Mutant tRNA Research Articles

Mitochondrial tRNAAsn, tRNACys and tRNATyr encoding genes mutations landscape in colorectal cancer patient

The colorectal cancer (CRC) is one of the significant diseases implicated in high mortality rates in the world. Multi factors contributed in the cancer development and progression like tRNA encoding genes which have been linked with colorectal cancer. The present study aimed to report Mitochondrial tRNAAsn, tRNACys and tRNATyr encoding genes mutations landscape in colorectal cancer patients. A case control design depended on using PCR-sequencing of mtDNA region consisting of three tRNA genes. Data analysis was implemented using multiple alignment seq, tRNA secondary structure construction and phylogenic tree construction. The results showed that one case (female) was 25 years old and cancer stage T3N1MX has shown 6 mutations in tRNAAsn and 3 mutations in both tRNACys and tRNATyr while other samples did not show variations in the patients and control groups. The secondary structure of varied tRNAAsn, tRNACys and tRNATyr in this case might have been affected. The phylogenetic tree showed the conservative sequence of all CRC patients except the case that has a single cluster. The present finding concluded that there was non-association between tRNAAsn, tRNACys and tRNATyr encoding genes in mtDNA with colorectal cancer. One patient has mutations in tRNAs that may be affected in tRNA secondary structure and may be contributed for cancer progression.

Bacterial persistence to antibiotics activated by tRNA mutations.

Bacterial persistence is a significant cause of the intractability of chronic and relapsing infections. Despite its importance, many of the underlying mechanisms are still not well understood. Antibiotic-tolerant mutants of Burkholderia thailandensis were isolated through exposure to lethal doses of AMP or MEM, followed by whole-genome sequencing to identify mutations. Subsequently, these mutants underwent comprehensive characterization via killing curves, growth curves, and persistence-fraction plots. Northern blot analysis was employed to detect uncharged tRNA, while the generation of relA and spoT null mutations served to confirm the involvement of the stringent response in this persistence mechanism. Phenotypic reversion of the persistence mutation was demonstrated by incubating the mutants without antibiotics for 2 weeks. We have discovered a novel mechanism of persistence triggered by specific mutations at positions 32 or 38 within the anticodon loop of tRNAAsp. This leads to heightened persistence through a RelA-dependent stringent response. Notably, this persistence can be easily reverted to wild-type physiology by losing the mutant tRNA allele within the tRNA gene cluster when persistence is no longer essential for survival. This distinct form of persistence underscores the novel function of tRNA mutations at positions 32 or 38 within the anticodon loop, as well as the significance of the tRNA gene cluster in conferring adaptability to regulate persistence for enhanced survival.

Unilateral Hearing Loss and Auditory Asymmetry in Mitochondrial Disease: A Scoping Review.

Background/Objectives: Mitochondrial transfer RNA mutations are one of the most important causes of hereditary hearing loss in humans. In most cases, its presentation is bilateral and symmetrical; however, there are numerous cases of single-sided presentation or asymmetrical onset described in the literature that may represent a diagnostic challenge. The aim of this review is to present the evidence of auditory asymmetry in mitochondrial diseases, highlighting the possible presence of cases with atypical presentation. Methods: A review of the English literature to date on hearing loss and mitochondrial diseases was performed using PubMed, Scopus, and Google Scholar databases. The literature review was performed using the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) guidelines for scoping review. Results: A total of 10 full-text articles were included in this review, comprising 25 patients with single-sided or asymmetrical hearing loss associated with mitochondrial disease. Conclusions: Sensorineural hearing loss due to mitochondrial disease can represent a complex diagnostic challenge in cases of asymmetric or unilateral presentation. It is critical to recognize this clinical variant and to diagnose it in daily clinical practice.

Functional analysis of the novel mitochondrial tRNATrp and tRNASer(AGY) variants associated with type 2 diabetes mellitus.

