tri seeds. However, the delayed germination of the tri mutant seeds was not fully complemented even by the addition of 10 µM GA. Moreover, irrespective of the presence or absence of GA in the medium, the slow growth of the tri mutant was equally observed (Supplementary Figure S2B). Therefore, the slow growth of the tri mutant seedlings did not result solely from delayed germination or a defect in GA biosynthesis during germination.

3.2 Lack of both wobbleU₃₄ modifications strongly affects ribosome occupancy in many transcripts especially in RNA-binding protein-related genes

RNA-seq and Ribo-seq were performed on 11 DAS Arabidopsis seedlings to analyze differentially expressed genes between wobbleU₃₄ mutants and WT. We calculated the log₂-fold-change in mRNA expression levels (mRNA-FClog₂) for each mutant relative to WT to indicate gene expression levels that differed in the mutants during transcription. The log₂-fold-change in ribosome occupancy (ribo-FClog₂) was similarly calculated to detect alterations in gene expression levels during translation in the mutants.

We defined differential ribosome occupancy for each transcript as the TE (Fujita et al., 2019). We calculated the TE by dividing the normalized Ribo-seq read counts by the normalized RNA-seq read counts for each transcript. A total of 1,123 genes were significantly differentially expressed in the tri mutant dataset (WT_tri), 121 in the elo strain (WT_elo), and 22 in the urm strain (WT_urm) compared with the TE values obtained from the WT (adjusted p-value of TE <0.05) (Figure 2A; Supplementary Table S1). Most of the transcripts that exhibited significant expression changes in the tri mutant were not affected in either the urm or elo mutants and may be responsible for the severe phenotypic changes caused by the lack of both wobbleU₃₄ modifications.

Figure 2

Figure 2. Differential gene expression analysis between the WT and mutants. (A) Overlap of transcripts with significant TE_FClog₂ values between WT and each mutant. Numbers indicate number of significantly differed transcripts in each comparison. (B) Log₂-fold change in mRNA expression (mRNA-FClog₂) versus log₂-fold change in ribosome occupancy (ribo_FClog₂) in each mutant relative to WT. Plots show data for transcripts that exhibited only significant log₂-fold changes. (C) Log₂-fold change value of change in TE versus normalized log₂ values of mRNA read counts (mRNA_NormClog₂) for comparison of variation in expression between strains. (D) Gene Ontology analysis of the 1,123 transcripts that significantly differed in tri. The X-axis presents the z-score, where positive values indicate that GO terms of each category were most likely to be enriched. The y-axis indicates the significance of extracted GO terms as negative log₁₀ values of adjusted p-values, with higher values indicating greater significance. The threshold was set at an adjusted p-value < 0.05. Bubble size indicates number of transcripts, and bubble color indicates type of each GO term: indicated biological process, cellular component, and molecular function, respectively. GO terms are listed in Supplementary Table S2. (E) Among the 1,123 transcripts (light blue circles), 145 transcripts belonging to the RNA-binding category identified by GO analysis are highlighted by pink and light-green circles in the same plots shown in (B, C) (WT_tri).

We found more significant changes in ribosome occupancy (ribo-FClog₂) than in transcriptional alterations (mRNA-FClog₂) in all three wobbleU₃₄-deficient mutant strains, indicating that the loss of either or both of the wobbleU₃₄ modifications affected translation more than transcription (Figure 2B). The expression profiles of many more genes were altered in the tri mutant than in the urm or elo mutant (Figure 2B), indicating that the two distinct modifications in the wobbleU₃₄ work together to properly translate many gene transcripts.

Transcripts showing significant expression changes with the log₂ fold-change in TE (TE_FClog₂) value >0 in the tri mutant accounted for two-thirds (755/1,123), which may have contributed to the large phenotypic changes observed in the tri mutants (Figure 2C). TE_FClog₂ values >0 can be interpreted as ribosome occupancy on the transcript increasing during translation. However, this does not necessarily mean that their translation is accelerated; it may indicate ribosome pausing on these transcripts, leading to delayed or inefficient translation (Nedialkova and Leidel, 2015). The defects in the wobbleU₃₄ modification might specifically affect the decoding of the cognate codons AAA, GAA, and CAA (referred to as [A/G/C]AA in this article) for Lys, Glu, and Gln, respectively. However, the frequency of [A/G/C]AA codon usage as well as the combined codon usage of each transcript did not correlate with their TE_FClog₂ value in the tri mutant in any of the cases (Supplementary Figure S3).

Gene Ontology analysis showed that 145 of the 1,123 transcripts with significant TE_FClog₂ values in the tri mutant were most remarkably enriched with the RNA-binding GO term (GO: 0003723) (Figure 2D, Supplementary Tables S2 and S3). Genes belonging to the RNA-binding category encode RNA-binding proteins (RBPs) involved in various RNA-related functions, such as ribosome biosynthesis and the posttranscriptional gene expression regulation (Glisovic et al., 2008; Bailey-Serres et al., 2009; Lee and Kang, 2016; Bazin et al., 2018; Chantarachot and Bailey-Serres, 2018; Dedow and Bailey-Serres, 2019; Muthusamy et al., 2021).

