Stop Codon Research Articles

Characterization and phylogenetic analysis of the mitochondrial genome of Cylicostephanus longibursatus.

Cylicostephanus longibursatus is a common parasite in equine animals. Hosts infected by these nematodes might face disease or death. This study utilized next-generation sequencing technology to sequence the complete mitochondrial genome (mt genome) of C. longibursatus. Through bioinformatics techniques, the genomic base composition, codon usage, tRNA secondary structures, evolutionary relationships, and taxonomic status were analyzed. The results revealed that the mitochondrial genome of C. longibursatus is a double-stranded, 13,807-bp closed circular molecule with an AT content of 76.0%, indicating a clear preference for AT bases. The mitochondrial genome consisted of a total of 12 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and 2 non-coding regions. Among the 12 protein-coding genes, TTG and ATT were the common start codons. TAA was the predominant termination codon, except for the ND3 and ND6 coding genes, and the COШ genes used TAG and "T" as termination codons, respectively. All tRNAs exhibited atypical clover-leaf secondary structures, except for tRNALys and tRNALeu2, where two tRNASer genes lacked DHU arms and DHU loops, tRNAmet lacked the TΨC-arm, tRNAIle lacked the TΨC-loop, and the remaining 16 tRNAs lacked the TΨC-arm and TΨC loop, which were substituted by the "TV-replacement loop". Phylogenetic analyses, based on the 12 protein-coding genes and utilizing maximum likelihood (ML) and Bayesian inference (BI) analyses, indicated that C. longibursatus did not form a monophyletic group with other Cylicostephanus but was instead more closely related to Cyathostomum. These research findings provide fundamental data for exploring the population classification and phylogeny of strongylid nematodes.

The Complete Mitochondrial Genomes of Five Nivanini Species (Hemiptera: Cicadellidae: Evacanthinae) With Phylogenetic Analysis.

To delve deeper into the phylogenetic relationships within Cicadellidae and the taxonomic arrangement of Evacanthinae, our study focuses on the mitochondrial genome sequencing of five Nivanini species: Extensus latus, Concavepiana hamulusa, Sophonia nigrilineata, Sophonia microstaina, and Sophonia fuscomarginata. The results showed that the length of the five mitochondrial genomes ranged from 15,610 to 16,032 bp and included 37 typical genes. The A + T content of Nivanini ranged from 72.5% to 78.7%, which is consistent with that of other sequenced Evacanthinae species. All transfer RNAs (tRNAs) exhibit the typical cloverleaf secondary structure, except for trnS (AGY), which lacks the dihydrouracil (DHU) arm. With regard to protein-coding genes, all started with ATN codons, except for atp8, and most of them use TAA or TAG as termination codons. Using the Bayesian inference and maximum likelihood methods, a phylogenetic tree based on all 37 genes was constructed, with a total of 57 Cicadellidae species and two outgroups included as research objects. The analyses confirmed the monophyletic nature of each subfamily, highlighting Deltocephalinae as the oldest, distinctively parallel to the others.

Two novel deep intronic variants cause Duchenne muscular dystrophy by splice-altering mechanism

Duchenne muscular dystrophy (DMD) is a genetic disorder characterized by progressive muscle degeneration and weakness, due to mutations in the DMD gene, which encodes the dystrophin protein. While mutations within the coding regions of DMD have been extensively studied, recent focus has shifted to deep intronic variants for their potential impact on disease severity. Here, we characterize two deep intronic variants, c.8669-19_8669-24del and c.6439-1016_6439-3376del, in unrelated DMD patients. These variants were identified using targeted long-read sequencing on patients' DNA. RNA sequencing/reverse transcription polymerase chain reaction on RNA extracted from muscle biopsies revealed the presence of a pseudoexon or retention of part of the intron in the transcript, resulting in the introduction of premature termination codons. This study enhances our understanding of pseudoexon activation mechanisms in DMD and underscores the diverse genetic abnormalities contributing to the disease's complexity.

Exonisation of an intronic L1 element in the dystrophin gene associated with X-linked muscular dystrophy in a Border Collie dog.