Mutations in mitochondrial tRNA (mt-tRNA) genes that result in mitochondrial dysfunction play important roles in type 2 diabetes mellitus (T2DM). We pre-viously reported a large Chinese pedigree with maternally inherited T2DM that harbors novel mt-tRNA Trp A5514G and tRNA Ser(AGY) C12237T variants, however, the effects of these mt-tRNA variants on T2DM progression are largely unknown. To assess the potential pathogenicity of T2DM-associated m.A5514G and m.C12237T variants at genetic, molecular, and biochemical levels. Cytoplasmic hybrid (cybrid) cells carrying both m.A5514G and m.C12237T variants, and healthy control cells without these mitochondrial DNA (mtDNA) variants were generated using trans-mitochondrial technology. Mitochondrial features, including mt-tRNA steady-state level, levels of adenosine triphosphate (ATP), mitochondrial membrane potential (MMP), reactive oxygen species (ROS), mtDNA copy number, nicotinamide adenine dinucleotide (NAD+)/NADH ratio, enzymatic activities of respiratory chain complexes (RCCs), 8-hydroxy-deo-xyguanine (8-OhdG), malondialdehyde (MDA), and superoxide dismutase (SOD) were examined in cell lines with and without these mt-tRNA variants. Compared with control cells, the m.A5514G variant caused an approximately 35% reduction in the steady-state level of mt-tRNA Trp (P < 0.0001); however, the m.C12237T variant did not affect the mt-tRNA Ser(AGY) steady-state level (P = 0.5849). Biochemical analysis revealed that cells with both m.A5514G and m.C12237T variants exhibited more severe mitochondrial dysfunctions and elevated oxidative stress than control cells: ATP, MMP, NAD+/NADH ratio, enzyme activities of RCCs and SOD levels were markedly decreased in mutant cells (P < 0.05 for all measures). By contrast, the levels of ROS, 8-OhdG and MDA were significantly increased (P < 0.05 for all measures), but mtDNA copy number was not affected by m.A5514G and m.C12237T variants (P = 0.5942). The m.A5514G variant impaired mt-tRNA Trp metabolism, which subsequently caused mitochondrial dysfunction. The m.C12237T variant did not alter the steady-state level of mt-tRNA Ser(AGY), indicating that it may be a modifier of the m.A5514G variant. The m.A5514G variant may exacerbate the pathogenesis and progression of T2DM in this Chinese pedigree.

Mistranslating tRNA variants have anticodon- and sex-specific impacts on Drosophila melanogaster.

Transfer RNAs (tRNAs) are vital in determining the specificity of translation. Mutations in tRNA genes can result in the misincorporation of amino acids into nascent polypeptides in a process known as mistranslation. Since mistranslation has different impacts, depending on the type of amino acid substitution, our goal here was to compare the impact of different mistranslating tRNASer variants on fly development, lifespan, and behaviour. We established two mistranslating fly lines, one with a tRNASer variant that misincorporates serine at valine codons (V→S) and the other that misincorporates serine at threonine codons (T→S). While both mistranslating tRNAs increased development time and developmental lethality, the severity of the impacts differed depending on amino acid substitution and sex. The V→S variant extended embryonic, larval, and pupal development whereas the T→S only extended larval and pupal development. Females, but not males, containing either mistranslating tRNA presented with significantly more anatomical deformities than controls. Mistranslating females also experienced extended lifespan whereas mistranslating male lifespan was unaffected. In addition, mistranslating flies from both sexes showed improved locomotion as they aged, suggesting delayed neurodegeneration. Therefore, although mistranslation causes detrimental effects, we demonstrate that mistranslation also has positive effects on complex traits such as lifespan and locomotion. This has important implications for human health given the prevalence of tRNA variants in humans.

Mechanisms and Delivery of tRNA Therapeutics.

Transfer ribonucleic acid (tRNA) therapeutics will provide personalized and mutation specific medicines to treat human genetic diseases for which no cures currently exist. The tRNAs are a family of adaptor molecules that interpret the nucleic acid sequences in our genes into the amino acid sequences of proteins that dictate cell function. Humans encode more than 600 tRNA genes. Interestingly, even healthy individuals contain some mutant tRNAs that make mistakes. Missense suppressor tRNAs insert the wrong amino acid in proteins, and nonsense suppressor tRNAs read through premature stop signals to generate full length proteins. Mutations that underlie many human diseases, including neurodegenerative diseases, cancers, and diverse rare genetic disorders, result from missense or nonsense mutations. Thus, specific tRNA variants can be strategically deployed as therapeutic agents to correct genetic defects. We review the mechanisms of tRNA therapeutic activity, the nature of the therapeutic window for nonsense and missense suppression as well as wild-type tRNA supplementation. We discuss the challenges and promises of delivering tRNAs as synthetic RNAs or as gene therapies. Together, tRNA medicines will provide novel treatments for common and rare genetic diseases in humans.