We compared expression levels of the 145 RBP transcripts between WT and the tri mutant (Figure 2E, left panel). The transcripts with mRNA-FClog₂ values between −1 and 1 accounted for 88.3% (128/145) of the total. However, their ribo-FClog₂ values ranged from −4.03 to 4.66, indicating that the differences observed in the TE_FClog₂ values of these RBPs in the tri mutant could be primarily attributed to changes in ribosome occupancy. In addition, 82.1% (119) of the 145 RBP genes had TE_FClog₂ values >0, suggesting that ribosome occupancy was substantially higher on those RBP transcripts in the tri mutant compared with the overall proportion of transcripts with TE_FClog₂ values >0, which was 67.2% (755/1123) (Figure 2E, right panel). Genes that showed higher TE_FClog₂ values >1 include several factors involved in rRNA biosynthesis, such as DEAD-box RNA helicase 1, DRH1 (AT3G01540, also named IRP6), which is involved in 25S rRNA maturation (Palm et al., 2019); RNA helicase gene ATRH7 (AT5G62190), which is also related to rRNA maturation (Huang et al., 2016); and the G-patch domain-containing protein gene GDP1 (AT1G63980) (Kojima et al., 2018). Several rRNA-biosynthesis-related genes were also found among those with TE_FClog₂ value <0 in the tri mutant, such as IRP3 (AT4G17720), which is involved in pre-rRNA processing (Palm et al., 2019), and GAF1 (RRP30, AT5G59980), which is a putative subunit of RNase P/MRP (Figure 2E) (Wang et al., 2012; Shaukat et al., 2021).

3.3 Deletion of both wobbleU₃₄ modifications leads to accumulation of rRNA processing precursors

To explore the direct effects of the lack of both wobbleU₃₄ modifications on rRNA biosynthesis, we performed northern blot analyses with total RNAs extracted from 11 DAS seedlings of the WT and the tri mutant to detect pre-rRNA precursors to be excised from the 35S pre-rRNA (Figure 3A). The results of northern blot analysis using probes p1, p2, and p3, which detect rRNA precursors retaining the internal transcribed spacer (ITS) 1 (ITS1) region, revealed that the tri mutant accumulated large amounts of 32S, 27SA₂, 27SA₃, P’-A₃, and 18S-A₃ pre-rRNA precursors (Figure 3B). This accumulation was not observed in the urm or elo mutants (Supplementary Figure S4). An additional rRNA processing intermediate, 27SB pre-rRNA, accumulated markedly in the tri mutant when using probe p4 (Figure 3B), whereas comparable levels of accumulation of the final product of 5.8S rRNA was detected in both the WT and the tri mutant samples. The accumulation of several distinct pre-rRNA processing intermediates indicated that rRNA maturation was significantly impaired in the tri mutant. rRNA can be biosynthesized through multiple pathways in Arabidopsis (Palm et al., 2019; Sáez-Vásquez and Delseny, 2019) (Supplementary Figure S5). The most common pathway involves the first cleavage within the ITS1 region of the 35S pre-rRNA precursor. Another rRNA biosynthesis pathway begins with cleavage of the 5′-external transcribed spacer (5′-ETS) region, which is similar to the rRNA biosynthesis pathway in yeast, and occurs in Arabidopsis. The 27SB pre-rRNA, consisting of the 5.8S-ITS2-25S region in both processes, is temporarily produced, cleaved within the ITS2 region, and produces mature 5.8S and 25S rRNAs. The accumulation of pre-rRNA processing intermediates in the tri mutant shown in Figure 3B indicates that the lack of both wobbleU₃₄ modifications impairs rRNA maturation, especially the ITS2 cleavage step.

Figure 3

Figure 3. Accumulation of pre-rRNA intermediates in tri mutant lacking both wobbleU₃₄ modifications. (A) Overview of rRNA maturation processes and pre-rRNA intermediates found in Arabidopsis. p1-p6 indicates the positions of the probes used in the Northern analysis. (B) Northern blot hybridization analysis of total RNA extracted from 11DAS seedlings of Arabidopsis tri and the WT. The positions of the final products and various intermediates are indicated. Abundant 28S rRNA bands detected under the UV light were shown as loading controls (the bottom panels). The abundant 5.8S rRNA bands in the right-most panel for the probe p4 serve as internal control. (C) Outline of method for detecting the 5′ end of pre-rRNA intermediates containing the 25S rRNA region using cRT-PCR. (D) Results of cRT-PCR analysis of tri mutant and WT. PCR-amplified fragments were separated via agarose gel electrophoresis. Deduced PCR fragments are schematically depicted at positions of expected sizes.