X-linked recessive dystrophinopathies are the most common muscular dystrophies (MDs) in humans and dogs. To date, 20 breed-specific MD-associated variants are described in the canine dystrophin gene (DMD), including one associated with dystrophin-deficient MD in the Border Collie mixed breed. Here, we report the diagnosis and follow-up of mild dystrophin-deficient MD in a 5-month-old male Border Collie, associated with a novel DMD variant. Diagnosis was based on neurological examination and laboratory evaluations including creatine kinase activity, electromyography and muscle biopsies with immunofluorescent staining. Inspection of the Sashimi plots of the RNA-seq data from the affected muscle biopsy led to the discovery of a 162-bp L1 pseudoexon in DMD intron 63, introducing a frameshift and a premature stop codon (NM_001003343.1: c.9271_9272insN[162] p.(Ala3091fs*21)). Reduced DMD mRNA levels were detected for both the non-pseudoexon (50× less) and pseudoexon (3× less) containing transcripts in the affected muscle, compared with the level of the non-pseudoexon containing transcript in a control muscle, resulting in very low dystrophin protein levels and the upregulation of utrophin. Because the variant was only found in the affected dog, not in the healthy mother and grandmother, or in 108 unrelated Border Collies from the Belgian population (46 males and 62 females), it was considered a de novo variant. Although the prognosis for dystrophinopathy is generally regarded as poor, the dog stabilised at the age of 6 months and is still clinically stable at the age of 2 years.

A cell-free bacteriophage synthesis system for directed evolution

Efficient phage production has always been an urgent need in fields such as drug discovery, disease treatment, and gene evolution. To meet this demand, we constructed a robust cell-free synthesis system for generating M13 phage by simplifying its genome, enabling a three-times faster efficiency compared with the traditional method in vivo. We further developed a cell-free directed evolution system in droplets, comprising a modified helper plasmid (ΔPS-ΔgIII-ΔgVI) and the simplified M13 genome-carrying gene mutation library. This system was greatly improved when coupled with fluorescence-activated droplet sorting (FADS). We successfully evolved the T7 RNA polymerase (RNAP), achieving a twofold higher activity to read through the T7 terminator. Moreover, we evolved the tryptophan tRNA into a suppressor tRNA with an eightfold increase in activity to read through the stop codon UAG.

Dominant stop-loss HNRNPA1 variants in juvenile-onset myopathy.

Heterogeneous nuclear ribonucleoprotein A1 is involved in nucleic acid homeostatic functions. The encoding gene HNRNPA1 has been associated with several neuromuscular disorders including an amyotrophic lateral sclerosis-like phenotype, distal hereditary motor neuropathy, multisystem proteinopathy, and various myopathies. We report two unrelated individuals with monoallelic stop loss variants affecting the same codon of HNRNPA1. Two individuals with unsolved juvenile-onset myopathy were enrolled under approved institutional protocols. Phenotype data were collected and genetic analyses were performed, including whole-exome sequencing (WES). The two probands (MNOT002-01 and K1440-01) showed a similar onset of slowly progressive extremity and facial weakness in early adolescence. K1440-01 presented with facial weakness, winged scapula, elevated serum creatine kinase (CK) levels, and mild neck weakness. MNOT002-01 also exhibited elevated CK levels along with facial weakness, cardiomyopathy, respiratory dysfunction, pectus excavatum, a mildly rigid spine, and loss of ambulation. On quadriceps muscle biopsy, K1440-01 displayed rounded myofibers, mild variation in fiber diameter, and type 2 fiber hypertrophy, while MNOT002-01 displayed rimmed vacuoles. Monoallelic stop-loss variants in HNRNPA1 were identified for both probands: c.1119A>C p.*373Tyrext*6 (K1440-01) and c.1118A>C p.*373Serext*6 (MNOT002-01) affect the same codon and are both predicted to lead to the addition of six amino acids before termination at an alternative stop codon. Both stop-loss variants in our probands are likely pathogenic. Our findings contribute to the disease characterization of pathogenic variants in HNRNPA1. This gene should be screened in clinical diagnostic testing of unsolved cases of sporadic or dominant juvenile-onset myopathy.

