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One gene does not equal one RNA or protein. The genomic revolution has revealed numerous different RNA and protein molecules that can be produced from one gene, such as circular RNAs generated by back-splicing, proteins with residues mismatching the genomic encoding because of RNA editing, and proteins extended in the C terminus via stop codon readthrough in translation. Are these diverse products results of exquisite gene regulations or imprecise biological processes? While there are cases where the gene product diversity appears beneficial, genome-scale patterns suggest that much of this diversity arises from nonadaptive, molecular errors. This finding has important implications for studying the functions of diverse gene products and for understanding the fundamental properties and evolution of cellular life.

Nonsense-mediated mRNA decay (NMD) protects cells from the toxic and potentially dominant effects of truncated proteins. Targeting of mRNAs with early stop codons is mediated by the ribosome and spatiotemporally aligned with translation termination. Previously we identified a novel NMD intermediate: ribosomes stalled on cleaved stop codons, raising the possibility that NMD begins even prior to ribosome removal from the stop codon. Here we show that this intermediate is the result of mRNA cleavage by the endonuclease SMG-6. Our work supports a model in which ribosomes stall secondary to SMG-6 mRNA cleavage in Caenorhabditis elegans and humans, i.e. that the novel NMD intermediate occurs after a prior ribosome elicits NMD. Our genetic analysis of C. elegans’ SMG-6 supports a central role for SMG-6 in metazoan NMD, and provides a context for evaluating its function in other metazoans.

Because DNA metabarcoding typically employs sequence diversity among mitochondrial amplicons to estimate species composition, nuclear mitochondrial pseudogenes (NUMTs) can inflate diversity. This study quantifies the incidence and attributes of NUMTs derived from the 658-bp barcode region of cytochrome c oxidase I (COI) in 156 marine animal genomes. NUMTs were examined to ascertain if they could be recognized by their possession of indels or stop codons. In total, 309 NUMTs ≥150 bp were detected, with an average of 1.98 per species (range = 0-33) and a mean length of 391 ± 200 bp. Among this total, 75 (24.3%) lacked indels or stop codons. NUMTs appear to pose the greatest interpretational risk when short (313 bp) amplicons are used, such as in environmental DNA studies, dietary analyses or processed fish identification. Employing the standard amplicon length (313 bp) for marine metabarcoding, NUMTs could potentially inflate the operational taxonomic unit (OTU) count by 21% above the true species count while also raising intraspecific variation at COI by 15%. However, when both amplicon length and position are considered, inflation in OTU counts and in barcode variation were just 9% and 10%, respectively, suggesting NUMTs will not seriously distort biodiversity assessments. There was a weak positive correlation between genome size and NUMT count but no variation among phyla or trophic groups. Until bioinformatic advances improve NUMT detection, the best defence involves targeting long amplicons and developing reference databases that include both mitochondrial sequences and their NUMT derivatives.

Cell-cell signaling in microorganisms is still poorly characterized. In this Methods paper, we describe a genetic procedure for detecting cell-nonautonomous genetic effects, and in particular cell-cell signaling, termed the chimeric colony assay (CCA). The CCA measures the effect of a gene on a biological response in a neighboring cell. This assay can measure cell autonomy for range of biological activities including transcript or protein accumulation, subcellular localization, and cell differentiation. To date, the CCA has been used exclusively to investigate colony patterning in the budding yeast Saccharomyces cerevisiae. To demonstrate the wider potential of the assay, we applied this assay to two other systems: the effect of Grr1 on glucose repression of GAL1 transcription in yeast and the effect of rpsL on stop-codon translational readthrough in Escherichia coli. We also describe variations of the standard CCA that address specific aspects of cell-cell signaling, and we delineate essential controls for this assay. Finally, we discuss complementary approaches to the CCA. Taken together, this Methods paper demonstrates how genetic assays can reveal and explore the roles of cell-cell signaling in microbial processes.

Nonsense-mediated mRNA decay (NMD)-mediated degradation of BrFLC2 transcripts is the main cause of rapid flowering of oilseed-type B. rapa 'LP08' plants. Many Brassica species require vernalization (long-term winter-like cooling) for transition to the reproductive stage. In the past several decades, scientific efforts have been made to discern the molecular mechanisms underlying vernalization in many species. Thus, to identify the key regulators required for vernalization in Brassica rapa L., we constructed a linkage map composed of 7833 single nucleotide polymorphism markers using the late-flowering Chinese cabbage (B. rapa L. ssp. pekinensis) inbred line 'Chiifu' and the early-flowering yellow sarson (B. rapa L. ssp. trilocularis) line 'LP08' and identified a single major QTL on the upper-arm of the chromosome A02. In addition, we compared the transcriptomes of the lines 'Chiifu' and 'LP08' at five vernalization time points, including both non-vernalized and post-vernalization conditions. We observed that BrFLC2 was significantly downregulated in the early flowering 'LP08' and had two deletion sites (one at 4th exon and the other at 3' downstream region) around the BrFLC2 genomic region compared with the BrFLC2 genomic region in 'Chiifu'. Large deletion at 3' downstream region did not significantly affect transcription of both sense BrFLC2 transcript and antisense transcript, BrFLC2as along vernalization time course. However, the other deletion at 4th exon of BrFLC2 resulted in the generation of premature stop codon in BrFLC2 transcript in LP08 line. Cycloheximide treatment of LP08 line showed the de-repressed level of BrFLC2 in LP08, suggesting that low transcript level of BrFLC2 in LP08 might be caused by nonsense-mediated mRNA decay removing the nonsense transcript of BrFLC2. Collectively, this study provides a better understanding of the molecular mechanisms underlying floral transition in B. rapa.

BACKGROUND Familial exudative vitreoretinopathy (FEVR) is a group of inherited eye diseases characterized by premature arrest of retinal vessel development. The purpose of our study was to characterize the genetic causes and clinical features in eight Chinese families with FEVR using next-generation sequencing (NGS) technology. MATERIALS AND METHODS Eight families with FEVR were included in genetic and clinical analyses. We screened the proband and the parents in eight pedigrees with FEVR using targeted NGS approach and in silico analysis to determine the causative mutation for their family's phenotype. RESULTS Four cases (4/8, 50.0%) were confirmed to harbor mutations in known genes, including 3 novel mutations and one previously reported mutation. Among the detected mutations, TSPAN12 accounted for 75% (3/4). We identified a novel stop codon of TSPAN12, a heterozygous missense mutation NM_012338.4:c.633T>A, NP_036470.1:p.Tyr211Ter involved in highly conserved residues in the proband. Retrospective analysis of its clinical manifestation showed that the mutant carrier presented mild clinical features. CONCLUSIONS We found the novel stop codon mutation p.Tyr211Ter in the TSPAN12, which creates a milder phenotype. Discovery of this novel mutation expands the mutation spectrum of TSPAN12, and would be valuable for future genetic disease diagnosis.