Next, we performed circular RT-PCR (cRT-PCR) analysis (Figure 3C) to precisely identify the rRNA intermediates accumulated in the tri mutant. With the single-stranded cDNA generated using a primer that bound near the 3′ end of the 25S sequence in the reverse direction, PCR was performed with another primer that bound near the 3′ end of 25S sequence (primer f) combined either with a primer (primer r1) that bound to the 5′ end of 25S or with a primer (primer r2) that bound to the ITS2 region using the obtained cDNAs as the template DNAs (Figure 3D). PCR amplified a 460-bp product with primers f and r1 corresponding to that derived from the 27SB pre-rRNA according to the results of DNA sequencing (Figure 3D, left). The 27SB pre-rRNA-derived PCR fragment was more strongly amplified using first-strand cDNAs prepared from the tri mutant RNAs than from the WT RNAs (Figure 3D, left). In addition, a small amount of the 708-bp PCR product containing the A₂ site in the ITS1 region was detected only when using the cDNA from the tri mutant RNA, indicating 27SA₂ pre-rRNA intermediate accumulation in the tri mutant. Furthermore, the predicted shorter fragments of the relevant sizes, 373 and 621 bp, were amplified with primers f and r2 (Figure 3D, right), further confirming the accumulation of 27SB and 27SA₂ rRNA processing intermediates in the tri mutant.

3.4 Lack of both wobbleU₃₄ modifications leads to ribosome pausing when the cognate [A/G/C]AA codon is positioned at the A site during translation of several rRNA maturation-related genes

DRH1, ATRH7, and GDP1 were included in the list of 145 RBP transcripts with significantly different TE_FClog₂ values in tri compared with WT (Figure 2E) and were involved in rRNA biosynthesis (Palm et al., 2019; Huang et al., 2016; Kojima et al., 2018). To explore the direct effects of the wobbleU₃₄ modifications on the translation of these transcripts, we first extracted the 100%-matched Ribo-seq records of 28- and 29-nt-long ribosome-protected mRNA fragments corresponding to the DRH1 transcript. We generated a P site plot with 12 or 13 nt offset, respectively (Figure 4A). We thus confirmed the 3-nt periodicity starting from the ATG start codon to a codon just before the stop codon, which justified the P site assignments (Supplementary Figure S6). The number of ribosome-occupied reads containing [A/G/C]AA or [A/G/C]AG at the P site was then normalized to the total Ribo-seq read counts and compared between the WT and wobbleU₃₄ modification mutants. Similarly, the ribosome-occupied reads containing [A/G/C]AA or [A/G/C]AG, either at their A or E site, as well as −6 or +6 nt relative to the P site, were separately counted and compared between the WT and mutants (Figure 4A). We found that the number of ribosome-occupied reads on the DRH1 transcript was substantially higher especially when the [A/G/C]AA codon was positioned at the A site in the tri mutant (Figure 4B, top left). A similar trend was also observed for the transcripts of ATRH7 and GDP1 (Figure 4B, middle and bottom left panels). In contrast, this trend was not observed for the [A/G/C]GA codons unrelated to the wobbleU₃₄ modification (Figure 4B, right panels).

Figure 4

Figure 4. Ribosome footprints in relation to codons at different positions on translating ribosomes. (A) Methods to deduce the distinct site positions in ribosome footprints. (B) The ribosome footprint counts for the cognate [A/G/C]AA and [A/G/C]AG codons corresponding to the wobbleU₃₄ containing tRNAs positioned at six different positions on translating ribosomes, including the P site and the A site, throughout the coding sequence of DRH1 (top), ATRH7 (middle), or GDP1 (bottom) were compared between the WT and the tri, urm, or elo mutants, and their differences are plotted (left panels). As controls, the unrelated [A/G/C]GA and [A/G/C]GG codons were also analyzed and plotted (right panels). Values are shown as the difference in counts observed in the mutant relative to those observed in WT.

4 Discussion

The simultaneous lack of both wobbleU₃₄ modifications, mcm⁵ and s², in A. thaliana was not lethal. However, the mutant plants lacking both wobbleU₃₄ modifications, the tri mutant, exhibited severe developmental defects with severe growth retardation and morphological abnormalities in many tissues. These findings indicate that the complete absence of the two wobbleU₃₄ modifications had a broadly adverse effect on cellular functions compared with single wobbleU₃₄ modification-deficient mutants.