Complete mitochondrial genomes of two Phoretic mites (Parasitidae: Poecilochirus) on burying beetle: Genome characterization and phylogenetic analysis

A pair of intriguing phoretic instances are the burying beetle and Poecilochirus mites. Their interaction is environment-dependent and can be either mutualistic or antagonistic hostile depending on various biological and physical circumstances. In this study, we sequenced the complete mitogenomes of two Poecilochirus mite species, P. davydovae and P. mrciaki, which are associated with burying beetles, using Illumina HiSeq sequencing technology. Each mitochondrial genome comprised 37 typical genes, including 13 protein-coding genes (PCGs), 22 tRNA genes, two rRNA genes, and one control region (CR). The size of these mitogenomes were determined to be 14,489 bp and 14,517 bp, respectively. These two mitochondrial genomes contain a notable AT bias in their nucleotide composition, with positive AT and negative GC skews in the deflection index of mitochondrial genomes, and a unique incomplete stop codon T- in both protein-coding genes. The DHU arm of tRNA-S1 (AGN) only forms a simple loop structure, while the secondary structures of 21 tRNA genes in both species showed the conventional cloverleaf structures, like those of most metazoans. The phylogenetic tree of Gamasina was constructed using Maximum likelihood and Bayesian inference based on two rRNA genes and 13 PCGs. Based on the existing data, the Parasitidae forms a monophyletic, and Poecilochirus and Parasitus are closely related, possibly belonging to the same genus.

Positive Selection of Mitochondrial cytochrome b Gene in the Marine Bivalve Keenocardium buelowi (Bivalvia, Cardiidae)

The mitochondrial genome provides valuable data for phylogenetic analysis and evolutionary research. In this study, we sequenced, assembled, and annotated the mitochondrial genome of Keenocardium buelowi using the Illumina platform. The genome spanned 16,967 bp and included 13 protein-coding genes (PCGs), two ribosomal RNAs, and 22 transfer RNAs. All PCGs utilized standard ATN start codons and TAN stop codons. The phylogenetic tree based on maximum likelihood and Bayesian inference analyses revealed Clinocardiinae as the sister group to Trachycardiinae, with the estimated divergence time being 44.5 million years ago (MYA) between K. buelowi and Vasticardium flavum. Notably, the cytochrome b gene (cob) exhibited a positive selection signal. Our findings provide valuable insights into the evolutionary history and molecular phylogeny of K. buelowi.

Structure of the Nmd4-Upf1 complex supports conservation of the nonsense-mediated mRNA decay pathway between yeast and humans.

The nonsense-mediated mRNA decay (NMD) pathway clears eukaryotic cells of mRNAs containing premature termination codons (PTCs) or normal stop codons located in specific contexts. It therefore plays an important role in gene expression regulation. The precise molecular mechanism of the NMD pathway has long been considered to differ substantially from yeast to metazoa, despite the involvement of universally conserved factors such as the central ATP-dependent RNA-helicase Upf1. Here, we describe the crystal structure of the yeast Upf1 bound to its recently identified but yet uncharacterized partner Nmd4, show that Nmd4 stimulates Upf1 ATPase activity and that this interaction contributes to the elimination of NMD substrates. We also demonstrate that a region of Nmd4 critical for the interaction with Upf1 in yeast is conserved in the metazoan SMG6 protein, another major NMD factor. We show that this conserved region is involved in the interaction of SMG6 with UPF1 and that mutations in this region affect the levels of endogenous human NMD substrates. Our results support the universal conservation of the NMD mechanism in eukaryotes.

Spastin accumulation and motor neuron defects caused by a novel SPAST splice site mutation

BackgroundHereditary spastic paraplegia (HSP) is a rare genetically heterogeneous neurodegenerative disorder. The most common type of HSP is caused by pathogenic variants in the SPAST gene. Various hypotheses regarding the pathogenic mechanisms of HSP-SPAST have been proposed. However, a single hypothesis may not be sufficient to explain HSP-SPAST.ObjectiveTo determine the causative gene of autosomal dominant HSP-SPAST in a pure pedigree and to study its underlying pathogenic mechanism.MethodsA four-generation Chinese family was investigated. Genetic testing was performed for the causative gene, and a splice site variant was identified. In vivo and in vitro experiments were conducted separately. Western blotting and immunofluorescence were performed after transient transfection of cells with the wild-type (WT) or mutated plasmid. The developmental expression pattern of zebrafish spasts was assessed via whole-mount in situ hybridization. The designed guide RNA (gRNA) and an antisense oligo spast-MO were microinjected into Tg(hb9:GFP) zebrafish embryos, spinal cord motor neurons were observed, and a swimming behavioral analysis was conducted.ResultsA novel heterozygous intron variant, c.1004 + 5G > A, was identified in a pure HSP-SPAST pedigree and shown to cosegregate with the disease phenotypes. This intron splice site variant skipped exon 6, causing a frameshift mutation that resulted in a premature termination codon. In vitro, the truncated protein was evenly distributed throughout the cytoplasm, formed filamentous accumulations around the nucleus, and colocalized with microtubules. Truncated proteins diffusing in the cytoplasm appeared denser. No abnormal microtubule structures were observed, and the expression levels of α-tubulin remained unchanged. In vivo, zebrafish larvae with this mutation displayed axon pathfinding defects, impaired outgrowth, and axon loss. Furthermore, spast-MO larvae exhibited unusual behavioral preferences and increased acceleration.ConclusionThe adverse effects of premature stop codon mutations in SPAST result in insufficient levels of functional protein, and the potential toxicity arising from the intracellular accumulation of spastin serves as a contributing factor to HSP-SPAST.