Rapid and sensitive detection of Salmonella is a critical step in routine food quality control, outbreak investigation, and food recalls. Although various genes have been the targets in the design of rapid molecular detection methods for Salmonella, there is limited information on the diversity of these target genes at the level of DNA sequence and the encoded protein structures. In this study, we investigated the diversity of ten target genes (invA, fimA, phoP, spvC, and agfA; ttrRSBCA operon including 5 genes) commonly used in the detection and identification of Salmonella. To this end, we performed whole genome sequencing of 143 isolates of Salmonella serotypes (Enteritidis, Typhimurium, and Heidelberg) obtained from poultry (eggs and chicken). Phylogenetic analysis showed that Salmonella ser. Typhimurium was more diverse than either Enteritidis or Heidelberg. Forty-five non-synonymous mutations were identified in the target genes from the 143 isolates, with the two most common mutations as T ↔ C (15 times) and A ↔ G (13 times). The gene spvC was primarily present in Salmonella ser. Enteritidis isolates and absent from Heidelberg isolates, whereas ttrR was more conserved (0 non-synonymous mutations) than ttrS, ttrB, ttrC, and ttrA (7, 2, 2, and 7 non-synonymous mutations, respectively). Notably, we found one non-synonymous mutation (fimA-Mut.6) across all Salmonella ser. Enteritidis and Salmonella ser. Heidelberg, C → T (496 nt postion), resulting in the change at AA 166 position, Glutamine (Q) → Stop condon (TAG), suggesting that the fimA gene has questionable sites as a target for detection. Using Phyre2 and SWISS-MODEL software, we predicted the structures of the proteins encoded by some of the target genes, illustrating the positions of these non-synonymous mutations that mainly located on the α-helix and β-sheet which are key elements for maintaining the conformation of proteins. These results will facilitate the development of sensitive molecular detection methods for Salmonella.

The complete mitochondrial genome (mitogenome) of Saturnia japonica (Lepidoptera: Saturniidae) was sequenced and annotated. It is a circular molecule of 15, 376 bp, composed of 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (rRNA), and an adenine (A) + thymine (T)-rich region. All protein-coding genes (PCGs) are initiated by the ATN codon except for cytochrome c oxidase subunit 1 (cox1) gene that is seemingly initiated by the CGA codon. Except for cox2 and nad4, which were terminated by incomplete stop codon T or TA, the rest were terminated by canonical stop codon TAA. The A + T-rich region is high conservative, including ‘ATAGA’ motif followed by a 19 bp poly-T stretch, a microsatellite-like element (AT)9 and also a poly-A element, with a total length of 332 bp. The Asn codon was the most frequently used codon, followed by Ile, Leu2, Lys, Met, Phe, and Tyr, while Cys was the least frequently used codon. Phylogenetic relationships analysis based on the 13 PCGs by using maximum likelihood (ML) and neighbor Joining (NJ) revealed that S. japonica belongs to the Saturniidae family. In this study, the annotation and characteristics of the mitogenome of S. japonica were resolved for the first time, which laid a foundation for species classification and the molecular evolution of Lepidoptera: Saturniidae.

Protein coding genes terminate with one of three stop codons (TAA, TGA, or TAG) that, like synonymous codons, are not employed equally. With TGA and TAG having identical nucleotide content, analysis of their differential usage provides an unusual window into the forces operating on what are ostensibly functionally identical residues. Across genomes and between isochores within the human genome, TGA usage increases with G + C content but, with a common G + C → A + T mutation bias, this cannot be explained by mutation bias-drift equilibrium. Increased usage of TGA in G + C-rich genomes or genomic regions is also unlikely to reflect selection for the optimal stop codon, as TAA appears to be universally optimal, probably because it has the lowest read-through rate. Despite TAA being favored by selection and mutation bias, as with codon usage bias G + C pressure is the prime determinant of between-species TGA usage trends. In species with strong G + C-biased gene conversion (gBGC), such as mammals and birds, the high usage and conservation of TGA is best explained by an A + T → G + C repair bias. How to explain TGA enrichment in other G + C-rich genomes is less clear. Enigmatically, across bacterial and archaeal species and between human isochores TAG usage is mostly unresponsive to G + C pressure. This unresponsiveness we dub the TAG paradox as currently no mutational, selective, or gBGC model provides a well-supported explanation. That TAG does increase with G + C usage across eukaryotes makes the usage elsewhere yet more enigmatic. We suggest resolution of the TAG paradox may provide insights into either an unknown but common selective preference (probably at the DNA/RNA level) or an unrecognized complexity to the action of gBGC.

Different essential viral proteins are translated via programmed stop codon readthrough. Pea enation mosaic virus 1 (PEMV1) and potato leafroll virus (PLRV) are related positive-sense RNA plant viruses in the family Solemoviridae, and are type members of the Enamovirus and Polerovirus genera, respectively. Both use translational readthrough to express a C-terminally extended minor capsid protein (CP), termed CP-readthrough domain (CP-RTD), from a viral subgenomic mRNA that is transcribed during infections. Limited incorporation of CP-RTD subunits into virus particles is essential for aphid transmission, however the functional readthrough structures that mediate CP-RTD translation have not yet been defined. Through RNA solution structure probing, RNA secondary structure modeling, site-directed mutagenesis, and functional in vitro and in vivo analyses, we have investigated in detail the readthrough elements and complex structure involved in expression of CP-RTD in PEMV1, and assessed and deduced a comparatively simpler readthrough structure for PLRV. Collectively, this study has (i) generated the first higher-order RNA structural models for readthrough elements in an enamovirus and a polerovirus, (ii) revealed a stark contrast in the complexity of readthrough structures in these two related viruses, (iii) provided compelling experimental evidence for the strict requirement for long-distance RNA-RNA interactions in generating the active readthrough signals, (iv) uncovered what could be considered the most complex readthrough structure reported to date, that for PEMV1, and (v) proposed plausible assembly pathways for the formation of the elaborate PEMV1 and simple PLRV readthrough structures. These findings notably advance our understanding of this essential mode of gene expression in these agriculturally important plant viruses.

The SARS-CoV-2 Variant of Concern 202012/01 (VOC-202012/01) emerged in southeast England and rapidly spread worldwide. This variant is believed to be more transmissible, with all attention being given to its spike mutations. However, VOC-202012/01 has also a mutation (Q27stop) that truncates the ORF8, a likely immune evasion protein. Removal of ORF8 changes the clinical outset of the disease, which may affect the virus transmissibility. Here I provide a detailed analysis of all reported ORF8-deficient lineages found in the background of relevant spike mutations, identified among 231,433 SARS-CoV-2 genomes. I found 19 ORF8 nonsense mutations, most of them occurring in the 5’ half of the gene. The ORF8-deficient lineages were rare, representing 0.67% of sequenced genomes. Nevertheless, I identified two clusters of related sequences that emerged recently and spread in different countries. The widespread D614G spike mutation was found in most ORF-deficient lineages. Although less frequent, HV69-70del and L5F spike mutations occurred in the background of six different ORF8 nonsense mutations. I also confirmed that VOC-202012/01 is the ORF8-deficient variant with more spike mutations reported to date, although other variants could have up to six spike mutations, some of putative biological relevance. Overall, these results suggest that monitoring ORF8-deficient lineages is important for the progression of the COVID-19 pandemic, particularly when associated with relevant spike mutations.

