Translation velocity determines the efficacy of engineered suppressor tRNAs on pathogenic nonsense mutations.

Nonsense mutations - the underlying cause of approximately 11% of all genetic diseases - prematurely terminate protein synthesis by mutating a sense codon to a premature stop or termination codon (PTC). An emerging therapeutic strategy to suppress nonsense defects is to engineer sense-codon decoding tRNAs to readthrough and restore translation at PTCs. However, the readthrough efficiency of the engineered suppressor tRNAs (sup-tRNAs) largely varies in a tissue- and sequence context-dependent manner and has not yet yielded optimal clinical efficacy for many nonsense mutations. Here, we systematically analyze the suppression efficacy at various pathogenic nonsense mutations. We discover that the translation velocity of the sequence upstream of PTCs modulates the sup-tRNA readthrough efficacy. The PTCs most refractory to suppression are embedded in a sequence context translated with an abrupt reversal of the translation speed leading to ribosomal collisions. Moreover, modeling translation velocity using Ribo-seq data can accurately predict the suppression efficacy at PTCs. These results reveal previously unknown molecular signatures contributing to genotype-phenotype relationships and treatment-response heterogeneity, and provide the framework for the development of personalized tRNA-based gene therapies.

Extended stop codon context predicts nonsense codon readthrough efficiency in human cells.

Protein synthesis terminates when a stop codon enters the ribosome's A-site. Although termination is efficient, stop codon readthrough can occur when a near-cognate tRNA outcompetes release factors during decoding. Seeking to understand readthrough regulation we used a machine learning approach to analyze readthrough efficiency data from published HEK293T ribosome profiling experiments and compared it to comparable yeast experiments. We obtained evidence for the conservation of identities of the stop codon, its context, and 3'-UTR length (when termination is compromised), but not the P-site codon, suggesting a P-site tRNA role in readthrough regulation. Models trained on data from cells treated with the readthrough-promoting drug, G418, accurately predicted readthrough of premature termination codons arising from CFTR nonsense alleles that cause cystic fibrosis. This predictive ability has the potential to aid development of nonsense suppression therapies by predicting a patient's likelihood of improvement in response to drugs given their nonsense mutation sequence context.

Fluorescent reporters give new insights into antibiotics-induced nonsense and frameshift mistranslation.

We developed a reporter system based on simultaneous expression of two fluorescent proteins: GFP as a reporter of the capacity of protein synthesis and mutated mScarlet-I as a reporter of translational errors. Because of the unique stop codons or frameshift mutations introduced into the mScarlet-I gene, red fluorescence was produced only after a mistranslation event. These reporters allowed us to estimate mistranslation at a single cell level using either flow cytometry or fluorescence microscopy. We found that laboratory strains of Escherichia coli are more prone to mistranslation compared to the clinical isolates. As relevant for uropathogenic E. coli, growth in human urine elevated translational frameshifting compared to standard laboratory media, whereas different standard media had a small effect on translational fidelity. Antibiotic-induced mistranslation was studied by using amikacin (aminoglycoside family) and azithromycin (macrolide family). Bactericidal amikacin induced preferably stop-codon readthrough at a moderate level. Bacteriostatic azithromycin on the other hand induced both frameshifting and stop-codon readthrough at much higher level. Single cell analysis revealed that fluorescent reporter-protein signal can be lost due to leakage from a fraction of bacteria in the presence of antibiotics, demonstrating the complexity of the antimicrobial activity.

Stop codon variant in EFEMP1 is associated with primary open-angle glaucoma due to impaired regulation of aqueous humor outflow.