RNA-seq and Ribo-seq analyses indicated that the expression levels of many genes (>1,000) of the tri mutant significantly differed compared with those of the WT. Many of these genes were categorized as RBPs, being involved in various RNA-related processes such as ribosome biosynthesis and rRNA maturation. In addition to DRH1, ATRH7, and GDP1 mentioned above, RNA helicase gene RH5/STRS1(AT1G31970) (Huang et al., 2016), one of the two Arabidopsis Nucleolin genes, AtNUC-L2(AT3G18610) (Pontvianne et al., 2007), another nucleolar-localized protein gene, PESCADILLO (AT5G14520) (Cho et al., 2013), and IRP3/BPA1 (AT4G17720) were included in the list of 145 RBP transcripts. IRP3 and DRH1(also named IRP6) genes were identified as IRP genes whose mutants exhibited pre-rRNA accumulation patterns distinct from that of the wild type (Palm et al., 2019). In the present study, among these 145 RBPs, we specifically selected DRH1, ATRH7, and GDP1 for the detailed ribosome footprint analysis shown in Figure 4 because of the following two criteria: i) relatively higher TE_FClog₂l values (>1) and ii) a high enough number and coverage of Ribo-seq reads to be analyzed for ribosome profiling at single-base resolution throughout the coding sequences. Arabidopsis drh1 mutant accumulated 27SA₂ and 27SA₃ pre-rRNA intermediates, suggesting that the processing of 27SB pre-rRNA at the C₂ site was impaired in the drh1 mutant (Palm et al., 2019; Sáez-Vásquez and Delseny, 2019). The accumulation of 27SA₂ and 27SB pre-rRNA precursors was also detected in the tri mutant, which likely results from a delay in the cleavage of the ITS2 region in the pre-rRNA precursor (Figure 3). Ribosome footprint analysis indicated that, in the tri mutant, the ribosomes appeared to be pausing when the wobbleU₃₄ modification-related cognate [A/G/C]AA codons were positioned at the A site in the translating ribosomes on the DRH1 transcript (Figure 4). Thus, the observed increase in ribosome pausing during translation of the DRH1 transcript probably caused delayed or insufficient supply of functional DRH1 protein, resulting in the accumulation of the pre-rRNA intermediates. A similar ribosome pausing was observed when the cognate [A/G/C]AA codons were positioned at the A site in the tri mutant for the ATRH7 and GDP1 transcripts (Figure 4). These results show that this trend in ribosome pausing at the A site of the cognate [A/G/C]AA codon is most likely a direct result of the deletion of both wobbleU₃₄ modifications in the tri mutant. This biased ribosome pausing was more intense in the tri mutant than in the urm or elo mutants, indicating that the deletion of both wobbleU₃₄ modifications synergistically had a negative effect on the entry of aminoacylated-tRNALys(UUU), -tRNALGlu(UUC), and -tRNAGln(UUG) into the A site of the translating ribosomes. Our observations in Arabidopsis are consistent with previous findings that ribosome pausing occurred when the [A/G/C]AA codons were positioned at the ribosome A site in similar complete wobbleU₃₄ modification mutants of yeast and Caenorhabditis elegans (Nedialkova and Leidel, 2015).

Plant mutants in the biosynthesis of the translation apparatus, such as rRNA and ribosomal proteins (RPs), exhibit morphological and physiological abnormalities, often accompanied by an impaired auxin response (Bailey-Serres et al., 2009; Zhou et al., 2010; Bazin et al., 2018; Sáez-Vásquez and Delseny, 2019). The phenotypic defects observed in the tri mutant (Figure 1) are similar to those observed in mutant RP genes (Nishimura et al., 2005; Horiguchi et al., 2011). RPs account for a significant portion of RBPs in Arabidopsis (Bailey-Serres et al., 2009). However, only six RP genes were included in the list of 1,123 transcripts that showed significantly altered translation efficiencies due to the lack of wobbleU₃₄ modifications (Supplementary Figure S7). This is presumably because RP gene transcripts are relatively highly expressed but preferentially use AAG codon for Lys instead of AAA (Supplementary Figure S8) and that the presence or absence of wobbleU₃₄ modifications in the corresponding tRNA does not affect the decoding efficiency of AAG codon, as shown in the present study (Figure 4B) and the literature (Nedialkova and Leidel, 2015).

As mentioned above, similar to the tri mutant, the drh1 mutant accumulated pre-rRNA processing intermediates, and this accumulation was more pronounced after auxin treatment, which enhances pre-rRNA synthesis (Palm et al., 2019). This suggests a rate-limiting role of DRH1 in pre-rRNA processing under such conditions. In addition, AtRH7 is involved in rRNA processing, and the ATRH7 gene-knocked mutant exhibits similar phenotypes as the auxin-related mutants (Huang et al., 2016). These findings suggest a possible link between auxin response and rRNA biosynthesis. From this perspective, in the tri mutant, the lack of both wobbleU₃₄ modifications causes ribosome pausing during translation of proteins involved in rRNA maturation, including DRH1 and/or ATRH7, which delay rRNA biosynthesis and may ultimately affect the auxin response, resulting in morphological abnormalities in various organs and tissues.

Interestingly, some organelle-related gene transcripts, such as PPR2 (AT3G06430), which interact with chloroplast 23S rRNA (Lu et al., 2011), and RNR1 (AT5G02250), which is involved in both mitochondrial and chloroplast rRNA maturation (Bollenbach et al., 2005), are included in the list of the 145 RBP transcripts. Although their TE_FClog₂ values were lower than those of DRH1, ATRH7, and GDP1, organellar rRNA biosynthesis may also be affected in the tri mutant.