SUMMARYPIWI-interacting RNAs (piRNAs) are small RNAs required to recognize and silence transposable elements. The 5’ ends of mature piRNAs are defined through cleavage of long precursor transcripts, primarily by Zucchini (Zuc). Zuc-dependent cleavage typically occurs immediately upstream of a uridine. However, Zuc lacks sequence preference in vitro, pointing towards additional unknown specificity factors. We examined murine piRNAs and revealed a strong and specific enrichment of three sequences (UAA, UAG, UGA)— corresponding to stop codons—at piRNA 5’ ends. Stop codon sequences were also enriched immediately after piRNA processing intermediates, reflecting their Zuc-dependent tail-to-head arrangement. Further analyses revealed that a Zuc in vivo cleavage preference at four sequences (UAA, UAG, UGA, UAC) promotes 5’ end stop codons. This observation was conserved across mammals and possibly further. Our work provides new insights into Zuc-dependent cleavage and may point to a previously unrecognized connection between piRNA biogenesis and the translational machinery.

Pseudogenes (genes disrupted by frameshift or in-frame stop codons) are ubiquitously present in the bacterial genome and considered as nonfunctional fossil. Here, we used RNA-seq and mass-spectrometry technologies to measure the transcriptomes and proteomes of Salmonella enterica serovars Paratyphi A and Typhi. All pseudogenes’ mRNA sequences remained disrupted, and were present at comparable levels to their intact homologs. At the protein level, however, 101 out of 161 pseudogenes suggested successful translation, with their low expression regardless of growth conditions, genetic background and pseudogenization causes. The majority of frameshifting detected was compensatory for -1 frameshift mutations. Readthrough of in-frame stop codons primarily involved UAG; and cytosine was the most frequent base adjacent to the codon. Using a fluorescence reporter system, fifteen pseudogenes were confirmed to express successfully in vivo in Escherichia coli. Expression of the intact copy of the fifteen pseudogenes in S. Typhi affected bacterial pathogenesis as revealed in human macrophage and epithelial cell infection models. The above findings suggest the need to revisit the nonstandard translation mechanism as well as the biological role of pseudogenes in the bacterial genome.

Recent studies have unequivocally confirmed the presence of 5-methylcytosine (m5C) in mammalian mRNAs while indicating significant functional roles for this internal base modification type. Here, a brief history of m5C epitranscriptome research and a discussion of the important ways in which the field may now progress is presented.

Amyotrophic lateral sclerosis (ALS) is the most common and severe adult-onset motoneuron disease and has currently no effective therapy. Approximately 20% of familial ALS cases are caused by dominantly-inherited mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1), which represents one of the most frequent genetic cause of ALS. Despite the overwhelming majority of ALS-causing missense mutations in SOD1, a minority of premature termination codons (PTCs) have been identified. mRNA harboring PTCs are known to be rapidly degraded by nonsense-mediated mRNA decay (NMD), which limits the production of truncated proteins. The rules of NMD surveillance varying with PTC location in mRNA, we analyzed the localization of PTCs in SOD1 mRNA to evaluate whether or not those PTCs can be triggered to degradation by the NMD pathway. Our study shows that all pathogenic PTCs described in SOD1 so far can theoretically escape the NMD, resulting in the production of truncated protein. This finding supports the hypothesis that haploinsufficiency is not an underlying mechanism of SOD1 mutant-associated ALS and suggests that PTCs found in the regions that trigger NMD are not pathogenic. Such a consideration is particularly important since the availability of SOD1 antisense strategies, in view of variant treatment assignment.

Background Selenium is an essential trace element, and selenocysteine (Sec, U) is its predominant form in vivo. Proteins that contain Sec are selenoproteins, whose special structural features include not only the TGA codon encoding Sec but also the SECIS element in mRNA and the conservation of the Sec-flanking region. These unique features have led to the development of a series of bioinformatics methods to predict and research selenoprotein genes. There have been some studies and reports on the evolution and distribution of selenoprotein genes in prokaryotes and multicellular eukaryotes, but the systematic analysis of single-cell eukaryotes, especially algae, has been very limited. Results In this study, we predicted selenoprotein genes in 137 species of algae by using a program we previously developed. More than 1000 selenoprotein genes were obtained. A database website was built to record these algae selenoprotein genes (www.selenoprotein.com). These genes belong to 42 selenoprotein families, including three novel selenoprotein gene families. Conclusions This study reveals the primordial state of the eukaryotic selenoproteome. It is an important clue to explore the significance of selenium for primordial eukaryotes and to determine the complete evolutionary spectrum of selenoproteins in all life forms.

A couple with five adverse pregnancy history required prenatal diagnosis. The fetus of this study was their fifth pregnancy. The fetus was found NT thickening at 12 weeks and 4 days gestation and the average long bone of limbs retardation 4SD at 27 weeks and 4 days gestation. Karyotype was normal. The next-generation sequencing (NGS) and Sanger sequencing were conducted of this fetus. The compound heterozygous mutations c.3722_3749dup[p.V1252fs*23] and c.3355 + 5 G > A at CUL7 gene were detected. The mutation c.3355 + 5 G > A was a novel mutation within intron 17 of the CUL7 gene. Minigene array was used to verify whether the novel mutation c.3355 + 5 G > A really affected the splicing of CUL7gene. The results showed that the mutation could result in the appearance of premature termination codon. The fetus could be diagnosed as 3 M syndrome. We suggested that close attention needed to be paid to fetuses with intrauterine growth restriction only by ultrasonic and avoid misdiagnosis and missed diagnosis of 3 M syndrome. In addition, our study enriched gene mutations of 3 M syndrome.

Adenine base editor (ABE) is a new generation of genome-editing technology through fusion of Cas9 nickase with an evolved E. coli TadA (TadA∗) and holds great promise as novel genome-editing therapeutics for treating genetic disorders. ABEs can directly convert A-T to G-C in specific genomic DNA targets without introducing double-strand breaks (DSBs). We recently showed that computer program-assisted analysis of Sanger sequencing traces can be used as a low-cost and rapid alternative of deep sequencing to assess base-editing outcomes. Here we developed a rapid fluorescence-based reporter assay (Base Editing ON [BEON]) to quantify ABE efficiency. The assay relies on the restoration of the downstream green fluorescent protein (GFP) in ABE-mediated editing of a stop codon located within the guide RNA (gRNA). We showed that this assay can be used to screen for effective ABE variants, characterize the protospacer adjacent motif (PAM) requirement of a novel NNG-targeting ABE based on ScCas9, and enrich for edited cells. Finally, we demonstrated that the reporter assay allowed us to assess the feasibility of ABE editing to correct point mutations associated with dysferlinopathy. Taken together, the BEON assay would facilitate and simplify the studies with ABEs.