It is known that the actin cytoskeleton and its associated cellular interactions in the trabecular meshwork (TM) and juxtacanalicular tissues mainly contribute to the formation of resistance to aqueous outflow of the eye. Fibulin-3, encoded by EFEMP1 gene, has a role in extracellular matrix (ECM) modulation, and interacts with enzymatic ECM regulators, but the effects of fibulin-3 on TM cells has not been explored. Here, we report a stop codon variant (c.T1480C, p.X494Q) of EFEMP1 that co-segregates with primary open angle glaucoma (POAG) in a Chinese pedigree. In the human TM cells, overexpression of wild-type fibulin-3 reduced intracellular actin stress fibers formation and the extracellular fibronectin levels by inhibiting Rho/ROCK signaling. TGFß1 up-regulated fibulin-3 protein levels in human TM cells by activating Rho/ROCK signaling. In rat eyes, overexpression of wild-type fibulin-3 decreased the intraocular pressure and the fibronectin expression of TM, however, overexpression of mutant fibulin-3 (c.T1480C, p.X494Q) showed opposite effects in cells and rat eyes. Taken together, the EFEMP1 variant may impair the regulatory capacity of fibulin-3 which has a role for modulating the cell contractile activity and ECM synthesis in TM cells, and in turn may maintain normal resistance of aqueous humor outflow. This study contributes to the understanding of the important role of fibulin-3 in TM pathophysiology and provides a new possible POAG therapeutic approach.

Transcript-specific induction of stop codon readthrough using a CRISPR-dCas13 system.

Stop codon readthrough (SCR) is the process where translation continues beyond a stop codon on an mRNA. Here, we describe a strategy to enhance or induce SCR in a transcript-selective manner using a CRISPR-dCas13 system. Using specific guide RNAs, we target dCas13 to the region downstream of canonical stop codons of mammalian AGO1 and VEGFA mRNAs, known to exhibit natural SCR. Readthrough assays reveal enhanced SCR of these mRNAs (both exogenous and endogenous) caused by the dCas13-gRNA complexes. This effect is associated with ribosomal pausing, which has been reported for several SCR events. Our data show that CRISPR-dCas13 can also induce SCR across premature termination codons (PTCs) in the mRNAs of green fluorescent protein and TP53. We demonstrate the utility of this strategy in the induction of readthrough across the thalassemia-causing PTC in HBB mRNA and hereditary spherocytosis-causing PTC in SPTA1 mRNA. Thus, CRISPR-dCas13 can be programmed to enhance or induce SCR in a transcript-selective and stop codon-specific manner.

Blastocrithidia nonstop mitochondrial genome and its expression are remarkably insulated from nuclear codon reassignment.

The canonical stop codons of the nuclear genome of the trypanosomatid Blastocrithidia nonstop are recoded. Here, we investigated the effect of this recoding on the mitochondrial genome and gene expression. Trypanosomatids possess a single mitochondrion and protein-coding transcripts of this genome require RNA editing in order to generate open reading frames of many transcripts encoded as 'cryptogenes'. Small RNAs that can number in the hundreds direct editing and produce a mitochondrial transcriptome of unusual complexity. We find B. nonstop to have a typical trypanosomatid mitochondrial genetic code, which presumably requires the mitochondrion to disable utilization of the two nucleus-encoded suppressor tRNAs, which appear to be imported into the organelle. Alterations of the protein factors responsible for mRNA editing were also documented, but they have likely originated from sources other than B. nonstop nuclear genome recoding. The population of guide RNAs directing editing is minimal, yet virtually all genes for the plethora of known editing factors are still present. Most intriguingly, despite lacking complex I cryptogene guide RNAs, these cryptogene transcripts are stochastically edited to high levels.

Engineered mRNA-ribosome fusions for facile biosynthesis of selenoproteins.

Ribosomes are often used in synthetic biology as a tool to produce desired proteins with enhanced properties or entirely new functions. However, repurposing ribosomes for producing designer proteins is challenging due to the limited number of engineering solutions available to alter the natural activity of these enzymes. In this study, we advance ribosome engineering by describing a novel strategy based on functional fusions of ribosomal RNA (rRNA) with messenger RNA (mRNA). Specifically, we create an mRNA-ribosome fusion called RiboU, where the 16S rRNA is covalently attached to selenocysteine insertion sequence (SECIS), a regulatory RNA element found in mRNAs encoding selenoproteins. When SECIS sequences are present in natural mRNAs, they instruct ribosomes to decode UGA codons as selenocysteine (Sec, U) codons instead of interpreting them as stop codons. This enables ribosomes to insert Sec into the growing polypeptide chain at the appropriate site. Our work demonstrates that the SECIS sequence maintains its functionality even when inserted into the ribosome structure. As a result, the engineered ribosomes RiboU interpret UAG codons as Sec codons, allowing easy and site-specific insertion of Sec in a protein of interest with no further modification to the natural machinery of protein synthesis. To validate this approach, we use RiboU ribosomes to produce three functional target selenoproteins in Escherichia coli by site-specifically inserting Sec into the proteins' active sites. Overall, our work demonstrates the feasibility of creating functional mRNA-rRNA fusions as a strategy for ribosome engineering, providing a novel tool for producing Sec-containing proteins in live bacterial cells.