In addition to these rRNA biosynthesis-related gene transcripts, splicing factor PRP4, which is involved in seed development (Raab and Hoth, 2007), PPR4 (AT5G04810), which modulates the trans-splicing of rps12 transcripts in chloroplasts (Lee et al., 2019), and HOG1 (AT4G13940), which is required for DNA methylation-dependent gene silencing (Rocha et al., 2005), are also included in the 145 RBP list, suggesting that other RNA-related functions such as mRNA-splicing or RNA silencing might also be affected in the tri mutant. Further work will be needed to clarify this point.

A recent report by Linder et al (Linder et al., 2025). proposed that interactions between m₆A-modified codons in mRNAs and wobbleU₃₄ modifications in tRNAs may affect mRNA decay in cultured human tumor cells. It remains unclear whether this interaction also affects mRNA decay in plants, such as Arabidopsis, and this issue awaits future studies.

It should be noted that there are interesting differences in the observed phenotypes between mice/flies and Arabidopsis caused by the lack of two wobbleU₃₄ modifications. Regarding cognate codon usage, mice and flies exhibit a relatively higher usage of XAG codons over XAA codons, whereas Arabidopsis shows nearly equal usage of these codons (Supplementary Figure S9). Although it is unclear whether these differences in codon usage are correlated with differences in the lethality of the mutants, animals and plants exhibit many differences during development and growth. Considering that the loss of tRNA wobbleU₃₄ modifications in plants, although not lethal, causes severe growth retardation and morphological abnormalities, such effects, if they occur during the development and growth of animals, might be more critical for their survival.

Our findings highlight the physiological importance of the wobbleU₃₄ modification in plants and further provide a clue to elucidate a close link between translational capacity and the development of tissues and organs in multicellular organisms.

Data availability statement

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

Author contributions

YN: Investigation, Supervision, Writing – review & editing, Validation, Data curation, Conceptualization, Formal Analysis, Writing – original draft, Methodology, Visualization. YK: Methodology, Validation, Formal Analysis, Data curation, Writing – original draft. YM: Writing – original draft, Formal Analysis, Validation, Data curation, Methodology. GH: Data curation, Investigation, Methodology, Validation, Formal Analysis, Writing – original draft, Visualization. KI: Validation, Methodology, Writing – original draft, Visualization. AH: Writing – original draft, Resources. MN: Writing – review & editing, Validation, Formal Analysis, Writing – original draft, Conceptualization, Methodology, Data curation, Visualization, Investigation. TY: Validation, Resources, Writing – original draft.

Funding

The author(s) declare that financial support was received for the research and/or publication of this article. This study was supported in part by the Collaborative Research Program (CR-19-01) of the Institute for Protein Research, Osaka University, an intramural project of Osaka University of Medical and Pharmaceutical Sciences, and by Grant-in-Aid for Challenging Research (Pioneering) (17K19340).

Acknowledgments

We thank Masaharu Kawauchi for statistical processing of our RNA-seq and Ribo-seq data, and Kazuyo Mihara for assistance with our plant cultures.

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Generative AI statement

The author(s) declare that no Generative AI was used in the creation of this manuscript.

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Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2025.1681927/full#supplementary-material

References

Abbassi, N. E., Jaciuk, M., Scherf, D., Böhnert, P., Rau, A., Hammermeister, A., et al. (2024). Cryo-EM structures of the human Elongator complex at work. Nat. Commun. 15, 4094. doi: 10.1038/s41467-024-48251-y

PubMed Abstract | Crossref Full Text | Google Scholar

Bailey-Serres, J., Sorenson, R., and Juntawong, P. (2009). Getting the message across: cytoplasmic ribonucleoprotein complexes. Trends Plant Sci. 14, 443–453. doi: 10.1016/j.tplants.2009.05.004

PubMed Abstract | Crossref Full Text | Google Scholar

Bazin, J., Romero, N., Rigo, R., Charon, C., Blein, T., Ariel, F., et al. (2018). Nuclear speckle RNA binding proteins remodel alternative splicing and the non-coding arabidopsis transcriptome to regulate a cross-talk between auxin and immune responses. Front. Plant Sci. 9. doi: 10.3389/fpls.2018.01209

PubMed Abstract | Crossref Full Text | Google Scholar

Bollenbach, T. J., Lange, H., Gutierrez, R., Erhardt, M., Stern, D. B., and Gagliardi, D. (2005). RNR1, a 3’-5’ exoribonuclease belonging to the RNR superfamily, catalyzes 3’ maturation of chloroplast ribosomal RNAs in Arabidopsis thaliana. Nucleic Acids Res. 33, 2751–2263. doi: 10.1093/nar/gki576

PubMed Abstract | Crossref Full Text | Google Scholar

Calviello, L., Mukherjee, N., Wyler, E., Zauber, H., Hirsekorn, A., Selbach, M., et al. (2016). Detecting actively translated open reading frames in ribosome profiling data. Nat. Methods 13, 165–170. doi: 10.1038/nmeth.3688

PubMed Abstract | Crossref Full Text | Google Scholar

Chantarachot, T. and Bailey-Serres, J. (2018). Polysomes, stress granules, and processing bodies: A dynamic triumvirate controlling cytoplasmic mRNA fate and function. Plant Physiol. 176, 254–269. doi: 10.1104/pp.17.01468