HPLC for at-line reaction monitoring and purification improves yield and purity of tRNA.

Engineered transfer RNA is an emerging therapeutic modality, particularly suited to treatment of diseases caused by genetic disorders based on premature termination codons, frameshifts, or missense mutations. It is also extensively used in reprogramming of in vitro translation systems to generate non-canonical amino acid-containing proteins and peptides, such as in mRNA display. Due to its length, chemical synthesis of tRNA is challenging and production of engineered tRNA at scale is currently limited to in vitro transcription from a DNA template. Previously, the highest reported in vitro transcription yield was 2.5 g/L, significantly below the industry standard for mRNA production of 7-10 g/L. To improve this process, we implemented monitoring of nucleoside triphosphate consumption and tRNA production during in vitro transcription, using at-line high-performance liquid chromatography, with a monolithic solid phase. This allowed for optimization of nucleoside triphosphate concentration, reduction of the in vitro transcription time to <4 h, and improvement of yield up to 4.7 g/L. A step-elution purification on a DEAE chromatographic monolith with >90% step yield was then developed. These improvements in the production and purification of tRNA represent an important step in facilitating production of tRNA for research purposes, and provide a method for purification of therapeutic tRNAs that is scalable and compatible with Good Manufacturing Practice requirements for clinical production.

Mitochondrial Genome Characteristics and Phylogenetic Analysis of Fulmekiola serrata (Kobus) (Thysanoptera: Thripidae)

Sugarcane thrips, Fulmekiola serrata (Kobus) (Thysanoptera: Thripidae), is a common foliar pest that infests sugarcane and is found throughout tropical and subtropical countries. In this study, we obtained and analyzed the complete mitochondrial genome of F. serrata for the first time and explored the phylogenetic relationships of the higher-order elements of Thysanoptera members at the mitochondrial level. The complete mitochondrial genome of F. serrata is 16,596 bp in length and includes 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, 2 ribosomal RNA (rRNA) genes, and 1 noncoding control region. A+T accounted for 75% of the total bases in the mitochondrial genome of F. serrata, revealing an obvious AT bias. Among the 13 PCGs, except for nad5, which had a start codon of TTG, the remaining genes had ATNs typical of insects (ATA, ATT, ATC, and ATG); nad1, nad2, nad3, and atp8 had incomplete termination codons of TA or T. The remaining nine PCGs were complete with the termination codon TAA. Of the 22 tRNA secondary structures, all were typical cloverleaf secondary structures except for trnS1, which was missing the DHU arm. Compared with the hypothetical ancestral gene arrangement of arthropods, F. serrata presented extensive gene rearrangement, with 23 translocated genes, 8 inverted genes, and 5 shuffled genes. Both maximum likelihood (ML) and Bayesian inference (BI) phylogenetic trees resulted in similar topologies: ((Thripidae + (Stenurothripidae + Aeolothripidae)) + Phlaeothripidae), with Thripidae, Aeolothripidae and Phlaeothripidae being monophyletic groups, whereas F. serrata is closely related to Thrips palmi, and the two are sister groups.

Directed evolution of aminoacyl-tRNA synthetases through in vivo hypermutation.