The first mitochondrial genome of Calophyllum soulattri Burm.f.

Calophyllum soulattri Burm.f. is traditionally used to treat skin infections and reduce rheumatic pain, yet genetic and genomic studies are still limited. Here, we present the first complete mitochondrial genome of C. soulattri. It is 378,262 bp long with 43.97% GC content, containing 55 genes (30 protein-coding, 5 rRNA, and 20 tRNA). Repeat analysis of the mitochondrial genome revealed 194 SSRs, mostly mononucleotides, and 266 pairs of dispersed repeats ( ≥ 30 bp) that were predominantly palindromic. There were 23 homologous fragments found between the mitochondrial and plastome genomes. We also predicted 345 C-to-U RNA editing sites from 30 protein-coding genes (PCGs) of the C. soulatrii mitochondrial genome. These RNA editing events created the start codon of nad1 and the stop codon of ccmFc. Most PCGs of the C. soulattri mitochondrial genome underwent negative selection, but atp4 and ccmB experienced positive selection. Phylogenetic analyses showed C. soulattri is a sister taxon of Garcinia mangostana. This study has shed light on C. soulattri's evolution and Malpighiales' phylogeny. As the first complete mitochondrial genome in Calophyllaceae, it can be used as a reference genome for other medicinal plant species within the family for future genetic studies.

Stop codon readthrough as a treatment option for epidermolysis bullosa-Where we are and where we are going.

In the context of rare genetic diseases caused by nonsense mutations, the concept of induced stop codon readthrough (SCR) represents an attractive avenue in the ongoing search for improved treatment options. Epidermolysis bullosa (EB)-exemplary for this group of diseases-describes a diverse group of rare, blistering genodermatoses. Characterized by extreme skin fragility upon minor mechanical trauma, the most severe forms often result from nonsense mutations that lead to premature translation termination and loss of function of essential proteins at the dermo-epidermal junction. Since no curative interventions are currently available, medical care is mainly limited to alleviating symptoms and preventing complications. Complementary to attempts of gene, cell and protein therapy in EB, SCR represents a promising medical alternative. While gentamicin has already been examined in several clinical trials involving EB, other potent SCR inducers, such as ataluren, may also show promise in treating the hitherto non-curative disease. In addition to the extensively studied aminoglycosides and their derivatives, several other substance classes-non-aminoglycoside antibiotics and non-aminoglycoside compounds-are currently under investigation. The extensive data gathered in numerous in vitro experiments and the perspectives they reveal in the clinical setting will be discussed in this review.

Mitochondrial genome features and systematic evolution of diospyros kaki thunb 'Taishuu'.

