Role In Protein Translation Research Articles

EIF4G2 Promotes Hepatocellular Carcinoma Progression via IRES-dependent PLEKHA1 Translation Regulation.

Hepatocellular carcinoma (HCC) is a highly lethal cancer, and proteomic studies have shown increased protein diversity and abundance in HCC tissues, whereas the role of protein translation has not been extensively explored in HCC. Our research focused on key molecules in the translation process to identify a potential contributor in HCC. We discovered that EIF4G2, a crucial translation initiation factor, is significantly upregulated in HCC tissues and associated with poor prognosis. This study uniquely highlights the impact of EIF4G2 deletion, which suppresses tumor growth and metastasis both in vitro and in vivo. Furthermore, polysome analysis and nascent protein synthesis assays revealed EIF4G2's role in regulating protein translation, specifically identifying PLEKHA1 as a key translational product. This represents a novel mechanistic insight into HCC malignancy. RNA immunoprecipitation (RIP) and Dual-luciferase reporter assays further revealed that EIF4G2 facilitates PLEKHA1 translation via an IRES-dependent manner. Importantly, the synergistic effects of EIF4G2 depletion and PLEKHA1 reduction in inhibiting cell migration and invasion underscore the therapeutic potential of targeting this axis. This study not only advances our understanding of translational regulation in HCC but also identifies the EIF4G2-PLEKHA1 axis as a promising therapeutic target, offering new avenues for intervention in HCC treatment.

Translation elongation factor-1α is pivotal for plant heat tolerance despite its pronounced heat-induced aggregation

The translation elongation factor 1α (EF1α) protein is a highly conserved G protein that is crucial for protein translation in all eukaryotic organisms. EF1α quickly became insoluble at temperatures 42 °C treatment for 2h in vitro, but generally remained soluble in vivo even after being exposed to temperatures as high as 45 °C for an extended period, which suggests that protective mechanisms exist for keeping EF1α soluble in plant cells under heat stress. EF1α had fast in vivo insolubilization when exposed to 45 °C, resulting in about 40% of the protein aggregating after 9 h. Given its established role in protein translation, heat-induced aggregation is most likely to impact the function of the elongation factor. Overexpression of constitutive mutants in both GTP-bound and GDP-bound forms of EF1α resulted in significantly decreased heat tolerance. These findings provide evidence to support the critical role of EF1α, a thermosensitive protein, in the heat tolerance of plants.

YTHDF1 in Tumor Cell Metabolism: An Updated Review.

With the advancement of research on m6A-related mechanisms in recent years, the YTHDF protein family within m6A readers has garnered significant attention. Among them, YTHDF1 serves as a pivotal member, playing a crucial role in protein translation, tumor proliferation, metabolic reprogramming of various tumor cells, and immune evasion. In addition, YTHDF1 also exerts regulatory effects on tumors through multiple signaling pathways, and numerous studies have confirmed its ability to assist in the reprogramming of the tumor cell-related metabolic processes. The focus of research on YTHDF1 has shifted in recent years from its m6A-recognition and -modification function to the molecular mechanisms by which it regulates tumor progression, particularly by exploring the regulatory factors that interact with YTHDF1 upstream and downstream. In this review, we elucidate the latest signaling pathway mechanisms of YTHDF1 in various tumor cells, with a special emphasis on its distinctive characteristics in tumor cell metabolic reprogramming. Furthermore, we summarize the latest pathological and physiological processes involving YTHDF1 in tumor cells, and analyze potential therapeutic approaches that utilize YTHDF1. We believe that YTHDF1 represents a highly promising target for future tumor treatments and a novel tumor biomarker.

Break and accelerator-The mechanics of Tau (and amyloid) toxicity.

Aggregates of the microtubule-associated protein Tau define more than a dozen primary tauopathies, and together with amyloid-β, the secondary tauopathy Alzheimer's disease (AD). Historically, Tau has been viewed as executor of amyloid-β toxicity, with the two molecules working together as "trigger and bullet." Given the two protein's opposing roles in protein translation, we wish to introduce another metaphor, borrowing from the mechanics of a car, with amyloid-β boosting Tau translation, whereas Tau puts a break on global translation. The underlying studies entail an alternative hypothesis regarding Tau's subcellular accumulation in AD, namely its de novo synthesis in the somatodendritic domain rather than the relocalization from the axon upon dissociation from microtubules. We contest that it may be worth (given Tau's 50th birthday) to revisit some entrenched dogmas about Tau's pathophysiology.