Genetic code expansion (GCE) has become a critical tool in biology by enabling the site-specific incorporation of non-canonical amino acids (ncAAs) into proteins. Central to GCE is the development of orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pairs wherein engineered aaRSs recognize chosen ncAAs and charge them onto tRNAs that decode blank codons ( e.g ., the amber stop codon). Many orthogonal aaRS/tRNA pairs covering a wide range of ncAAs have been generated by directed evolution, yet the evolution of new aaRS/tRNA pairs by standard strategies remains a labor-intensive process that often produces aaRS/tRNA pairs with suboptimal ncAA incorporation efficiencies. In this study, we present a strategy for evolving aaRSs that leverages OrthoRep to drive their continuous hypermutation in yeast. We demonstrate our strategy in 8 independent aaRS evolution campaigns starting from 4 different aaRS/tRNA parents targeting 7 distinct ncAAs. We observed the rapid evolution of multiple novel aaRSs capable of incorporating an overall range of 13 ncAAs tested into proteins in response to the amber codon. Some evolved systems reached efficiencies for amber codon-specified ncAA-dependent translation comparable to translation with natural amino acids specified by sense codons in yeast. Additionally, we discovered a surprising aaRS that evolved to self-regulate its own expression for greater dependency on ncAAs for translation. These findings demonstrate the potential of OrthoRep-driven aaRS evolution platforms in supporting the continued growth of GCE technologies.

Tyrp1 is the mendelian determinant of the Axolotl (Ambystoma mexicanum) copper mutant

Several dozen Mendelian mutants have been discovered in axolotl (Ambystoma mexicanum) populations, including several that affect pigmentation. Four recessive mutants have been described in the scientific literature and genes for three of these have been identified. Here we describe and genetically dissect copper, a mutant with an albino-like phenotype known only from the pet trade. We performed a cross segregating copper and wildtype color phenotypes and used bulked segregant RNA-Seq to identify a region on chromosome 6 that was enriched for single-nucleotide polymorphisms (SNPs) between the color phenotypes. This region included Tyrosinase-like Protein 1 (Tyrp1), a melanin synthesis protein that when mutated, is associated with lighter than black melanin coloration in animal models and oculocutaneous albinism in humans. Inspection of RNA-Seq reads identified a single nucleotide deletion that is predicted to change the coding frame, introduce a premature stop codon in exon 6 and yield a truncated Tyrp1 protein in copper individuals. Using CRISPR-Cas9 editing, we show that wildtype Tyrp1 crispants exhibit copper pigmentation, thus confirming Tyrp1 as the copper locus. Our results suggest that commercial and hobbyist axolotl populations may harbor useful mutants for biological research.

The evolution and functional significance of the programmed ribosomal frameshift in prfB.

Release Factor 2 (RF2) is one of two peptide release factors that terminate translation in bacteria. In Escherichia coli, the gene encoding RF2, prfB, contains an in-frame premature RF2-specific stop codon. Therefore, a programmed ribosomal frameshift is required to translate full-length RF2. Here, we investigate the diversity of prfB frameshifting through bioinformatic analyses of >12,000 genomes. We present evidence that prfB frameshifting autoregulates RF2 levels throughout the bacterial domain since (i) the prfB in-frame stop codon is always TGA or TAA, both of which are recognized by RF2, and never the RF1-specific TAG stop codon, and (ii) species that lack the autoregulatory programmed frameshift likely need higher RF2 levels since, on average, they have significantly higher RF2-specific stop codon usage. Overexpression of prfB without the autoregulatory frameshift motif is toxic to Bacillus subtilis, an organism with intermediate RF2-specific stop codon usage. We did not detect the programmed frameshift in any Actinobacteriota. Consistent with this finding, we observed very low frameshift efficiency at the prfB frameshift motif in the Actinobacterium Mycobacterium smegmatis. Our work provides a more complete picture of the evolution of the RF2 programmed frameshifting motif, and its usage to prevent toxic overexpression of RF2.

Generation of XPA p.Arg228T mutant LUMCi004-A cell line for modeling Xeroderma pigmentosum group A

Xeroderma pigmentosum group A (XPA) is an inherited skin disorder characterized by sensitivity to ultraviolet radiation. In Maghrebi patients, a homozygous mutation in exon 6 of the XPA gene (c.682C>T) results in the introduction of a premature termination codon. Using CRISPR/Cas9-mediated gene editing, this mutation was introduced into the well-characterized LUMCi004-A line. The resulting hiPSC line showed typical morphology, expressed markers of the undifferentiated state, was able to differentiate into the three germ layers in vitro and displayed a normal karyotype. When paired with its isogenic counterpart, this line represents a valuable resource to model the disease.

Overcoming Challenges with Biochemical Studies of Selenocysteine and Selenoproteins.