BACKGROUND: 'Taishuu' has a crisp texture, abundant juice, and sweet flavor with hints of cantaloupe. The availability of mitochondrial genome data of Diospyros species is far from the known number of species. RESULTS: The sequencing data were assembled into a closed circular mitochondrial chromosome with a 421,308 bp length and a 45.79% GC content. The mitochondrial genome comprised 40 protein-coding, 24 tRNA, and three rRNA genes. The most common codons for arginine (Arg), proline (Pro), glycine (Gly), tryptophan (Trp), valine (Val), alanine (Ala), and leucine (Leu) were AGA, CCA, GGA, UGG, GUA, GCA, and CUA, respectively. The start codon for cox1 and nad4L protein-coding genes was ACG (ATG), whereas the remaining protein-coding genes started with ATG. There are four types of stop codons: CGA, TAA, TAG, and TGA, with TAA being the most frequently used stop codon (45.24%). In the D. kaki Thunb. 'Taishuu' mitochondrial genome, a total of 645 repeat sequences were identified, including 125 SSRs, 7 tandem repeats, and 513 dispersed repeats. Collinearity analysis revealed a close relationship between D. kaki Thunb. 'Taishuu' and Diospyros oleifera, with conserved homologous gene fragments shared among these species in large regions of the mitochondrial genome. The protein-coding genes ccmB and nad4L were observed to undergo positive selection. Analysis of homologous sequences between chloroplasts and mitochondria identified 28 homologous segments, with a total length of 24,075 bp, accounting for 5.71% of the mitochondrial genome. These homologous segments contain 8 annotated genes, including 6 tRNA genes and 2 protein-coding genes (rrn18 and ccmC). There are 23 homologous genes between chloroplasts and nuclei. Mitochondria, chloroplasts, and nuclei share two homologous genes, which are trnV-GAC and trnW-CCA. CONCLUSION: In conclusion, a high-quality chromosome-level draft genome for D. kaki was generated in this study, which will contribute to further studies of major economic traits in the genus Diospyros.

Noncanonical usage of stop codons in ciliates expands proteins with structurally flexible Q-rich motifs.

Serine(S)/threonine(T)-glutamine(Q) cluster domains (SCDs), polyglutamine (polyQ) tracts and polyglutamine/asparagine (polyQ/N) tracts are Q-rich motifs found in many proteins. SCDs often are intrinsically disordered regions that mediate protein phosphorylation and protein-protein interactions. PolyQ and polyQ/N tracts are structurally flexible sequences that trigger protein aggregation. We report that due to their high percentages of STQ or STQN amino acid content, four SCDs and three prion-causing Q/N-rich motifs of yeast proteins possess autonomous protein expression-enhancing activities. Since these Q-rich motifs can endow proteins with structural and functional plasticity, we suggest that they represent useful toolkits for evolutionary novelty. Comparative Gene Ontology (GO) analyses of the near-complete proteomes of 26 representative model eukaryotes reveal that Q-rich motifs prevail in proteins involved in specialized biological processes, including Saccharomyces cerevisiae RNA-mediated transposition and pseudohyphal growth, Candida albicans filamentous growth, ciliate peptidyl-glutamic acid modification and microtubule-based movement, Tetrahymena thermophila xylan catabolism and meiosis, Dictyostelium discoideum development and sexual cycles, Plasmodium falciparum infection, and the nervous systems of Drosophila melanogaster, Mus musculus and Homo sapiens. We also show that Q-rich-motif proteins are expanded massively in 10 ciliates with reassigned TAAQ and TAGQ codons. Notably, the usage frequency of CAGQ is much lower in ciliates with reassigned TAAQ and TAGQ codons than in organisms with expanded and unstable Q runs (e.g. D. melanogaster and H. sapiens), indicating that the use of noncanonical stop codons in ciliates may have coevolved with codon usage biases to avoid triplet repeat disorders mediated by CAG/GTC replication slippage.

Readthrough events in plants reveal plasticity of stop codons.

Stop codon readthrough (SCR) has important biological implications but remains largely uncharacterized. Here, we identify 1,009 SCR events in plants using a proteogenomic strategy. Plant SCR candidates tend to have shorter transcript lengths and fewer exons and splice variants than non-SCR transcripts. Mass spectrometry evidence shows that stop codons involved in SCR events can be recoded as 20 standard amino acids, some of which are also supported by suppressor tRNA analysis. We also observe multiple functional signals in 34 maize extended proteins and characterize the structural and subcellular localization changes in the extended protein of basic transcription factor 3. Furthermore, the SCR events exhibit non-conserved signature, and the extensions likely undergo protein-coding selection. Overall, our study not only characterizes that SCR events are commonly present in plants but also identifies the recoding plasticity of stop codons, which provides important insights into the flexibility of genetic decoding.

In silico prediction of the metabolism of Blastocrithidia nonstop, a trypanosomatid with non-canonical genetic code.