PubMed Abstract | Crossref Full Text | Google Scholar

Cho, H. K., Ahn, C. S., Lee, H.-S., Kim, J.-K., and Pai, H.-S. (2013). Pescadillo plays an essential role in plant cell growth and survival by modulating ribosome biogenesis. Plant J. 76, 393–405. doi: 10.1111/tpj.12302

PubMed Abstract | Crossref Full Text | Google Scholar

Dedow, L. K. and Bailey-Serres, J. (2019). Searching for a match: structure, function and application of sequence-specific RNA-binding proteins. Plant Cell Physiol. 60, 1927–1938. doi: 10.1093/pcp/pcz072

PubMed Abstract | Crossref Full Text | Google Scholar

Delaunay, S. and Frye, M. (2019). RNA modifications regulating cell fate in cancer. Nat. Cell Biol. 21, 552–559. doi: 10.1038/s41556-019-0319-0

PubMed Abstract | Crossref Full Text | Google Scholar

Dobin, A., Davis, C. A., Schlesinger, F., Drenkow, J., Zaleski, C., Jha, S., et al. (2013). STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21. doi: 10.1093/bioinformatics/bts635

PubMed Abstract | Crossref Full Text | Google Scholar

Duechler, M., Leszczyńska, G., Sochacka, E., and Nawrot, B. (2016). Nucleoside modifications in the regulation of gene expression: focus on tRNA. Cell Mol. Life Sci. 73, 3075–3095. doi: 10.1007/s00018-016-2217-y

PubMed Abstract | Crossref Full Text | Google Scholar

Fujita, T., Kurihara, Y., and Iwasaki, S. (2019). The plant translatome surveyed by ribosome profiling. Plant Cell Physiol. 60, 1917–1926. doi: 10.1093/pcp/pcz059

PubMed Abstract | Crossref Full Text | Google Scholar

Glisovic, T., Bachorik, J. L., Yong, J., and Dreyfuss, G. (2008). RNA-binding proteins and post-transcriptional gene regulation. FEBS Lett. 582, 1977–1986. doi: 10.1016/j.febslet.2008.03.004

PubMed Abstract | Crossref Full Text | Google Scholar

Horiguchi, G., Mollá-Morales, A., Pérez-Pérez, J. M., Kojima, K., Robles, P., Ponce, M. R., et al. (2011). Differential contributions of ribosomal protein genes to Arabidopsis thaliana leaf development. Plant J. 65, 724–736. doi: 10.1111/j.1365-313X.2010.04457.x

PubMed Abstract | Crossref Full Text | Google Scholar

Huang, C.-K., Shen, Y.-L., Huang, L.-F., Wu, S.-J., Yeh, C.-H., and Lu, C.-A. (2016). The DEAD-box RNA helicase atRH7/PRH75 participates in pre-rRNA processing, plant development and cold tolerance in arabidopsis. Plant Cell Physiol. 57, 174–191. doi: 10.1093/pcp/pcv188

PubMed Abstract | Crossref Full Text | Google Scholar

Jaciuk, M., Scherf, D., Kaszuba, K., Gaik, M., Rau, A., Kościelniak, A., et al. (2023). Cryo-EM structure of the fully assembled Elongator complex. Nucleic Acids Res. 51, 2011–2032. doi: 10.1093/nar/gkac1232

PubMed Abstract | Crossref Full Text | Google Scholar

Kojima, K., Tamura, J., Chiba, H., Fukada, K., Tsukaya, H., and Horiguchi, G. (2018). Two Nucleolar Proteins, GDP1 and OLI2, Function as Ribosome Biogenesis Factors and Are Preferentially Involved in Promotion of Leaf Cell Proliferation without Strongly Affecting Leaf Adaxial-Abaxial Patterning in Arabidopsis thaliana. Front. Plant Sci. 8. doi: 10.3389/fpls.2017.02240

PubMed Abstract | Crossref Full Text | Google Scholar

Kurihara, Y., Makita, Y., Kawashima, M., Fujita, T., Iwasaki, S., and Matsui, M. (2018). Transcripts from downstream alternative transcription start sites evade uORF-mediated inhibition of gene expression in Arabidopsis. Proc. Natl. Acad. Sci. U S A. 115, 7831–7836. doi: 10.1073/pnas.1804971115

PubMed Abstract | Crossref Full Text | Google Scholar

Larsen, A. T., Fahrenbach, A. C., Sheng, J., Pian, J., and Szostak, J. W. (2015). Thermodynamic insights into 2-thiouridine-enhanced RNA hybridization. Nucleic Acids Res. 18, 7675–7687. doi: 10.1093/nar/gkv761

PubMed Abstract | Crossref Full Text | Google Scholar

Lee, K. and Kang, H. (2016). Emerging roles of RNA-binding proteins in plant growth, development, and stress responses. Mol. Cells 39, 179–185. doi: 10.14348/molcells.2016.2359