Selenocysteine (Sec) is an essential amino acid that distinguishes itself from cysteine by a selenium atom in place of a sulfur atom. This single change imparts distinct chemical properties to Sec which are crucial for selenoprotein (Sec-containing protein) function. These properties include a lower pKa, enhanced nucleophilicity, and reversible oxidation. However, studying Sec incorporation in proteins is a complex process. While we find Sec in all domains of life, each domain has distinct translation mechanisms. These mechanisms are unique to canonical translation and are composed of Sec-specific enzymes and an mRNA hairpin to drive recoding of the UGA stop codon with Sec. In this review, we highlight the obstacles that arise when investigating Sec insertion, and the role that Sec has in proteins. We discuss the strategic methods implemented in this field to address these challenges. Though the Sec translation system is complex, a remarkable amount of information has been obtained and specialized tools have been developed. Continued studies in this area will provide a deeper understanding on the role of Sec in the context of proteins, and the necessity that we have for maintaining this complex translation machinery to make selenoproteins.

Calorie restriction and rapamycin distinctly restore non-canonical ORF translation in the muscles of aging mice

Loss of protein homeostasis is one of the hallmarks of aging. As such, interventions that restore proteostasis should slow down the aging process and improve healthspan. Two of the most broadly used anti-aging interventions that are effective in organisms from yeast to mammals are calorie restriction (CR) and rapamycin (RM) treatment. To identify the regulatory mechanisms by which these interventions improve the protein homeostasis, we carried out ribosome footprinting in the muscle of mice aged under standard conditions, or under long-term treatment with CR or RM. We found that the treatments distinctly impact the non-canonical translation, RM primarily remodeling the translation of upstream open reading frames (uORFs), while CR restores stop codon readthrough and the translation of downstream ORFs. Proteomics analysis revealed the expression of numerous non-canonical ORFs at the protein level. The corresponding peptides may provide entry points for therapies aiming to maintain muscle function and extend health span.

The first report of complete mitogenomes of two endangered species of genus Propomacrus (Coleoptera: Scarabaeidae: Euchirinae) and phylogenetic implications.

To understand the mitochondrial genome structure of two endangered and long-armed scarab beetles, Propomacrus davidi and Propomacrus bimucronatus, their complete mitogenomes were sequenced for the first time in this study. The complete mitogenomes of P. davidi and P. bimucronatus were 18, 042 bp and 18, 104 bp in length, respectively. The gene orders of their mitogenomes were highly consistent with other Coleopteran species, and the typical ATN was used as the start codon in most protein coding genes. The incomplete stop codon T was used in cox1, cox2, and nad5, and TAN was used as a complete stop codon in most protein coding genes. All predicted tRNAs could form a typical cloverleaf secondary structure, except that trnS1 lacked the dihydrouridine arm. Based on the maximum likelihood and the Bayesian inference methods, phylogenetic trees of 50 species were reconstructed. The results showed that P. davidi, P. bimucronatus, Cheirotonus jansoni and Cheirotonus gestroi clustered in the same branch, and were the most closely related. The results supported that subfamily Euchirinae is a monophyletic group of Scarabaeidae, which was consistent with the morphological classification. These molecular data enriched the complete mitogenome database of Euchirinae, and improved our understanding of the phylogenetic relationship and evolutionary characteristics of these two endangered species.

NMDtxDB: data-driven identification and annotation of human NMD target transcripts.

The nonsense-mediated RNA decay (NMD) pathway is a crucial mechanism of mRNA quality control. Current annotations of NMD substrate RNAs are rarely data-driven, but use generally established rules. We present a data set with four cell lines and combinations for SMG5, SMG6, and SMG7 knockdowns or SMG7 knockout. Based on this data set, we implemented a workflow that combines Nanopore and Illumina sequencing to assemble a transcriptome, which is enriched for NMD target transcripts. Moreover, we use coding sequence information (CDS) from Ensembl, Gencode consensus Ribo-seq ORFs, and OpenProt to enhance the CDS annotation of novel transcript isoforms. In summary, 302,889 transcripts were obtained from the transcriptome assembly process, out of which 24% are absent from Ensembl database annotations, 48,213 contain a premature stop codon, and 6433 are significantly upregulated in three or more comparisons of NMD active versus deficient cell lines. We present an in-depth view of these results through the NMDtxDB database, which is available at https://shiny.dieterichlab.org/app/NMDtxDB, and supports the study of NMD-sensitive transcripts. We open sourced our implementation of the respective web-application and analysis workflow at https://github.com/dieterich-lab/NMDtxDB and https://github.com/dieterich-lab/nmd-wf.