BACKGROUND: Almost all extant organisms use the same, so-called canonical, genetic code with departures from it being very rare. Even more exceptional are the instances when a eukaryote with non-canonical code can be easily cultivated and has its whole genome and transcriptome sequenced. This is the case of Blastocrithidia nonstop, a trypanosomatid flagellate that reassigned all three stop codons to encode amino acids. RESULTS: We in silico predicted the metabolism of B. nonstop and compared it with that of the well-studied human parasites Trypanosoma brucei and Leishmania major. The mapped mitochondrial, glycosomal and cytosolic metabolism contains all typical features of these diverse and important parasites. We also provided experimental validation for some of the predicted observations, concerning, specifically presence of glycosomes, cellular respiration, and assembly of the respiratory complexes. CONCLUSIONS: In an unusual comparison of metabolism between a parasitic protist with a massively altered genetic code and its close relatives that rely on a canonical code we showed that the dramatic differences on the level of nucleic acids do not seem to be reflected in the metabolisms. Moreover, although the genome of B. nonstop is extremely AT-rich, we could not find any alterations of its pyrimidine synthesis pathway when compared to other trypanosomatids. Hence, we conclude that the dramatic alteration of the genetic code of B. nonstop has no significant repercussions on the metabolism of this flagellate.

CdsA, a CDP-diacylglycerol synthase involved in phospholipid and glycolipid MPIase biosynthesis, possesses multiple initiation codons.

CdsA is a CDP-diacylglycerol synthase essential for phospholipid and glycolipid MPIase biosynthesis, and therefore for growth. The initiation codon of CdsA has been assigned as "TTG," while methionine at the 37th codon was reported to be an initiation codon in the original report. Since a vector containing the open reading frame starting with "TTG" under a controllable promoter complemented the cdsA knockout, "TTG" could function as an initiation codon. However, no evidence supporting that this "TTG" is the sole initiation codon has been reported. We determined the initiation codon by examining the ability of mutants around the N-terminal region to complement cdsA mutants. Even if the "TTG" was substituted with a stop codon, the clear complementation was observed. Moreover, the clones with multiple mutations of stop codons complemented the cdsA mutant up to the 37th codon, indicating that cdsA possesses multiple codons that can function as initiation codons. We constructed an experimental system in which the chromosomal expression of cdsA can be analyzed. By means of this system, we found that the cdsA mutant with substitution of "TTG" with a stop codon is fully functional. Thus, we concluded that CdsA contains multiple initiation codons.

The Deficiency of Hypusinated eIF5A Decreases the Putrescine/Spermidine Ratio and Inhibits +1 Programmed Ribosomal Frameshifting during the Translation of Ty1 Retrotransposon in Saccharomyces cerevisiae.

Programmed ribosomal frameshifting (PRF) exists in all branches of life that regulate gene expression at the translational level. The eukaryotic translation initiation factor 5A (eIF5A) is a highly conserved protein essential in all eukaryotes. It is identified initially as an initiation factor and functions broadly in translation elongation and termination. The hypusination of eIF5A is specifically required for +1 PRF at the shifty site derived from the ornithine decarboxylase antizyme 1 (OAZ1) in Saccharomyces cerevisiae. However, whether the regulation of +1 PRF by yeast eIF5A is universal remains unknown. Here, we found that Sc-eIF5A depletion decreased the putrescine/spermidine ratio. The re-introduction of Sc-eIF5A in yeast eIF5A mutants recovered the putrescine/spermidine ratio. In addition, the Sc-eIF5A depletion decreases +1 PRF during the decoding of Ty1 retrotransposon mRNA, but has no effect on -1 PRF during the decoding of L-A virus mRNA. The re-introduction of Sc-eIF5A in yeast eIF5A mutants restored the +1 PRF rate of Ty1. The inhibition of the hypusine modification of yeast eIF5A by GC7 treatment or by mutating the hypusination site Lys to Arg caused decreases of +1 PRF rates in the Ty1 retrotransposon. Furthermore, mutational studies of the Ty1 frameshifting element support a model where the efficient removal of ribosomal subunits at the first Ty1 frame 0 stop codon is required for the frameshifting of trailing ribosomes. This dependency is likely due to the unique position of the frame 0 stop codon distance from the slippery sequence of Ty1. The results showed that eIF5A is a trans-regulator of +1 PRF for Ty1 retrotransposon and could function universally in yeast.