PubMed Abstract | Crossref Full Text | Google Scholar

Lee, K., Park, S. J., Colas des Francs-Small, C., Whitby, M., Small, I., and Kang, H. (2019). The coordinated action of PPR4 and EMB2654 on each intron half mediates trans-splicing of rps12 transcripts in plant chloroplasts. Plant J. 100, 1193–1207. doi: 10.1111/tpj.14509

PubMed Abstract | Crossref Full Text | Google Scholar

Leihne, V., Kirpekar, F., Vågbø, C. B., van den Born, E., Krokan, H. E., Grini, P. E., et al. (2011). Roles of Trm9- and ALKBH8-like proteins in the formation of modified wobble uridines in Arabidopsis tRNA. Nucleic Acids Res. 39, 7688–7701. doi: 10.1093/nar/gkr406

PubMed Abstract | Crossref Full Text | Google Scholar

Linder, B., Sharma, P., Wu, J., Birbaumer, T., Eggers, C., Murakami, S., et al. (2025). tRNA modifications tune m₆A-dependent mRNA decay. Cell 188, 3715–3727.e13. doi: 10.1016/j.cell.2025.04.013

PubMed Abstract | Crossref Full Text | Google Scholar

Lu, Y., Li, C., Wang, H., Chen, H., Berg, H., and Xia, Y. (2011). AtPPR2, an Arabidopsis pentatricopeptide repeat protein, binds to plastid 23S rRNA and plays an important role in the first mitotic division during gametogenesis and in cell proliferation during embryogenesis. Plant J. 67, 13–25. doi: 10.1111/j.1365-313X.2011.04569.x

PubMed Abstract | Crossref Full Text | Google Scholar

Maekawa, S., Ishida, T., and Yanagisawa, S. (2018). Reduced Expression of APUM24, Encoding a Novel rRNA Processing Factor, Induces Sugar-Dependent Nucleolar Stress and Altered Sugar Responses in Arabidopsis thaliana. Plant Cell. 30, 209–227. doi: 10.1105/tpc.17.00778

PubMed Abstract | Crossref Full Text | Google Scholar

Mehlgarten, C., Jablonowski, D., Wrackmeyer, U., Tschitschmann, S., Sondermann, D., Jäger, G., et al. (2010). Elongator function in tRNA wobble uridine modification is conserved between yeast and plants. Mol. Microbiol. 76, 1082–1094. doi: 10.1111/j.1365-2958.2010.07163.x

PubMed Abstract | Crossref Full Text | Google Scholar

Murashige, T. and Skoog, F. (1962). A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia Plantarum 15, 473–497. doi: 10.1111/j.1399-3054.1962.tb08052.x

Crossref Full Text | Google Scholar

Murphy, F. V., IV, Ramakrishnan, V., Malkiewicz, A., and Agris, P. F. (2004). The role of modifications in codon discrimination by tRNALysUUU. Nat. Struct. Mol. Biol. 11, 1186–1191. doi: 10.1038/nsmb861

PubMed Abstract | Crossref Full Text | Google Scholar

Muthusamy, M., Kim, J.-H., Kim, J. A., and Lee, S.-I. (2021). Plant RNA binding proteins as critical modulators in drought, high salinity, heat, and cold stress responses. Int. J. Mol. Sci. 22, 6731. doi: 10.3390/ijms22136731

PubMed Abstract | Crossref Full Text | Google Scholar

Nakai, Y., Harada, A., Hashiguchi, Y., Nakai, M., and Hayashi, H. (2012). Arabidopsis molybdopterin biosynthesis protein Cnx5 collaborates with the ubiquitin-like protein Urm11 in the thio-modification of tRNA. J. Biol. Chem. 28, 30874–30884. doi: 10.1074/jbc.M112.350090

PubMed Abstract | Crossref Full Text | Google Scholar

Nakai, Y., Horiguchi, G., Iwabuchi, K., Harada, A., Nakai, M., Hara-Nishimura, I., et al. (2019). tRNA wobble modification affects leaf cell development in arabidopsis thaliana. Plant Cell Physiol. 60, 2026–2039. doi: 10.1093/pcp/pcz064

PubMed Abstract | Crossref Full Text | Google Scholar

Nakai, Y., Nakai, M., and Hayashi, H. (2008). Thio-modification of yeast cytosolic tRNA requires a ubiquitin-related system that resembles bacterial sulfur transfer systems. J. Biol. Chem. 283, 27469–27476. doi: 10.1074/jbc.M804043200

PubMed Abstract | Crossref Full Text | Google Scholar

Nedialkova, D. D. and Leidel, S. A. (2015). Optimization of Codon Translation Rates via tRNA Modifications Maintains Proteome Integrity. Cell 161, 1606–1618. doi: 10.1016/j.cell.2015.05.022

PubMed Abstract | Crossref Full Text | Google Scholar

Nishimura, T., Wada, T., Yamamoto, K. T., and Okada, K. (2005). The Arabidopsis STV1 protein, responsible for translation reinitiation, is required for auxin-mediated gynoecium patterning. Plant Cell. 17, 2940–2953. doi: 10.1105/tpc.105.036533