Characterization of GEXP15 as a Potential Regulator of Protein Phosphatase 1 in Plasmodium falciparum.

The Protein Phosphatase type 1 catalytic subunit (PP1c) (PF3D7_1414400) operates in combination with various regulatory proteins to specifically direct and control its phosphatase activity. However, there is little information about this phosphatase and its regulators in the human malaria parasite, Plasmodium falciparum. To address this knowledge gap, we conducted a comprehensive investigation into the structural and functional characteristics of a conserved Plasmodium-specific regulator called Gametocyte EXported Protein 15, GEXP15 (PF3D7_1031600). Through in silico analysis, we identified three significant regions of interest in GEXP15: an N-terminal region housing a PP1-interacting RVxF motif, a conserved domain whose function is unknown, and a GYF-like domain that potentially facilitates specific protein-protein interactions. To further elucidate the role of GEXP15, we conducted in vitro interaction studies that demonstrated a direct interaction between GEXP15 and PP1 via the RVxF-binding motif. This interaction was found to enhance the phosphatase activity of PP1. Additionally, utilizing a transgenic GEXP15-tagged line and live microscopy, we observed high expression of GEXP15 in late asexual stages of the parasite, with localization predominantly in the nucleus. Immunoprecipitation assays followed by mass spectrometry analyses revealed the interaction of GEXP15 with ribosomal- and RNA-binding proteins. Furthermore, through pull-down analyses of recombinant functional domains of His-tagged GEXP15, we confirmed its binding to the ribosomal complex via the GYF domain. Collectively, our study sheds light on the PfGEXP15-PP1-ribosome interaction, which plays a crucial role in protein translation. These findings suggest that PfGEXP15 could serve as a potential target for the development of malaria drugs.

Dual targeting of protein translation and nuclear protein export results in enhanced antimyeloma effects.

Selinexor (KPT-330) is a small molecule inhibitor of XPO1, which mediates the transport of tumor suppressor proteins, oncogene messenger RNAs, and other proteins involved in governing cell growthfrom the cell nucleus to the cytoplasm. It is overexpressed in many cancer types. Because eukaryotic translation initiator factor 4E (eIF4E) plays a critical role in protein translation in cancer cells in multiple myeloma (MM), we evaluated the effectiveness of combined inhibition of protein translation and nuclear export in MM. Selinexor, an inhibitor of nuclear protein export, dose-dependently decreased eIF4E, IKZF1, and c-MYC protein levels. Using a doxycycline-inducible-pLKO-Tet-On vector, knockdown of eIF4E significantly enhanced the antiproliferative effects of selinexor, sensitized resistant MM cells to selinexor, and increased apoptosis in MM cells. Immunofluorescent analysis of MM cells showed that the combined treatment increased the localization of residual eIF4E to the nucleus compared with selinexor-only treatment. The overexpression of eIF4E at least partially rescued the effects of selinexor in MM cells by reducing G1 cell cycle arrest and increasing the selinexor-IC50 10-fold. Moreover, the combination of selinexor with pharmacologic inhibitors of protein translation showed synergistic anti-MM effects. These results suggest a synergistic anti-MM effect of selinexor combined with eIF4E inhibitors invitro. Our work provides a better understanding of the potential mechanism of resistance to selinexor and a rationale for combining selinexor with eIF4E inhibitors for the treatment of MM.

MRGPRX2 signaling involves the Lysyl-tRNA synthetase and MITF pathway.

MRGPRX2, a G-protein-coupled-seven transmembrane domain receptor, is mainly expressed in mast cells and neurons and is involved in skin immunity and pain. It is implicated in the pathophysiology of non-IgE-mediated immediate hypersensitivity and has been related to adverse drug reactions. Moreover, a role has been proposed in asthma, atopic dermatitis, contact dermatitis, and chronic spontaneous urticaria. Although it has a prominent role in disease, its signaling transduction is poorly understood. This study shows that MRGPRX2 activation with substance P increased Lysyl t-RNA synthetase (LysRS) translocation to the nucleus. LysRS is a moonlighting protein with a dual role in protein translation and IgE signaling in mast cells. Upon allergen- IgE-FcεRI crosslinking, LysRS is translocated to the nucleus and activates microphthalmia-associated transcription factor (MITF) activity. In this study, we found that MRGPRX2 triggering led to MITF phosphorylation and increased MITF activity. Therefore, overexpression of LysRS increased MITF activity after MRGPRX2 activation. MITF silencing reduced MRGPRX2-dependent calcium influx and mast cell degranulation. Furthermore, a MITF pathway inhibitor, ML329, impaired MITF expression, calcium influx, and mast cell degranulation. Moreover, drugs such as atracurium, vancomycin, and morphine, reported to induce MRGPRX2-dependent degranulation, increased MITF activity. Altogether, our data show that MRGPRX2 signaling enhances MITF activity, and its abrogation by silencing or inhibition resulted in defective MRGPRX2 degranulation. We conclude that MRGPRX2 signaling involves the LysRS and MITF pathway. Thus, MITF and MITF-dependent targets may be considered therapeutic approaches to treat pathologies where MRGPRX2 is implicated.