Since 1992, China has promoted hepatitis B vaccination. Concurrently, during this period, increasing use of immunoglobulins and nucleoside analogues might have exerted selective pressure on the hepatitis B virus (HBV) S gene, driving mutations in the HBsAg and changed the subtype. Using the National Center for Biotechnology Information database, we obtained gene sequence information for HBV strains from China and analysed changes in HBsAg subtypes and substitution mutations in HBsAg in 5-year intervals over 25 years to identify potential challenges to the prevention and treatment of hepatitis B. Most HBV sequences from China were genotype C (1996/2833, 70.46%) or B (706/2833, 24.92%). During the implementation of hepatitis B vaccination (recombinant hepatitis B vaccine was subgenotype A2 and HBsAg subtype adw2), the proportion of subtypes ayw1 and adw3 in genotype B and ayw2 in genotype C increased over the programme period. The overall mutation rate in HBsAg tended to decrease for genotype B, whereas, for genotype C, the rate increased gradually and then decreased slightly. Moreover, the mutation rate at some HBsAg amino acid sites (such as sG145 of genotype B and sG130 and sK141 of genotype C) is gradually increasing. HBV strains with internal stop codons of HBsAg (e.g., sC69*) and additional N-glycosylation (e.g., sG130N) mutations should be studied extensively to prevent them from becoming dominant circulating strains. The development of HBV vaccines and antiviral immunoglobulins and use of antiviral drugs may require making corresponding changes.

The release of DNA into the cytoplasm upon damage to the nucleus or during viral infection triggers an interferon-mediated defense response, inflammation and cell death. In human cells cytoplasmic DNA is sensed by cyclic GMP-AMP Synthase (cGAS) and Absent In Melanoma 2 (AIM2). Here, we report the identification of a “natural knockout” model of cGAS. Comparative genomics of phylogenetically diverse mammalian species showed that cGAS and its interaction partner Stimulator of Interferon Genes (STING) have been inactivated by mutations in the Malayan pangolin whereas other mammals retained intact copies of these genes. The coding sequences of CGAS and STING1 are also disrupted by premature stop codons and frame-shift mutations in Chinese and tree pangolins, suggesting that expression of these genes was lost in a common ancestor of all pangolins that lived more than 20 million years ago. AIM2 is retained in a functional form in pangolins whereas it is inactivated by mutations in carnivorans, the phylogenetic sister group of pangolins. The deficiency of cGAS and STING points to the existence of alternative mechanisms of controlling cytoplasmic DNA-associated cell damage and viral infections in pangolins. Electronic supplementary material The online version of this article (10.1007/s10495-020-01614-4) contains supplementary material, which is available to authorized users.

Faithful translation of genetic information depends on the ability of the translational machinery to decode stop codons as termination signals. Although termination of protein synthesis is highly efficient, errors in decoding of stop codons may lead to the synthesis of C-terminally extended proteins. It was found that in eukaryotes such elongated proteins do not accumulate in cells. However, the mechanism for sequestration of C-terminally extended proteins is still unknown. Here we show that 3′-UTR-encoded polypeptides promote aggregation of the C-terminally extended proteins, and targeting to lysosomes. We demonstrate that 3′-UTR-encoded polypeptides can promote different levels of protein aggregation, similar to random sequences. We also show that aggregation of endogenous proteins can be induced by aminoglycoside antibiotics that promote stop codon read-through, by UAG suppressor tRNA, or by knokcdown of release factor 1. Furthermore, we find correlation between the fidelity of termination signals, and the predicted propensity of downstream 3′-UTR-encoded polypeptides to form intrinsically disordered regions. Our data highlight a new quality control mechanism for elimination of C-terminally elongated proteins.

Purpose Existing data do not explain the reason why some individuals homozygous for the hypomorphic FECH allele develop erythropoietic protoporphyria (EPP) while the majority are completely asymptomatic. This study aims to identify novel possible genetic variants contributing to this variable phenotype. Methods High-throughput resequencing of the FECH gene, qualitative analysis of RNA, and quantitative DNA methylation examination were performed on a cohort of 72 subjects. Results A novel deep intronic variant was found in four homozygous carriers developing a clinically overt disease. We demonstrate that this genetic variant leads to the insertion of a pseudo-exon containing a stop codon in the mature FECH transcript by the abolition of an exonic splicing silencer site and the concurrent institution of a new methylated CpG dinucleotide. Moreover, we show that the hypomorphic FECH allele is linked to a single haplotype of about 20 kb in size that encompasses three noncoding variants that were previously associated with expression quantitative trait loci (eQTLs). Conclusion This study confirms that intronic variants could explain the variability in the clinical manifestations of EPP. Moreover, it supports the hypothesis that the control of the FECH gene expression can be mediated through a methylation-dependent modulation of the precursor messenger RNA (pre-mRNA) splicing pattern.

Contains fulltext : 248221.pdf (Publisher’s version ) (Open Access) SUPV3L1 encodes a helicase that is mainly localized in the mitochondria. It has been shown in vitro to possess both double-stranded RNA and DNA unwinding activity that is ATP-dependent. Here we report the first two patients for this gene who presented with a homozygous preliminary stop codon resulting in a C-terminal truncation of the SUPV3L1 protein. They presented with a characteristic phenotype of neurodegenerative nature with progressive spastic paraparesis, growth restriction, hypopigmentation, and predisposition to autoimmune disease. Ophthalmological examination showed severe photophobia with corneal erosions, optic atrophy, and pigmentary retinopathy, while neuroimaging showed atrophy of the optic chiasm and the pons with calcification of putamina, with intermittent and mild elevation of lactate. We show that the amino acids that are eliminated by the preliminary stop codon are highly conserved and are predicted to form an amphipathic helix. To investigate if the mutation causes mitochondrial dysfunction, we examined fibroblasts of the proband. We observed very low expression of the truncated protein, a reduction in the mature ND6 mRNA species as well as the accumulation of double-stranded RNA. Lentiviral complementation with the full-length SUPV3L1 cDNA partly restored the observed RNA phenotypes, supporting that the SUPV3L1 mutation in these patients is pathogenic and the cause of the disease.

SummaryTranslational readthrough (TR) occurs when the ribosome decodes a stop codon as a sense codon, resulting in two protein isoforms synthesized from the same mRNA. TR is pervasive in eukaryotic organisms; however, its biological significance remains unclear. In this study, we quantify the TR potential of several candidate genes inDrosophila melanogasterand characterize the regulation of TR in the large Maf transcription factor Traffic jam (Tj). We used CRISPR/Cas9 generated mutant flies to show that the TR-generated Tj isoform is expressed in the nuclei of a subset of neural cells of the central nervous system and is excluded from the somatic cells of gonads, which express the short Tj isoform only. Translational control of TR is critical for preservation of neuronal integrity and maintenance of reproductive health. Fine-tuning of the gene regulatory functions of transcription factors by TR provides a new potential mechanism for cell-specific regulation of gene expression.HighlightsTj undergoes tissue-specific TR in neural cells of the central nervous system.Strict control of TR is crucial for neuroprotection and maintenance of reproductive capacity.TR selectively fine-tunes the gene regulatory functions of the transcription factor.TR in Tj links transcription and translation of tissue-specific control of gene expression.