Identification of a Novel Mitochondrial tRNA Mutation in Chinese Family with Type 2 Diabetes Mellitus.

Mutations in mitochondrial tRNA (mt-tRNA) could be the origin of some type 2 diabetes mellitus (T2DM) cases, but the mechanism remained largely unknown. The aim of this study was to assess the impact of a novel mitochondrial tRNACys/tRNATyr A5826G mutation on the development and progression of T2DM. A four-generation Han Chinese family with maternally inherited diabetes underwent clinical, genetic and biochemical analyses. The mitochondrial DNA (mtDNA) mutations of three matrilineal relatives were screened by PCR-Sanger sequencing. Furthermore, to see whether m.A5826G mutations affected mitochondrial functions, the cybrid cell lines were derived from three subjects with m.A5826G mutation and three controls without this mutation. ATP was evaluated by luminescent cell viability assay, mitochondrial membrane potential (MMP), and reactive oxygen species (ROS) were determined by flow cytometry. The student's two-tailed, unpaired t-test was used to assess the statistical significance between the control and mutant results. The age at onset of diabetes in this pedigree varied from 40 to 63 years, with an average of 54 years. Mutational analysis of mitochondrial genomes revealed the presence of a novel m.A5826G mutation. Interestingly, the m.A5826G mutation occurred at the conjunction between tRNACys and tRNATyr, a very conserved position that was critical for tRNAs processing and functions. Using trans-mitochondrial cybrid cells, we found that mutant cells carrying the m.A5826G showed approximately 36.5% and 22.4% reductions in ATP and MMP, respectively. By contrast, mitochondrial ROS levels increased approximately 33.3%, as compared with the wild type cells. A novel m.A5826G mutation was identified in a pedigree with T2DM, and this mutation would lead to mitochondrial dysfunction. Thus, the genetic spectrum of mitochondrial diabetes was expanded by including m.A5826G mutation in tRNACys/tRNATyr, our study provided novel insight into the molecular pathogenesis, early diagnosis, prevention and clinical treatment for mitochondrial diabetes.

The protein-only RNase Ps, endonucleases that cleave pre-tRNA: Biological relevance, molecular architectures, substrate recognition and specificity, and protein interactomes.

Protein-only RNase P (PRORP) is an essential enzyme responsible for the 5' maturation of precursor tRNAs (pre-tRNAs). PRORPs are classified into three categories with unique molecular architectures, although all three classes of PRORPs share a mechanism and have similar active sites. Single subunit PRORPs, like those found in plants, have multiple isoforms with different localizations, substrate specificities, and temperature sensitivities. Most recently, Arabidopsis thaliana PRORP2 was shown to interact with TRM1A and B, highlighting a new potential role between these enzymes. Work with At PRORPs led to the development of a ribonuclease that is being used to protect against plant viruses. The mitochondrial RNase P complex, found in metazoans, consists of PRORP, TRMT10C, and SDR5C1, and has also been shown to have substrate specificity, although the cause is unknown. Mutations in mitochondrial tRNA and mitochondrial RNase P have been linked to human disease, highlighting the need to continue understanding this complex. The last class of PRORPs, homologs of Aquifex RNase P (HARPs), is found in thermophilic archaea and bacteria. This most recently discovered type of PRORP forms a large homo-oligomer complex. Although numerous structures of HARPs have been published, it is still unclear how HARPs bind pre-tRNAs and in what ratio. There is also little investigation into the substrate specificity and ideal conditions for HARPs. Moving forward, further work is required to fully characterize each of the three classes of PRORP, the pre-tRNA binding recognition mechanism, the rules of substrate specificity, and how these three distinct classes of PRORP evolved. This article is categorized under: RNA Structure and Dynamics > RNA Structure, Dynamics and Chemistry RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems.