An asymmetric structure of bacterial TrpRS supports the half-of-the-sites catalytic mechanism and facilitates antimicrobial screening.

Tryptophanyl-tRNA synthetase (TrpRS) links tryptophan to tRNATrp, thereby playing an indispensable role in protein translation. Unlike most class I aminoacyl-tRNA synthetases (AARSs), TrpRS functions as a homodimer. Herein, we captured an 'open-closed' asymmetric structure of Escherichia coli TrpRS (EcTrpRS) with one active site occupied by a copurified intermediate product and the other remaining empty, providing structural evidence for the long-discussed half-of-the-sites reactivity of bacterial TrpRS. In contrast to its human counterpart, bacterial TrpRS may rely on this asymmetric conformation to functionally bind with substrate tRNA. As this asymmetric conformation is probably a dominant form of TrpRS purified from bacterial cells, we performed fragment screening against asymmetric EcTrpRS to support antibacterial discovery. Nineteen fragment hits were identified, and 8 of them were successfully cocrystallized with EcTrpRS. While a fragment named niraparib bound to the L-Trp binding site of the 'open' subunit, the other 7 fragments all bound to an unprecedented pocket at the interface between two TrpRS subunits. Binding of these fragments relies on residues specific to bacterial TrpRS, avoiding undesired interactions with human TrpRS. These findings improve our understanding of the catalytic mechanism of this important enzyme and will also facilitate the discovery of bacterial TrpRS inhibitors with therapeutic potential.

Quantitative analysis of tRNA abundance and modifications by nanopore RNA sequencing

Transfer RNAs (tRNAs) play a central role in protein translation. Studying them has been difficult in part because a simple method to simultaneously quantify their abundance and chemical modifications is lacking. Here we introduce Nano-tRNAseq, a nanopore-based approach to sequence native tRNA populations that provides quantitative estimates of both tRNA abundances and modification dynamics in a single experiment. We show that default nanopore sequencing settings discard the vast majority of tRNA reads, leading to poor sequencing yields and biased representations of tRNA abundances based on their transcript length. Re-processing of raw nanopore current intensity signals leads to a 12-fold increase in the number of recovered tRNA reads and enables recapitulation of accurate tRNA abundances. We then apply Nano-tRNAseq to Saccharomyces cerevisiae tRNA populations, revealing crosstalks and interdependencies between different tRNA modification types within the same molecule and changes in tRNA populations in response to oxidative stress.

Effects of P70 S6 kinase on the ageing process and reproduction of Daphnia pulex (Branchiopoda, Anomopoda)

Abstract Daphnia pulex is a natural food widely used in aquaculture, and S6K plays an important role in protein translation and ageing. To explore the function of S6K in the ageing process of D. pulex, we cloned and analysed the full-length cDNA of S6K. With the growth of D. pulex, the expression of S6K mRNA first increased, then peaked at 10 days, and then decreased. Western blot analysis showed that the S6K protein expression increased from 1 to 5 days of age, but did not change significantly from 5 to 25 days of age. RNAi knockdown of S6K, reduced the expression of S6K mRNA and S6K protein, and it causes changes in other genes associated with ageing in both sexes. S6K may be involved in reproductive transformation, affecting reproductive capacity, thus significantly affecting the ageing of D. pulex. miRNA with potential regulation of the S6K gene were screened by the bioinformatics method and validated using a dual luciferase reporter gene system. This study deepens our understanding of the mechanisms of ageing in D. pulex and expands our understanding of the role of S6K in the ageing process.

Men who inject opioids exhibit altered tRNA-Gly-GCC isoforms in semen.