PubMed Abstract | Crossref Full Text | Google Scholar

Palm, D., Streit, D., Shanmugam, T., Weis, B. L., Ruprecht, M., Simm, S., et al. (2019). Plant-specific ribosome biogenesis factors in Arabidopsis thaliana with essential function in rRNA processing. Nucleic Acids Res. 47, 1880–1895. doi: 10.1093/nar/gky1261

PubMed Abstract | Crossref Full Text | Google Scholar

Pontvianne, F., Matía, I., Douet, J., Tourmente, S., Medina, F. J., Echeverria, M., et al. (2007). Characterization of atNUC-L1 reveals a central role of nucleolin in nucleolus organization and silencing of atNUC-L2 gene in arabidopsis. Mol. Biol. Cell 18, 369–379. doi: 10.1091/mbc.E06–08–0751

PubMed Abstract | Crossref Full Text | Google Scholar

Raab, S. and Hoth, S. (2007). A mutation in the AtPRP4 splicing factor gene suppresses seed development in Arabidopsis. Plant Biol. 9, 447–452. doi: 10.1055/s-2006-924726

PubMed Abstract | Crossref Full Text | Google Scholar

Rocha, P. S. C. F., Sheikh, M., Melchiorre, R., Fagard, M., Boutet, S., Loach, R., et al. (2005). The Arabidopsis HOMOLOGY-DEPENDENT GENE SILENCING1 gene codes for an S-adenosyl-L-homocysteine hydrolase required for DNA methylation-dependent gene silencing. Plant Cell 17, 404–417. doi: 10.1105/tpc.104.028332

PubMed Abstract | Crossref Full Text | Google Scholar

Sáez-Vásquez, J. and Delseny, M. (2019). Ribosome biogenesis in plants: from functional 45S ribosomal DNA organization to ribosome assembly factors. Plant Cell. 31, 1945–1967. doi: 10.1105/tpc.18.00874

PubMed Abstract | Crossref Full Text | Google Scholar

Shaukat, A.-N., Kaliatsi, E. G., Skeparnias, I., and Stathopoulos, C. (2021). The dynamic network of RNP RNase P subunits. Int. J. Mol. Sci. 22, 10307. doi: 10.3390/ijms221910307

PubMed Abstract | Crossref Full Text | Google Scholar

Sokołowski, M., Kwasna, D., Ravichandran, K. E., Eggers, C., Krutyhołowa, R., Kaczmarczyk, M., et al. (2024). Molecular basis for thiocarboxylation and release of Urm1 by its E1-activating enzyme Uba4. Nucleic Acids Res. 52, 13980–13995. doi: 10.1093/nar/gkae1111

PubMed Abstract | Crossref Full Text | Google Scholar

Suzuki, T. (2021). The expanding world of tRNA modifications and their disease relevance. Nat. Rev. Mol. Cell Biol. 22, 375–392. doi: 10.1038/s41580-021-00342-0

PubMed Abstract | Crossref Full Text | Google Scholar

Trapnell, C., Roberts, A., Goff, L., Pertea, G., Kim, D., Kelley, D. R., et al. (2012). Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat. Protoc. 7, 562–578. doi: 10.1038/nprot.2012.016

PubMed Abstract | Crossref Full Text | Google Scholar

Vendeix, F. A. P., Murphy, F. V., IV, Cantara, W. A., Leszczyńska, G., Gustilo, E. M., Sproat, B., et al. (2012). Human tRNA(Lys3)(UUU) is pre-structured by natural modifications for cognate and wobble codon binding through keto-enol tautomerism. J. Mol. Biol. 416, 467–485. doi: 10.1016/j.jmb.2011.12.048

PubMed Abstract | Crossref Full Text | Google Scholar

Wang, S.-Q., Shi, D.-Q., Long, Y.-P., Liu, J., and Yang, W.-C. (2012). GAMETOPHYTE DEFECTIVE 1, a putative subunit of RNases P/MRP, is essential for female gametogenesis and male competence in Arabidopsis. PloS One 7, e33595. doi: 10.1371/journal.pone.0033595

PubMed Abstract | Crossref Full Text | Google Scholar

Yarian, C., Marszalek, M., Sochacka, E., Malkiewicz, A., Guenther, R., Miskiewicz, A., et al. (2000). Modified nucleoside dependent Watson-Crick and wobble codon binding by tRNALysUUU species. Biochemistry 39, 13390–13395. doi: 10.1021/bi001302g

PubMed Abstract | Crossref Full Text | Google Scholar

Zhou, F., Roy, B., and von Arnim, A. G. (2010). Translation reinitiation and development are compromised in similar ways by mutations in translation initiation factor eIF3h and the ribosomal protein RPL24. BMC Plant Biol. 10, 193. doi: 10.1186/1471-2229-10-193

PubMed Abstract | Crossref Full Text | Google Scholar