PRFect: a tool to predict programmed ribosomal frameshifts in prokaryotic and viral genomes.

BACKGROUND: One of the stranger phenomena that can occur during gene translation is where, as a ribosome reads along the mRNA, various cellular and molecular properties contribute to stalling the ribosome on a slippery sequence and shifting the ribosome into one of the other two alternate reading frames. The alternate frame has different codons, so different amino acids are added to the peptide chain. More importantly, the original stop codon is no longer in-frame, so the ribosome can bypass the stop codon and continue to translate the codons past it. This produces a longer version of the protein, a fusion of the original in-frame amino acids, followed by all the alternate frame amino acids. There is currently no automated software to predict the occurrence of these programmed ribosomal frameshifts (PRF), and they are currently only identified by manual curation. RESULTS: Here we present PRFect, an innovative machine-learning method for the detection and prediction of PRFs in coding genes of various types. PRFect combines advanced machine learning techniques with the integration of multiple complex cellular properties, such as secondary structure, codon usage, ribosomal binding site interference, direction, and slippery site motif. Calculating and incorporating these diverse properties posed significant challenges, but through extensive research and development, we have achieved a user-friendly approach. The code for PRFect is freely available, open-source, and can be easily installed via a single command in the terminal. Our comprehensive evaluations on diverse organisms, including bacteria, archaea, and phages, demonstrate PRFect's strong performance, achieving high sensitivity, specificity, and an accuracy exceeding 90%. The code for PRFect is freely available and installs with a single terminal command. CONCLUSION: PRFect represents a significant advancement in the field of PRF detection and prediction, offering a powerful tool for researchers and scientists to unravel the intricacies of programmed ribosomal frameshifting in coding genes.

Transcription Needs Translation Initiation of the Downstream Gene to Continue Downstream at Intercistronic Junctions in E. Coli.

We have reported a gal mutant called galE stop0, wherein the galE stop codon was changed to a sense codon. The experiment results demonstrated that preventing galE translation termination inhibited the production of galE 3' ends. This implies that when the galE translation termination was prevented, the galE 3' ends generation was reduced or impaired. We anticipated that the translation of galE would continue to galT, producing a chimeric protein GalE-GalT. This study verified that the chimeric protein was produced, but unexpectedly, we found that the GalT protein was also synthesized in the mutant, and its amount equaled that in the wild-type. In the wild-type, we also found that the GalE-GalT chimeric protein was produced in an amount equal to that of the GalE protein. These results suggest that translation termination of galE and translation initiation of galT occur independently, thus, corroborating our transcription-translation model: At the cistron junction, transcription, decoupled from translation due to the translation termination of galE, needs translation initiation of galT to continue downstream; otherwise, transcription would be terminated by Rho. RNase E-mediated transcript cleavage also produces the 3' ends of pre-galE mRNA. These findings indicated that RNase E produces the 3' end of mRNA and establishes gene expression polarity.

A homozygous stop codon in HORMAD2 in a patient with recurrent digynic triploid miscarriage.

BACKGROUND: Recurrent miscarriage (RM) affects 1% to 5% of couples trying to conceive. Despite extensive clinical and laboratory testing, half of the RM cases remain unexplained. We report the genetic analysis of a couple with eight miscarriages and the search for their potential genetic etiology. METHODS: Short tandem repeat (STR) markers, single nucleotide polymorphic (SNP) microarray, and human DNA methylation microarray were used to analyze the genotypes of two miscarriages. Exomes sequencing was performed on DNA from the two partners and identified variants were validated by Sanger sequencing. RESULTS: STR marker genotyping demonstrated that the two available miscarriages are triploid digynic and resulted from the failure of Meiosis II. SNP microarray analysis revealed an additional Meiosis I abnormality that is the segregation of the two maternal homologous chromosomes in one triploid miscarriage. Whole-exome sequencing on DNA from the two partners identified candidate variants only in the female partner in two genes with roles in female reproduction, a missense in EIF4ENIF1 (OMIM 607445) and a stop gain in HORMAD2 (OMIM 618842). EIF4ENIF1 is a eukaryotic translation initiation factor 4E nuclear import factor required for the oocyte germinal vesicle breakdown, and HORMAD2 is part of the synaptonemal complex that was hypothesized to act as a checkpoint mechanism to eliminate oocytes with asynapsis during meiotic prophase I in mice. CONCLUSION: While both genes may contribute to the phenotype, the Meiosis I abnormalities in the conceptions favor the causal role of HORMAD2 in the etiology of RM in this couple. This report illustrates the importance of comprehensively analyzing the products of conception to guide the search for the genetic causation of RM.