In addition to their role in protein translation, tRNAs can be cleaved into shorter, biologically active fragments called tRNA fragments (tRFs). Specific tRFs from spermatocytes can propagate metabolic disorders in second generations of mice. Thus, tRFs in germline cells are a mechanism of epigenetic inheritance. It has also been shown that stress and toxins can cause alterations in tRF patterns. We were therefore interested in whether injecting illicit drugs, a major stressor, impacts tRFs in germline cells. We sequenced RNA from spermatocytes and from semen-derived exosomes from people who inject illicit drugs (PWID) and from non-drug using controls, both groups of unknown fertility status. All PWID injected opioids daily, but most also used other illicit drugs. The tRF cleavage products from Gly-GCC tRNA were markedly different between spermatocytes from PWID compared to controls. Over 90% of reads in controls mapped to shorter Gly-GCC tRFs, while in PWID only 45% did. In contrast, only 4.1% of reads in controls mapped to a longer tRFs versus 45.6% in PWID. The long/short tRF ratio was significantly higher in PWID than controls (0.23 versus 0.16, P = 0.0128). We also report differential expression of a group of small nucleolar RNAs (snoRNAs) in semen-derived exosomes, including, among others, ACA14a, U19, and U3-3. Thus, PWID exhibited an altered cleavage pattern of tRNA-Gly-GCC in spermatocytes and an altered cargo of snoRNAs in semen-derived exosomes. Participants were not exclusively using opioids and were not matched with controls in terms of diet, chronic disease, or other stressors, so our finding are not conclusively linked to opioid use. However, all individuals in the PWID group did inject heroin daily. Our study indicates a potential for opioid injection and/or its associated multi-drug use habits and lifestyle changes to influence epigenetic inheritance.

TRNA fragment biomarkers of Neurological Disease: Challenges and Opportunities

Transfer RNAs play a crucial role in protein translation where they bring amino acids to the ribosome to be incorporated into nascent polypeptide chains. During stress conditions tRNAs can be cleaved to generate tRNA-derived fragments. Several ribonucleases have been identified that cleave tRNA, however mutations in the stress-induced ribonuclease Angiogenin have been identified in a range of neurological disorders including Amyotrophic Lateral Sclerosis, Parkinson’s Disease, and Alzheimer’s Disease, suggesting that tRNA cleavage may be dysregulated in neurological disease. tRNA fragments have been detected in biofluids indicating they may be of use as biomarkers for neurological diseases. There is considerable variability in the methods used to quantify tRFs from size selection, adapter ligation, removal of RNA modifications, and sequence analysis approaches which can make it difficult to reconcile multiple studies. Here we review the biology of transfer RNAs and the biogenesis of tRNA-derived fragments, with a focus on the methods used to identify and quantify tRNA fragments and how different methodological approaches can influence tRNA fragment detection. We provide an overview of current literature on the identification of tRNA fragments in neurological disease models and patient samples, with a focus on circulating tRNA fragments as potential biomarkers of neurological diseases.

Structure-Guided Design of Halofuginone Derivatives as ATP-Aided Inhibitors Against Bacterial Prolyl-tRNA Synthetase.

Aminoacyl-tRNA synthetases (aaRSs) are promising antimicrobial targets due to their essential roles in protein translation, and expanding their inhibitory mechanisms will provide new opportunities for drug discovery. We report here that halofuginone (HF), an herb-derived medicine, moderately inhibits prolyl-tRNA synthetases (ProRSs) from various pathogenic bacteria. A cocrystal structure of Staphylococcus aureus ProRS (SaProRS) with HF and an ATP analog was determined, which guided the design of new HF analogs. Compound 3 potently inhibited SaProRS at IC50 = 0.18 μM and Kd = 30.3 nM and showed antibacterial activities with an MIC of 1-4 μg/mL in vitro. The bacterial drug resistance to 3 only developed at a rate similar to or slower than those of clinically used antibiotics in vitro. Our study indicates that the scaffold and ATP-aided inhibitory mechanism of HF could apply to bacterial ProRS and also provides a chemical validation for using bacterial ProRS as an antibacterial target.

Genome-wide base editor screen identifies regulators of protein abundance in yeast.

Proteins are key molecular players in a cell, and their abundance is extensively regulated not just at the level of gene expression but also post-transcriptionally. Here, we describe a genetic screen in yeast that enables systematic characterization of how protein abundance regulation is encoded in the genome. The screen combines a CRISPR/Cas9 base editor to introduce point mutations with fluorescent tagging of endogenous proteins to facilitate a flow-cytometric readout. We first benchmarked base editor performance in yeast with individual gRNAs as well as in positive and negative selection screens. We then examined the effects of 16,452 genetic perturbations on the abundance of eleven proteins representing a variety of cellular functions. We uncovered hundreds of regulatory relationships, including a novel link between the GAPDH isoenzymes Tdh1/2/3 and the Ras/PKA pathway. Many of the identified regulators are specific to one of the eleven proteins, but we also found genes that, upon perturbation, affected the abundance of most of the tested proteins. While the more specific regulators usually act transcriptionally, broad regulators often have roles in protein translation. Overall, our novel screening approach provides unprecedented insights into the components, scale and connectedness of the protein regulatory network.