Mitochondrial tRNASer(UCN) mutations associated non-syndromic sensorineural hearing loss in Chinese families

Mitochondrial transfer RNA mutation is one of the most important causes of hereditary hearing loss in humans. Mitochondrial transfer RNASer (UCN) gene is another hot spot for mutations associated with non-syndromic hearing loss, besides the 12S ribosomal RNA gene. In this study, we assessed the clinical phenotype and the molecular characteristics of two Chinese families with non-syndromic hearing loss. Mutational analysis revealed that 7445A > G and 7510T > C mutations in the mitochondrial transfer RNASer (UCN) gene were the molecular etiology of Family 1 and Family 2, respectively. However, the clinical and genetic characteristics of the two families carrying the above mutations in the transfer RNASer (UCN) gene exhibited a variable expression of hearing loss and an incomplete penetrance. Sequencing analysis of the complete mitochondrial genome showed the presence of transfer RNATrp 5568A > G and NADH-ubiquinone oxidoreductase chain 4 11696G > A mutations in Family 1. The mitochondrial haplotype analysis showed that the two families belonged to Asian D4 and M80′D haplotypes, respectively, and no pathogenic variations were found in the nuclear genes. To our knowledge, our study is the first to report 7445A > G and 7510T > C mutations in the mitochondrial transfer RNASer (UCN) gene, in multi-generation non-syndromic hearing loss pedigrees from China. Our study suggests that 5568A > G and 11696G > A mutations may enhance the penetrance of hearing loss in Chinese Family 1, while mitochondrial haplotypes and known nuclear genes may not be modifiers for the phenotypic expression of 7445A > G and 7510T > C mutations in these Chinese families.

Enhanced Directed Evolution in Mammalian Cells Yields a Hyperefficient Pyrrolysyl tRNA for Noncanonical Amino Acid Mutagenesis.

Heterologous tRNAs used for noncanonical amino acid (ncAA) mutagenesis in mammalian cells typically show poor activity. We recently introduced a virus-assisted directed evolution strategy (VADER) that can enrich improved tRNA mutants from naïve libraries in mammalian cells. However, VADER was limited to processing only a few thousand mutants; the inability to screen a larger sequence space precluded the identification of highly active variants with distal synergistic mutations. Here, we report VADER2.0, which can process significantly larger mutant libraries. It also employs a novel library design, which maintains base-pairing between distant residues in the stem regions, allowing us to pack a higher density of functional mutants within a fixed sequence space. VADER2.0 enabled simultaneous engineering of the entire acceptor stem of M. mazei pyrrolysyl tRNA (tRNAPyl ), leading to a remarkably improved variant, which facilitates more efficient incorporation of a wider range of ncAAs, and enables facile development of viral vectors and stable cell-lines for ncAA mutagenesis.

Enhanced Directed Evolution in Mammalian Cells Yields a Hyperefficient Pyrrolysyl tRNA for Noncanonical Amino Acid Mutagenesis

AbstractHeterologous tRNAs used for noncanonical amino acid (ncAA) mutagenesis in mammalian cells typically show poor activity. We recently introduced a virus‐assisted directed evolution strategy (VADER) that can enrich improved tRNA mutants from naïve libraries in mammalian cells. However, VADER was limited to processing only a few thousand mutants; the inability to screen a larger sequence space precluded the identification of highly active variants with distal synergistic mutations. Here, we report VADER2.0, which can process significantly larger mutant libraries. It also employs a novel library design, which maintains base‐pairing between distant residues in the stem regions, allowing us to pack a higher density of functional mutants within a fixed sequence space. VADER2.0 enabled simultaneous engineering of the entire acceptor stem of M. mazei pyrrolysyl tRNA (tRNAPyl), leading to a remarkably improved variant, which facilitates more efficient incorporation of a wider range of ncAAs, and enables facile development of viral vectors and stable cell‐lines for ncAA mutagenesis.

Correction of a homoplasmic mitochondrial tRNA mutation in patient-derived iPSCs via a mitochondrial base editor

Pathogenic mutations in mitochondrial DNA cause severe and often lethal multi-system symptoms in primary mitochondrial defects. However, effective therapies for these defects are still lacking. Strategies such as employing mitochondrially targeted restriction enzymes or programmable nucleases to shift the ratio of heteroplasmic mutations and allotopic expression of mitochondrial protein-coding genes have limitations in treating mitochondrial homoplasmic mutations, especially in non-coding genes. Here, we conduct a proof of concept study applying a screened DdCBE pair to correct the homoplasmic m.A4300G mutation in induced pluripotent stem cells derived from a patient with hypertrophic cardiomyopathy. We achieve efficient G4300A correction with limited off-target editing, and successfully restore mitochondrial function in corrected induced pluripotent stem cell clones. Our study demonstrates the feasibility of using DdCBE to treat primary mitochondrial defects caused by homoplasmic pathogenic mitochondrial DNA mutations.