Rare but impaired flavin-containing monooxygenase 3 (FMO3) variants reported in a recently updated Japanese mega-databank of genome resources.

Genetic variants of human flavin-containing monooxygenase 3 (FMO3) were investigated using an updated Japanese population panel containing 54,000 subjects (the previous panel contained 38,000 subjects). One stop codon mutation and six amino acid-substituted FMO3 variants were newly identified in the updated databank. Of these, two substituted variants (p.Thr329Ala and p.Arg492Trp) were previously identified in compound haplotypes with p.[(Glu158Lys; Glu308Gly)] and were associated with the metabolic disorder trimethylaminuria. Three recombinant FMO3 protein variants (p.Ser137Leu, p.Ala334Val, and p.Ile426Val) expressed in bacterial membranes had similar activities toward trimethylamine N-oxygenation (∼75-125 %) as wild-type FMO3 (117 min-1); however, the recombinant novel FMO3 variant Phe313Ile showed moderately decreased FMO3 catalytic activity (∼20 % of wild-type). Because of the known deleterious effects of FMO3 C-terminal stop codons, the novel truncated FMO3 Gly184Ter variant was suspected to be inactive. To easily identify the four impaired FMO3 variants (one stop codon mutation and three amino-acid substitutions) in the clinical setting, simple confirmation methods for these FMO3 variants are proposed using polymerase chain reaction/restriction fragment length polymorphism or allele-specific PCR methods. The updated whole-genome sequence data and kinetic analyses revealed that four of the seven single-nucleotide nonsense or missense FMO3 variants had moderately or severely impaired activity toward trimethylamine N-oxygenation.

Novel insight into lepidopteran phylogenetics from the mitochondrial genome of the apple fruit moth of the family Argyresthiidae.

BACKGROUND: The order Lepidoptera has an abundance of species, including both agriculturally beneficial and detrimental insects. Molecular data has been used to investigate the phylogenetic relationships of major subdivisions in Lepidoptera, which has enhanced our understanding of the evolutionary relationships at the family and superfamily levels. However, the phylogenetic placement of many superfamilies and/or families in this order is still unknown. In this study, we determine the systematic status of the family Argyresthiidae within Lepidoptera and explore its phylogenetic affinities and implications for the evolution of the order. We describe the first mitochondrial (mt) genome from a member of Argyresthiidae, the apple fruit moth Argyresthia conjugella. The insect is an important pest on apples in Fennoscandia, as it switches hosts when the main host fails to produce crops. RESULTS: The mt genome of A. conjugella contains 16,044 bp and encodes all 37 genes commonly found in insect mt genomes, including 13 protein-coding genes (PCGs), two ribosomal RNAs, 22 transfer RNAs, and a large control region (1101 bp). The nucleotide composition was extremely AT-rich (82%). All detected PCGs (13) began with an ATN codon and terminated with a TAA stop codon, except the start codon in cox1 is ATT. All 22 tRNAs had cloverleaf secondary structures, except trnS1, where one of the dihydrouridine (DHU) arms is missing, reflecting potential differences in gene expression. When compared to the mt genomes of 507 other Lepidoptera representing 18 superfamilies and 42 families, phylogenomic analyses found that A. conjugella had the closest relationship with the Plutellidae family (Yponomeutoidea-super family). We also detected a sister relationship between Yponomeutoidea and the superfamily Tineidae. CONCLUSIONS: Our results underline the potential importance of mt genomes in comparative genomic analyses of Lepidoptera species and provide valuable evolutionary insight across the tree of Lepidoptera species.