Although the constraints on a gene’s sequence are often assumed to reflect the functioning of that gene, here we propose transfer selection, a constraint operating on one class of genes transferred to another, mediated by shared binding factors. We show that such transfer can explain an otherwise paradoxical depletion of stop codons in long intergenic noncoding RNAs (lincRNAs). Serine/arginine-rich proteins direct the splicing machinery by binding exonic splice enhancers (ESEs) in immature mRNA. As coding exons cannot contain stop codons in one reading frame, stop codons should be rare within ESEs. We confirm that the stop codon density (SCD) in ESE motifs is low, even accounting for nucleotide biases. Given that serine/arginine-rich proteins binding ESEs also facilitate lincRNA splicing, a low SCD could transfer to lincRNAs. As predicted, multiexon lincRNA exons are depleted in stop codons, a result not explained by open reading frame (ORF) contamination. Consistent with transfer selection, stop codon depletion in lincRNAs is most acute in exonic regions with the highest ESE density, disappears when ESEs are masked, is consistent with stop codon usage skews in ESEs, and is diminished in both single-exon lincRNAs and introns. Owing to low SCD, the maximum lengths of pseudo-ORFs frequently exceed null expectations. This has implications for ORF annotation and the evolution of de novo protein-coding genes from lincRNAs. We conclude that not all constraints operating on genes need be explained by the functioning of the gene but may instead be transferred owing to shared binding factors.

Splicing disruption is one type of mutation mechanism for disease-predisposing alleles. To date, less than 30 mutations in TTC8/BBS8 have been reported; however, mutations affecting the splice site are rare. Generally missense mutations are assumed to alter protein function; however, reports have shown that mutations in protein coding exons can disrupt splicing by altering exonic splicing silencer or enhancer motifs. Hence, a missense mutation c.1347G C (p.Q449H) involving final base of the exon 13 in the TTC8, previously identified by us to be linked with non-syndromic autosomal recessive retinitis pigmentosa (arRP), in an Indian family, that might deleteriously affect splicing has been functionally characterized. RNA was isolated, cDNA prepared and amplified using region-specific primers. PCR products were purified and sequenced bi-directionally by Sanger sequencing. Effect of mutation (c.1347G C) on mRNA splicing has been predicted using bioinformatics tools. We reported that missense mutation (c.1347G C) at the last base of exon 13 of TTC8 disrupted the canonical donor splice-site resulting in aberrant RNA splicing. A cryptic donor splice-site got activated 77 bases downstream of the authentic splice donor site in intron 13, resulting in the retention of 77 bases of intron 13, and a frameshift leading to pre-mature termination codon in exon 14 at codon 486. Further, duplication of exon 15 and fusion of its duplicated copy occurred with exon 13. The binding site for SC35 protein, normally involved in splicing, also got disrupted (as predicted by SpliceAid2 software), hence, leading to alternative splicing. Our findings strongly suggest that a missense mutation c.1347G C in TTC8 disrupted the splice donor site causing retention of 77 bases of intron 13, resulting in a frameshift and subsequently introduced a pre-mature termination codon into exon 14, hence creating an altered mRNA transcript. These findings emphasize the significance of examining missense mutations especially in TTC8, to determine their pathogenic role through alternative splicing. Present findings also reiterate the notion that mutations in the TTC8/BBS8 cause phenotypic heterogeneity and does not always follow Mendelian genetics in this ciliopathy.

Major advances have recently been made in the development and application of CFTR (cystic fibrosis transmembrane conductance regulator) mutation class-specific modulator therapies, but to date, there are no approved modulators for Class I mutations, i.e., those introducing a premature termination codon (PTC) into the CFTR mRNA. Such mutations induce nonsense-mediated decay (NMD), a cellular quality control mechanism that reduces the quantity of PTC bearing mRNAs, presumably to avoid translation of potentially deleterious truncated CFTR proteins. The NMD-mediated reduction of PTC-CFTR mRNA molecules reduces the efficacy of one of the most promising approaches to treatment of such mutations, namely, PTC readthrough therapy, using molecules that induce the incorporation of near-cognate amino acids at the PTC codon, thereby enabling translation of a full-length protein. In this study, we measure the effect of three different PTC mutations on the abundance, integrity, and stability of respective CFTR mRNAs, using CFTR specific RT-qPCR-based assays. Altogether, our data suggest that optimized rescue of PTC mutations has to take into account (1) the different steady-state levels of the CFTR mRNA associated with each specific PTC mutation, (2) differences in abundance between the 3′ and 5′ regions of CFTR mRNA, even following PTC readthrough or NMD inhibition, and (3) variable effects on CFTR mRNA stability for each specific PTC mutation.

Genetic code expansion in multicellular organisms is currently limited to the use of repurposed amber stop codons. Here we introduce a system for the use of quadruplet codons to direct incorporation of non-canonical amino acids in vivo in an animal, the nematode worm Caenorhabditis elegans. We develop hybrid pyrrolysyl tRNA variants to incorporate non-canonical amino acids in response to the quadruplet codon UAGA. We demonstrate the efficiency of the quadruplet decoding system by incorporating photocaged amino acids into two proteins widely used as genetic tools. We use photocaged lysine to express photocaged Cre recombinase for the optical control of gene expression and photocaged cysteine to express photo-activatable caspase for light inducible cell ablation. Our approach will facilitate the routine adoption of quadruplet decoding for genetic code expansion in eukaryotic cells and multicellular organisms.