Escherichia coli tRNA (Gm18) methyltransferase (TrmH) requires the correct localization of its methylation site (G18) in the D-loop for efficient methylation.

TrmH is a eubacterial tRNA methyltransferase responsible for formation of 2'-O-methylguaosine at position 18 (Gm18) in tRNA. In Escherichia coli cells, only 14 tRNA species possess the Gm18 modification. To investigate the substrate tRNA selection mechanism of E. coli TrmH, we performed biochemical and structural studies. Escherichia coli TrmH requires a high concentration of substrate tRNA for efficient methylation. Experiments using native tRNA SerCGA purified from a trmH gene disruptant strain showed that modified nucleosides do not affect the methylation. A gel mobility-shift assay reveals that TrmH captures tRNAs without distinguishing between relatively good and very poor substrates. Methylation assays using wild-type and mutant tRNA transcripts revealed that the location of G18 in the D-loop is very important for efficient methylation by E. coli TrmH. In the case of tRNASer, tRNATyrand tRNALeu, the D-loop structure formed by interaction with the long variable region is important. For tRNAGln, the short distance between G18 and A14 is important. Thus, our biochemical study explains all Gm18 modification patterns in E. coli tRNAs. The crystal structure of E. coli TrmH has also been solved, and the tRNA binding mode of E. coli TrmH is discussed based on the structure.

The sequence variation of mitochondrial tRNA tyrosine and cysteine among Iranian women with idiopathic recurrent miscarriage: A case-control study.

Recurrent miscarriage is one of the most prevalent reproductive diseases. This phenomenon has several reasons, including maternal, hormonal, immunological, and parental genetic factors. Idiopathic recurrent miscarriage (IRM), with no distinctive etiology, involves about half of the recurrent miscarriage cases. Some mutations in mitochondrial DNA can lead to miscarriage. Mitochondrial tRNA (mt-tRNA) mutations cause nearly half of the mitochondrial disorders. To identify mt- tRNA gene mutations in Iranian women with IRM. In this case-control study, 100 Iranian women with IRM and 100 women as control without any history of miscarriage were investigated by polymerase chain reaction-single strand conformation polymorphism technique followed by gene sequencing. Bioinformatics analysis were done using human mitochondrial genome database, molecular evolutionary genetics analysis, mammalian mitochondrial-tRNA, etc. Results showed 4 mt-tRNA mutations including 1 cysteine mt-tRNA mutation (5824CT) and 3 tyrosine mt-tRNA mutations (5868TA, 5849CT, and 5836TC) in our cases. Amongst the 4 mutations found, one was novel that is still not reported. Our bioinformatics analysis revealed that these mutations can be pathogenic. They occurred in tRNA-conserved regions and their secondary structure was changed, which can result in mitochondrial dysfunction. Mutations of these genes may help in the assessment of IRM. Further study of all 22 mt-tRNAs possible mutations is recommended to describe their etiologic role in IRM.

Role of TRDMT1/DNMT2 in stress adaptation and its influence on transcriptome and proteome dynamics under osmotic stress in Physcomitrium patens.

Early land plants such as the moss Physcomitrium patens lack several morphological traits that offer protection to tracheophytes from environmental stresses. These plants instead have evolved several physiological and biochemical mechanisms that facilitate them to adapt to terrestrial stresses such as drought. We have previously shown that loss-of-function mutants of tRNA (cytosine(38)-C(5))-methyltransferase TRDMT1/DNMT2 in P. patens are highly sensitive to oxidative and osmotic stress. To gain insight into the role of PpTRDMT1/PpDNMT2 in modulating genetic networks under osmotic stress, genome-wide transcriptome and proteome studies were undertaken in wild-type and ppdnmt2 plants. Transcriptome analysis revealed 375 genes to be differentially expressed in the ppdnmt2 under stress compared to the WT. Most of these genes are affiliated with carbohydrate metabolic pathways, photosynthesis, cell wall biogenesis, pathways related to isotropic and polarised cell growth and transcription factors among others. Histochemical staining showed elevated levels of reactive oxygen species in ppdnmt2 while transmission electron microscopy revealed no distinct defects in the ultrastructure of chloroplasts. Immunoprecipitation using PpDNMT2-specific antibody coupled with mass spectrometry revealed core proteins of the glycolytic pathway, antioxidant enzymes, proteins of amino acid biosynthetic pathways and photosynthesis-related proteins among others to co-purify with PpTRDMT1/PpDNMT2 under osmotic stress. Yeast two-hybrid assays, protein deletion and α-galactosidase assays showed the cytosol glycolytic protein glyceraldehyde 3-phosphate dehydrogenase to bind to the catalytic motifs in PpTRDMT1/PpDNMT2. Results presented in this study allow us to better understand genetic networks linking enzymes of energy metabolism, epigenetic processes and RNA pol II-mediated transcription during osmotic stress tolerance in P. patens.