MicroRNA-140-5p inhibitor attenuates memory impairment induced by amyloid-ß oligomer in vivo possibly through Pin1 regulation.

The peptidyl-prolyl cis/trans isomerase, Pin1, has a protective role in age-related neurodegeneration by targeting different phosphorylation sites of tau and the key proteins required to produce Amyloid-β, which are the well-known molecular signatures of Alzheimer's disease (AD) neuropathology. The direct interaction of miR-140-5p with Pin1 mRNA and its inhibitory role in protein translation has been identified. The main purpose of this study was to investigate the role of miRNA-140-5p inhibition in promoting Pin1 expression and the therapeutic potential of the AntimiR-140-5p in the Aß oligomer (AßO)-induced AD rat model. Spatial learning and memory were assessed in the Morris water maze. RT-PCR, western blot, and histological assays were performed on hippocampal samples at various time points after treatments. miRNA-140-5p inhibition enhanced Pin1 and ADAM10 mRNA expressions but has little effect on Pin1 protein level. The miRNA-140-5p inhibitor markedly ameliorated spatial learning and memory deficits induced by AßO, and concomitantly suppressed the mRNA expression of inflammatory mediators TNFα and IL-1β, and phosphorylation of tau at three key sites (thr231, ser396, and ser404) as well as increased phosphorylated Ser473-Akt. According to our results, Antimir-140-mediated improvement of AβO-induced neuronal injury and memory impairment in rats may provide an appropriate rationale for evaluating miR-140-5p inhibitors as a promising agent for the treatment of AD.

Mass spectrometry profiling analysis enables the identification of new modifications in ribosomal RNA

Ribosomal RNAs (rRNAs) provide the structural framework of ribosomes and play critical roles in protein translation. In ribosome biogenesis, rRNAs acquire various modifications that can influence the structure and catalytic activity of ribosomes. However, rRNA modifications in plants have yet to be fully defined. Herein, we proposed a method to purify rRNAs by a successive isolation with different strategies, including polyA-based mRNA depletion and agarose gel electrophoresis-based purification, with which highly pure rRNAs could be obtained. In addition, we developed a liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method to systematically profile and characterize modifications from the isolated highly pure plant 18S rRNA and 25S rRNA. LC-ESI-MS/MS analysis showed that 10 and 12 kinds of modifications were present in plant 18S rRNA and 25S rRNA, respectively. Notably, among these identified modifications, 2 kinds of modifications of N2,N2-dimethylguanosine (m2,2G) and N6,N6-dimethyladenosine (m6,6A) in 18S rRNA, and 4 kinds of modifications of m2,2G, m6,6A, N7-methylguanosine (m7G) and 3-methyluridin (m3U) in 25S rRNA, were first reported to be present in plants. Moreover, exposure of Arabidopsis thaliana to cadmium (Cd) led to significant changes of modifications in both 18S rRNA and 25S rRNA of plants, indicating that rRNA modifications play important roles in response to environmental stress. The discovery of new modifications in plant rRNAs improves the spectra of plant rRNA modifications and may promote the investigation of the functional roles of plant ribosomes in regulating gene expression.

Design, Synthesis, and Proof-of-Concept of Triple-Site Inhibitors against Aminoacyl-tRNA Synthetases.

Aminoacyl-tRNA synthetases (aaRSs) are promising drug targets due to their essential roles in protein translation. Although current inhibitors primarily occupy one or two of the three substrate binding sites on aaRSs, we report here the structure-based design of the first class of triple-site aaRS inhibitors by targeting Salmonella enterica threonyl-tRNA synthetase (SeThrRS). Competition of our compounds with all three substrates on SeThrRS binding was confirmed via isothermal titration calorimetry assays. Cocrystal structures of three compounds bound to SeThrRS unambiguously confirmed their substrate-mimicking triple-site binding mode. Compound 36j exhibited the best enzyme activity against SeThrRS with IC50 = 19 nM and Kd = 35.4 nM. Compounds 36b, 36k, and 36l exhibited antibacterial activities with minimum inhibitory concentration values of 2-8 μg/mL against the tested bacteria, which are superior to those of the reported dual-site ThrRS inhibitors. Our study provides the first proof-of-concept for developing triple-site inhibitors against aaRSs, inspiring future aaRS-based drug discoveries.