α1-anti-trypsin (A1AT), encoded by SERPINA1, is a neutrophil elastase inhibitor that controls the inflammatory response in the lung. Severe A1AT deficiency increases risk for Chronic Obstructive Pulmonary Disease (COPD), however, the role of A1AT in COPD in non-deficient individuals is not well known. We identify a 2.1-fold increase (p = 2.5x10-6) in the use of a distal poly-adenylation site in primary lung tissue RNA-seq in 82 COPD cases when compared to 64 controls and replicate this in an independent study of 376 COPD and 267 controls. This alternative polyadenylation event involves two sites, a proximal and distal site, 61 and 1683 nucleotides downstream of the A1AT stop codon. To characterize this event, we measured the distal ratio in human primary tissue short read RNA-seq data and corroborated our results with long read RNA-seq data. Integrating these results with 3’ end RNA-seq and nanoluciferase reporter assay experiments we show that use of the distal site yields mRNA transcripts with over 50-fold decreased translation efficiency and A1AT expression. We identified seven RNA binding proteins using enhanced CrossLinking and ImmunoPrecipitation precipitation (eCLIP) with one or more binding sites in the SERPINA1 3’ UTR. We combined these data with measurements of the distal ratio in shRNA knockdown experiments, nuclear and cytoplasmic fractionation, and chemical RNA structure probing. We identify Quaking Homolog (QKI) as a modulator of SERPINA1 mRNA translation and confirm the role of QKI in SERPINA1 translation with luciferase reporter assays. Analysis of single-cell RNA-seq showed differences in the distribution of the SERPINA1 distal ratio among hepatocytes, macrophages, αβ-Tcells and plasma cells in the liver. Alveolar Type 1,2, dendritic cells and macrophages also vary in their distal ratio in the lung. Our work reveals a complex post-transcriptional mechanism that regulates alternative polyadenylation and A1AT expression in COPD., Author summary In certain cases, the molecular mechanism underlying a human genetic disease association occurs at the level of the RNA transcript. The SERPINA1 gene is transcribed into multiple RNA isoforms that differ in their non-coding regions and influence production of the A1AT protein, which is associated with Chronic Obstructive Pulmonary Disease (COPD). Here we report a newly discovered alternative polyadenylation event as an underlying regulatory mechanism that influences A1AT protein expression. In the SERPINA1 mRNA there are two polyadenylation sites in the 3’ untranslated region (3’UTR), resulting in a short and a long transcript. We find that the longer 3’UTR strongly represses protein production, and is expressed at a higher level in individuals with COPD, in two independent studies of lung tissue. The inhibitory mechanism in the long transcript is likely caused by an interaction with the RNA binding protein Quaking, which is also differentially expressed in individuals with COPD. This work demonstrates how post-transcriptional regulation can affect disease and also identifies a potential target in the SERPINA1 3’ UTR for RNA targeted therapeutics.

BackgroundSince the first reported case of coronavirus disease 2019 (COVID-19) in China, SARS-CoV-2 has been spreading worldwide. Genomic surveillance of SARS-CoV-2 has had a critical role in tracking the emergence, introduction, and spread of new variants, which may affect transmissibility, pathogenicity, and escape from infection or vaccine-induced immunity. As anticipated, the rapid increase in COVID-19 infections in Iraq in February 2021 is due to the introduction of variants of concern during the second wave of the COVID-19 pandemic.AimTo understand the molecular epidemiology of SARS-CoV-2 during the second wave in Iraq (2021),MethodWe sequenced 76 complete SARS-CoV-2 genomes using NGS technology and identified genomic mutations and proportions of circulating variants among these. Also, we performed an in silico study to predict the effect of the truncation of NS7a protein (ORF7a) on its functionResultsWe detected nine different lineages of SARS-CoV-2. The B.1.1.7 lineage was predominant (78.9%) from February to May 2021, while only one B.1.351 strain was detected. Interestingly, the phylogenetic analysis showed that multiple strains of the B.1.1.7 lineage clustered closely with those from European countries. A high frequency (88%) of stop codon mutation (NS7a Q62stop) was detected among the B.1.1.7 lineage sequences. In silico analysis of NS7a with Q62stop found that this stop codon had no significant effect on the function of NS7a.ConclusionThis work provides molecular epidemiological insights into the spread variants of SARS-CoV-2 in Iraq, which are most likely imported from Europe.

Translation of messenger RNAs (mRNAs) with premature termination codons produces truncated proteins with potentially deleterious effects. This is prevented by nonsense-mediated mRNA decay (NMD) of these mRNAs. NMD is triggered by ribosomes terminating upstream of a splice site marked by an exon-junction complex (EJC), but also acts on many mRNAs lacking a splice junction after their termination codon. We developed a genome-wide CRISPR flow cytometry screen to identify regulators of mRNAs with premature termination codons in K562 cells. This screen recovered essentially all core NMD factors and suggested a role for EJC factors in degradation of PTCs without downstream splicing. Among the strongest hits were the translational repressors GIGYF2 and EIF4E2. GIGYF2 and EIF4E2 mediate translational repression but not mRNA decay of a subset of NMD targets and interact with NMD factors genetically and physically. Our results suggest a model wherein recognition of a stop codon as premature can lead to its translational repression through GIGYF2 and EIF4E2., Author summary A premature stop codon–a signal to abort production of a protein before the normal end has been reached—is treated differently by the cell than the corresponding normal stop codon. Many disease-causing mutations result in premature stop codons, and current research suggests that differences between stop codons could be exploited for clinical therapy. We have carried out a genetic screen to identify factors that regulate the production of protein from an messenger RNA (mRNA) with a premature stop codon. Our screen recovered previously known factors in this process, as well as unexpected roles for factors that normally mark the sites of RNA splicing, as well as a pair of proteins (GIGYF2 and EIF4E2) that mediate translational repression but not mRNA decay of mRNAs with a premature stop codon.

In Escherichia coli, elevated levels of free l-tryptophan (l-Trp) promote translational arrest of the TnaC peptide by inhibiting its termination. However, the mechanism by which translation-termination by the UGA-specific decoding release factor 2 (RF2) is inhibited at the UGA stop codon of stalled TnaC-ribosome-nascent chain complexes has so far been ambiguous. This study presents cryo-EM structures for ribosomes stalled by TnaC in the absence and presence of RF2 at average resolutions of 2.9 and 3.5 Å, respectively. Stalled TnaC assumes a distinct conformation composed of two small α-helices that act together with residues in the peptide exit tunnel (PET) to coordinate a single L-Trp molecule. In addition, while the peptidyl-transferase center (PTC) is locked in a conformation that allows RF2 to adopt its canonical position in the ribosome, it prevents the conserved and catalytically essential GGQ motif of RF2 from adopting its active conformation in the PTC. This explains how translation of the TnaC peptide effectively allows the ribosome to function as a L-Trp-specific small-molecule sensor that regulates the tnaCAB operon.

Rapidly appearing SARS-CoV-2 mutations can affect T cell epitopes, which can help the virus to evade either CD8 or CD4 T-cell responses. We developed T-cell COVID-19 Atlas (T-CoV, https://t-cov.hse.ru) – the comprehensive web portal, which allows one to analyze how SARS-CoV-2 mutations alter the presentation of viral peptides by HLA molecules. The data are presented for common virus variants and the most frequent HLA class I and class II alleles. Binding affinities of HLA molecules and viral peptides were assessed with accurate in silico methods. The obtained results highlight the importance of taking HLA alleles diversity into account: mutation-mediated alterations in HLA-peptide interactions were highly dependent on HLA alleles. For example, we found that the essential number of peptides tightly bound to HLA-B*07:02 in the reference Wuhan variant ceased to be tight binders for the Indian (Delta) and the UK (Alpha) variants. In summary, we believe that T-CoV will help researchers and clinicians to predict the susceptibility of individuals with different HLA genotypes to infection with variants of SARS-CoV-2 and/or forecast its severity.