Transfer RNA Mutation Associated with Type 2 Diabetes Mellitus.

Transfer RNA (tRNA) genes in the mitochondrial DNA genome play an important role in protein synthesis. The 22 tRNA genes carry the amino acid that corresponds to that codon but changes in the genetic code often occur such as gene mutations that impact the formation of adenosine triphosphate (ATP). Insulin secretion does not occur because the mitochondria cannot work optimally. tRNA mutation may also be caused by insulin resistance. In addition, the loss of tRNA modification can cause pancreatic β cell dysfunction. Therefore, both can be indirectly associated with diabetes mellitus because diabetes mellitus, especially type 2, is caused by insulin resistance and the body cannot produce insulin. In this review, we will discuss tRNA in detail, several diseases related to tRNA mutations, how tRNA mutations can lead to type 2 diabetes mellitus, and one example of a point mutation that occurs in tRNA.

A homozygous mutation in the human selenocysteine tRNA gene impairs UGA recoding activity and selenoproteome regulation by selenium.

The selenocysteine (Sec) tRNA (tRNA[Ser]Sec) governs Sec insertion into selenoproteins by the recoding of a UGA codon, typically used as a stop codon. A homozygous point mutation (C65G) in the human tRNA[Ser]Sec acceptor arm has been reported by two independent groups and was associated with symptoms such as thyroid dysfunction and low blood selenium levels; however, the extent of altered selenoprotein synthesis resulting from this mutation has yet to be comprehensively investigated. In this study, we used CRISPR/Cas9 technology to engineer homozygous and heterozygous mutant human cells, which we then compared with the parental cell lines. This C65G mutation affected many aspects of tRNA[Ser]Sec integrity and activity. Firstly, the expression level of tRNA[Ser]Sec was significantly reduced due to an altered recruitment of RNA polymerase III at the promoter. Secondly, selenoprotein expression was strongly altered, but, more surprisingly, it was no longer sensitive to selenium supplementation. Mass spectrometry analyses revealed a tRNA isoform with unmodified wobble nucleotide U34 in mutant cells that correlated with reduced UGA recoding activities. Overall, this study demonstrates the pleiotropic effect of a single C65G mutation on both tRNA phenotype and selenoproteome expression.

Mitochondrial Diabetes Is Associated with the ND4 G11696A Mutation.

Type 2 diabetes mellitus (T2DM) is a common endocrine disorder which remains a large challenge for clinicians. Previous studies have suggested that mitochondrial dysfunction plays an active role in T2DM progression, but a detailed mechanism is still elusive. In the current study, two Han Chinese families with maternally inherited T2DM were evaluated using clinical, genetic, molecular, and biochemical analyses. The mitochondrial genomes were PCR amplified and sequenced. Phylogenetic and bioinformatic analyses were used to assess the potential pathogenicity of mitochondrial DNA (mtDNA) mutations. Interestingly, the matrilineal relatives of these pedigrees exhibited variable severity of T2DM, in particular, the age at onset of T2DM varied from 26 to 65 years, with an average of 49 years. Sequence analysis revealed the presence of ND4 G11696A mutation, which resulted in the substitution of an isoleucine for valine at amino acid (AA) position 312. Indeed, this mutation was present in homoplasmy only in the maternal lineage, not in other members of these families, as well as 200 controls. Furthermore, the m.C5601T in the tRNAAla and novel m.T5813C in the tRNACys, showing high evolutional conservation, may contribute to the phenotypic expression of ND4 G11696A mutation. In addition, biochemical analysis revealed that cells with ND4 G11696A mutation exhibited higher levels of reactive oxygen species (ROS) productions than the controls. In contrast, the levels of mitochondrial membrane potential (MMP), ATP, mtDNA copy number (mtDNA-CN), Complex I activity, and NAD+/NADH ratio significantly decreased in cell lines carrying the m.G11696A and tRNA mutations, suggesting that these mutations affected the respiratory chain function and led to mitochondrial dysfunction that was involved in T2DM. Thus, our study broadened the clinical phenotypes of m.G11696A mutation.