Untranslated region-5’ and viral protein 1-based genetic stability analysis of bulk polio in Indonesia 2010-2019

Cases of vaccine-associated paralytic poliomyelitis (VAPP) continued increasingly from 2010-2019 in the world. Oral polio vaccine (OPV) is the live attenuated virus-based vaccine that could genetically revert to neurovirulent during the vaccine production process or when the virus replicates in the human body. The poliovirus neurovirulence is determined by the UTR-5' region and VP1 coding region. UTR-5' played a role in protein translation and VP1 was responsible for the immunogenicity of the virus. Some reported mutations in UTR-5' and VP1 could affect the neurovirulence of poliovirus. In this study, we analyzed the genetic stability of the UTR-5' and VP1 in the bulk of OPV types -1 and -3 produced in 2010 - 2019. The results of the analysis of UTR-5' sequences in Sabin strain types-1 and -3 and VP1 sequences on Sabin virus type 1 did not show any mutations. Meanwhile, the VP1 sequences in Sabin strain type 3 showed nucleotide mutation C2493U that caused the substitution amino acid Thr6Ile amino acid in all samples of the type 3 bulk polio test. Based on the results of in silico analysis, this mutation in VP1 did not contribute significantly to the neurovirulence of the virus.

The MERS-CoV N Protein Regulates Host Cytokinesis and Protein Translation via Interaction With EF1A.

Middle East respiratory syndrome coronavirus (MERS-CoV), a pathogen causing severe respiratory disease in humans that emerged in June 2012, is a novel beta coronavirus similar to severe acute respiratory syndrome coronavirus (SARS-CoV). In this study, immunoprecipitation and proximity ligation assays revealed that the nucleocapsid (N) protein of MERS-CoV interacted with human translation elongation factor 1A (EF1A), an essential component of the translation system with important roles in protein translation, cytokinesis, and filamentous actin (F-actin) bundling. The C-terminal motif (residues 359–363) of the N protein was the crucial domain involved in this interaction. The interaction between the MERS-CoV N protein and EF1A resulted in cytokinesis inhibition due to the formation of inactive F-actin bundles, as observed in an in vitro actin polymerization assay and in MERS-CoV-infected cells. Furthermore, the translation of a CoV-like reporter mRNA carrying the MERS-CoV 5′UTR was significantly potentiated by the N protein, indicating that a similar process may contribute to EF1A-associated viral protein translation. This study highlights the crucial role of EF1A in MERS-CoV infection and provides new insights into the pathogenesis of coronavirus infections.

Phylogeny and evolution of chloroplast tRNAs in Adoxaceae.

Chloroplasts are semiautonomous organelles found in photosynthetic plants. The major functions of chloroplasts include photosynthesis and carbon fixation, which are mainly regulated by its circular genomes. In the highly conserved chloroplast genome, the chloroplast transfer RNA genes (cp tRNA) play important roles in protein translation within chloroplasts. However, the evolution of cp tRNAs remains unclear. Thus, in the present study, we investigated the evolutionary characteristics of chloroplast tRNAs in five Adoxaceae species using 185 tRNA gene sequences. In total, 37 tRNAs encoding 28 anticodons are found in the chloroplast genome in Adoxaceae species. Some consensus sequences are found within the Ψ‐stem and anticodon loop of the tRNAs. Some putative novel structures were also identified, including a new stem located in the variable region of tRNATyr in a similar manner to the anticodon stem. Furthermore, phylogenetic and evolutionary analyses indicated that synonymous tRNAs may have evolved from multiple ancestors and frequent tRNA duplications during the evolutionary process may have been primarily caused by positive selection and adaptive evolution. The transition and transversion rates are uneven among different tRNA isotypes. For all tRNAs, the transition rate is greater with a transition/transversion bias of 3.13. Phylogenetic analysis of cp tRNA suggested that the type I introns in different taxa (including eukaryote organisms and cyanobacteria) share the conserved sequences “U‐U‐x2‐C” and “U‐x‐G‐x2‐T,” thereby indicating the diverse cyanobacterial origins of organelles. This detailed study of cp tRNAs in Adoxaceae may facilitate further investigations of the evolution, phylogeny, structure, and related functions of chloroplast tRNAs.