The genetic code of mammalian cells can be expanded to allow the incorporation of non-canonical amino acids (ncAAs) by suppressing in-frame amber stop codons (UAG) with an orthogonal pyrrolysyl-tRNA synthetase (PylRS)/tRNAPylCUA (PylT) pair. However, the feasibility of this approach is substantially hampered by unpredictable variations in incorporation efficiencies at different stop codon positions within target proteins. Here, we apply a proteomics-based approach to quantify ncAA incorporation rates at hundreds of endogenous amber stop codons in mammalian cells. With these data, we compute iPASS (Identification of Permissive Amber Sites for Suppression; available at www.bultmannlab.eu/tools/iPASS), a linear regression model to predict relative ncAA incorporation efficiencies depending on the surrounding sequence context. To verify iPASS, we develop a dual-fluorescence reporter for high-throughput flow-cytometry analysis that reproducibly yields context-specific ncAA incorporation efficiencies. We show that nucleotides up- and downstream of UAG synergistically influence ncAA incorporation efficiency independent of cell line and ncAA identity. Additionally, we demonstrate iPASS-guided optimization of ncAA incorporation rates by synonymous exchange of codons flanking the amber stop codon. This combination of in silico analysis followed by validation in living mammalian cells substantially simplifies identification as well as adaptation of sites within a target protein to confer high ncAA incorporation rates, Nucleic Acids Research, 49 (11), ISSN:1362-4962, ISSN:0301-5610

Ribosome was long considered as a critical yet passive player in protein synthesis. Only recently the role of its basic components, ribosomal RNAs and proteins, in translational control has begun to emerge. Here we examined function of the small ribosomal protein uS3/Rps3, earlier shown to interact with eukaryotic translation initiation factor eIF3, in termination. We identified two residues in consecutive helices occurring in the mRNA entry pore, whose mutations to the opposite charge either reduced (K108E) or increased (R116D) stop codon readthrough. Whereas the latter increased overall levels of eIF3-containing terminating ribosomes in heavy polysomes in vivo indicating slower termination rates, the former specifically reduced eIF3 amounts in termination complexes. Combining these two mutations with the readthrough-reducing mutations at the extreme C-terminus of the a/Tif32 subunit of eIF3 either suppressed (R116D) or exacerbated (K108E) the readthrough phenotypes, and partially corrected or exacerbated the defects in the composition of termination complexes. In addition, we found that K108 affects efficiency of termination in the termination context-specific manner by promoting incorporation of readthrough-inducing tRNAs. Together with the multiple binding sites that we identified between these two proteins, we suggest that Rps3 and eIF3 closely co-operate to control translation termination and stop codon readthrough.

The importance of the local sequence context in determining how efficiently aminoglycosides rescue nonsense mutations has been established previously in disease models. Different stop codons appear to facilitate the termination process with differing efficiencies. Furthermore, the efficiency with which termination is suppressed may also be influenced by the local sequence context surrounding the stop codon. The strongest bias has usually been identified with the nucleotide base that immediately follows the stop codon in the majority of experiments. However, how the sequence context influences the efficiency of aminoglycosides in rescuing the human ether-a-go-go-related (HERG) protein in mammalian cells remains to be fully elucidated. Therefore, the present study was devised to examine the susceptibility of different termination codons on the HERG gene and the +4 nucleotide immediately following them to be suppressed by aminoglyco-sides in 293 cells. The 293 cells were transiently transfected with the wild-type or mutant genes. The read-through effect was subsequently examined by adding aminoglycoside G418 into the culture medium, followed by incubation of the cells for 24 h. An immunofluorescence method was then used to observe the protein expression of HERG prior to and following drug treatment. Patch clamping was performed to evaluate the function of the HERG protein. These experiments revealed that stop codons TGA and TAA in the R1014X mutant were more susceptible to treatment with the drug G418. Similar results were observed with the W927X-TGA and W927X-TAA mutants. Subsequently, R1014X-TGAC, R1014X-TGAG and R1014X-TGAA mutants were constructed based on the R1014X-TGAT mutant. The level of red fluorescence was observed prior to and following the administration of G418 using antibodies targeting the N- or C-terminus of the HERG protein. However, the tail current density was found only to increase with the R1014X-TGAT mutant following G418 treatment. Taken together, the results of the present study suggest that the type of premature stop codon and the context of the nucleotide immediately following at the +4 position, may determine the pharmacological rescue efficiency of the HERG gene.

During canonical translation, the ribosome moves along an mRNA from the start to the stop codon in exact steps of one codon at a time. The collinearity of the mRNA and the protein sequence is essential for the quality of the cellular proteome. Spontaneous errors in decoding or translocation are rare and result in a deficient protein. However, dedicated recoding signals in the mRNA can reprogram the ribosome to read the message in alternative ways. This review summarizes the recent advances in understanding the mechanisms of three types of recoding events: stop-codon readthrough, –1 ribosome frameshifting and translational bypassing. Recoding events provide insights into alternative modes of ribosome dynamics that are potentially applicable to other non-canonical modes of prokaryotic and eukaryotic translation.

Selenoproteins are a unique class of proteins that contain the 21st amino acid, selenocysteine (Sec). Addition of Sec into a protein is achieved by recoding of the UGA stop codon. All 25 mammalian selenoprotein mRNAs possess a 3′ UTR stem-loop structure, the Selenocysteine Insertion Sequence (SECIS), which is required for Sec incorporation. It is widely believed that the SECIS is the major RNA element that controls Sec insertion, however recent findings in our lab suggest otherwise for Selenoprotein S (SelS). Here we report that the first 91 nucleotides of the SelS 3′ UTR contain a proximal stem loop (PSL) and a conserved sequence we have named the SelS Positive UGA Recoding (SPUR) element. We developed a SelS-V5/UGA surrogate assay for UGA recoding, which was validated by mass spectrometry to be an accurate measure of Sec incorporation in cells. Using this assay, we show that point mutations in the SPUR element greatly reduce recoding in the reporter; thus, the SPUR is required for readthrough of the UGA-Sec codon. In contrast, deletion of the PSL increased Sec incorporation. This effect was reversed when the PSL was replaced with other stem-loops or an unstructured sequence, suggesting that the PSL does not play an active role in Sec insertion. Additional studies revealed that the position of the SPUR relative to the UGA-Sec codon is important for optimal UGA recoding. Our identification of the SPUR element in the SelS 3′ UTR reveals a more complex regulation of Sec incorporation than previously realized.

Codon usage bias is a universal feature of eukaryotic and prokaryotic genomes and plays an important role in regulating gene expression levels. A major role of codon usage is thought to regulate protein expression levels by affecting mRNA translation efficiency, but the underlying mechanism is unclear. By analyzing ribosome profiling results, here we showed that codon usage regulates translation elongation rate and that rare codons are decoded more slowly than common codons in all codon families in Neurospora. Rare codons resulted in ribosome stalling in manners both dependent and independent of protein sequence context and caused premature translation termination. This mechanism was shown to be conserved in Drosophila cells. In both Neurospora and Drosophila cells, codon usage plays an important role in regulating mRNA translation efficiency. We found that the rare codon-dependent premature termination is mediated by the translation termination factor eRF1, which recognizes ribosomes stalled on rare sense codons. Silencing of eRF1 expression resulted in codon usage-dependent changes in protein expression. Together, these results establish a mechanism for how codon usage regulates mRNA translation efficiency.