Analysis of Mitochondrial Transfer RNA Mutations in Breast Cancer.

Damage of mitochondrial functions caused by mitochondrial DNA (mtDNA) pathogenic mutations had long been proposed to be involved in breast carcinogenesis. However, the detailed pathological mechanism remained deeply undetermined. In this case-control study, we screened the frequencies of mitochondrial tRNA (mt-tRNA) mutations in 80 breast cancer tissues and matched normal adjacent tissues. PCR and Sanger sequence revealed five possible pathogenic mutations: tRNAVal G1606A, tRNAIle A4300G, tRNASer(UCN) T7505C, tRNAGlu A14693G and tRNAThr G15927A. We noticed that these mutations resided at extremely conserved positions of tRNAs and would affect tRNAs transcription or modifications. Furthermore, functional analysis suggested that patients with these mt-tRNA mutations exhibited much lower levels of mtDNA copy number and ATP, as compared with controls (p<0.05). Therefore, it can be speculated that these mutations may impair mitochondrial protein synthesis and oxidative phosphorylation (OXPHOS) complexes, which caused mitochondrial dysfunctions that were involved in the breast carcinogenesis. Taken together, our data indicated that mutations in mt-tRNA were the important contributors to breast cancer, and mutational analyses of mt-tRNA genes were critical for prevention of breast cancer.

Abstract 3511: Melanoma-shed, lymph-borne CSPG4 conditions the pre-metastatic lymph node niche

Abstract Tissues are primed for metastasis prior to the arrival of tumor cells. Primary tumors drive this transformation by shedding tumor-derived factors (TDFs), including extracellular vesicles and secreted/shed proteins (TSPs) into tumor-associated blood and lymphatic vasculature. Despite being the most common site of metastasis across solid tumor types, we know very little about how the lymph node (LN) is primed for metastasis by TDFs. Here, we test the hypothesis that lymph-borne TDFs are sufficient to initiate the formation of the pre-metastatic LN niche. Using a murine melanoma model (YUMM1.7), we demonstrate primary melanomas initiate remodeling in their draining LNs that enhances metastatic outgrowth relative to contralateral, non-draining and naïve LN controls. Furthermore, we have demonstrated that TDFs isolated from YUMM1.7-conditioned media uniquely activate this pro-metastatic LN phenotype rather than factors derived from benign dermis. The focus of this field has been on the role tumor-derived extracellular vesicles play in establishing this pre-metastatic niche, however our data further demonstrates TSPs are also sufficient to activate this niche. To identify candidate TDFs in vivo that have the potential to initiate LN transformation, we adopted a novel chemical biology strategy to ubiquitously tag TDFs in vivo using non-canonical amino acids and engineered expression of mutant tRNA synthetases in YUMM1.7 melanoma cells. Using this approach, we identified several TDFs by mass spectrometry present in pre-metastatic LNs, and identified one candidate (CSPG4; Melanoma-Associated Chondroitin Sulfate Proteoglycan) as a potential mediator of pre-metastatic niche development. Shed CSPG4 is found within TSPs isolated from media conditioned by YUMM1.7, but not benign dermal, cells. We generated stable shRNA-expressing YUMM1.7 cell lines for efficient knockdown of CSPG4, and found that loss of CSPG4 confers loss of TSP-dependent pre-metastatic niche development in the draining LN. Furthermore, using an enzyme specific to chondroitins (the main glycan decorating CSPG4), we demonstrate that loss of chondroitin glycosylation of CSPG4 is sufficient to attenuate metastasis, even with the core protein present. We find that fibroblastic reticular cells in the draining LN respond to TSPs by becoming more myofibroblast like, resembling cancer-associated fibroblasts that provide direct trophic support to tumor cells. This phenotypic switch is partially rescued in the absence of CSPG4. Our results collectively indicate TSPs prime the LN for metastasis and we have identified melanoma-shed CSPG4 as a driver of pre-metastatic niche development, potentially through its influence on the LN-resident fibroblasts. Citation Format: Haley du Bois, Annie L. Chen, Katherine S. Ventre, Maria S. Steele, Triantafyllia Karakousi, Amanda W. Lund. Melanoma-shed, lymph-borne CSPG4 conditions the pre-metastatic lymph node niche [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3511.