The ambush hypothesis speculates that off-frame stop codons increase translational efficiency after ribosomal frameshifts by stopping early frameshifted translation. Some evidences fit this hypothesis: (1) synonymous codon usages increase with their potential contribution to off-frame stops; (2) the genetic code assigns frequent amino acids to codon families contributing to off-frame stops; (3) positive biases for off-frame stops (AT rich) occur despite adverse nucleotide (GC) biases; and (4) mitochondrial off-frame stop codon densities increase with ribosomal structural instability, potential proxy of frameshift frequencies. In this study, analyses of vertebrate mitogenes and tRNA synthetase genes from all superkingdoms and viruses test a new prediction of the ambush hypothesis: sequences immediately downstream of frameshift-inducing homopolymer codons (AAA, CCC, GGG, and TTT) are off-frame stop rich. Codons immediately downstream of homopolymer codons form more than average off-frame stops, biases are stronger than for corresponding upstream distances and for any other group of synonymous codons. Sequences downstream of that high-density region are off-frame stop depleted. This decrease suggests that off-frame stops, combined with suppressor tRNAs regulate translation of overlapping coding sequences. Results show the predictive power of the ambush hypothesis, from macroevolutionary (genetic code structure) to detailed gene sequence anatomy.

Single nucleotide polymorphisms in CCR6 (C-C chemokine receptor type 6) gene have been found to be the possible cause of many diseases like rheumatoid arthritis, psoriasis, lupus nephritis and systemic sclerosis and other autoimmune diseases. Therefore, identification of structurally and functionally important polymorphisms in CCR6 is important in order to study its potential malfunctioning and discovering therapeutic targets. Several bioinformatics tools were used to identify most damaging nsSNPs that might be vital for CCR6 structure and function. The in silico tools included PROVEAN, SIFT, SNP&GO and PolyPhen2 followed by I-Mutant MutPred and ConSurf. Phyre2 and I-TASSER were used for protein 3-D Modelling while gene-gene interaction was predicted by STRING and GeneMANIA. Our study suggested that three nsSNPs rs1376162684, rs751102128 and rs1185426631 are the most damaging in CCR6 gene while 7 missense SNPs rs1438637216, rs139697820, rs768420505, rs1282264186, rs1394647982, rs769360638 and rs1263402382 are found to revert into stop codons. Prediction of post-transcriptional modifications highlighted the significance of rs1376162684 because it effected potential phosphorylation site. Gene-gene interactions showed relation of CCR6 with other genes depicting its importance in several pathways and co-expressions. In future, studying diseases related to CCR6 should include investigation of these 10 nsSNPs. Being the first of its type, this study also proposes future perspectives that will help in precision medicines. For such purposes, CCR6 proteins from patients of autoimmune diseases should be explored. Animal models can also be of significance find out the effects of CCR6 in diseases.

The yeast ribosome-associated complex RAC and the Hsp70 homolog Ssb are anchored to the ribosome and together act as chaperones for the folding and co-translational assembly of nascent polypeptides. In addition, the RAC/Ssb system plays a crucial role in maintaining the fidelity of translation termination; however, the latter function is poorly understood. Here we show that the RAC/Ssb system promotes the fidelity of translation termination via two distinct mechanisms. First, via direct contacts with the ribosome and the nascent chain, RAC/Ssb facilitates the translation of stalling-prone poly-AAG/A sequences encoding for polylysine segments. Impairment of this function leads to enhanced ribosome stalling and to premature nascent polypeptide release at AAG/A codons. Second, RAC/Ssb is required for the assembly of fully functional ribosomes. When RAC/Ssb is absent, ribosome biogenesis is hampered such that core ribosomal particles are structurally altered at the decoding and peptidyl transferase centers. As a result, ribosomes assembled in the absence of RAC/Ssb bind to the aminoglycoside paromomycin with high affinity (KD = 76.6 nM) and display impaired discrimination between stop codons and sense codons. The combined data shed light on the multiple mechanisms by which the RAC/Ssb system promotes unimpeded biogenesis of newly synthesized polypeptides.

The authors report for the first time a novel mutation in the FLNC gene associated with cardiac arrhythmias in two half-siblings. The FLNC gene on chromosome 7q32 encodes filamin C, which stabilizes the actin network within the cardiomyocyte. The proband is an 8-year-old asymptomatic patient with frequent premature ventricular contractions noted on serial monitoring. Interestingly, the proband and his half-brother harbored a heterozygous 13 base pair deletion that resulted in a frameshift mutation and introduction of a premature stop codon. Notably, the proband also had a very tragic family history of sudden death in young individuals involving three generations and five family members. Because of their concerning family history and arrhythmias, both siblings underwent off-label implantable cardiac device placement for primary prevention of sudden cardiac death. Whether or not the FLNC mutation is associated with sudden cardiac death requires additional investigation and is beyond the scope of this manuscript. While previous studies have identified several mutations in the FLNC gene associated with dilated and hypertrophic cardiomyopathies, the goal of this study was to report a novel mutation in the FLNC gene that is associated with cardiac arrhythmias. The current study indicates that this mutation may help identify patients at risk for cardiac arrhythmias who would benefit from further cardiac evaluation.

The CGA arginine codon is a rare codon in Saccharomyces cerevisiae. Thus, full‐length mature protein synthesis from reporter genes with internal CGA codon repeats are markedly reduced, and the reporters, instead, produce short‐sized polypeptides via an unknown mechanism. Considering the product size and similar properties between CGA sense and UGA stop codons, we hypothesized that eukaryote polypeptide‐chain release factor complex eRF1/eRF3 catalyses polypeptide release at CGA repeats. Herein, we performed a series of analyses and report that the CGA codon can be, to a certain extent, decoded as a stop codon in yeast. This also raises an intriguing possibility that translation termination factors eRF1/eRF3 rescue ribosomes stalled at CGA codons, releasing premature polypeptides, and competing with canonical tRNAICG to the CGA codon. Our results suggest an alternative ribosomal rescue pathway in eukaryotes. The present results suggest that misdecoding of low efficient codons may play a novel role in global translation regulation in S. cerevisiae., One of the rare codons in Saccharomyces cerevisiae, CGA, was found to be misdecoded as stop‐codon to a certain extent by the eRF1/eRF3 translation termination complex. Our genetic study of eRF1, a protein tRNA mimic canonically recognizing all three stop codons, suggests a novel class of ribosome rescue pathway for the inefficient translation.

Site directed mutagenesis is a very effective approach to recode genetic information. Proper linking of the catalytic domain of the RNA editing enzyme adenosine deaminase acting on RNA (ADAR) to an antisense guide RNA can convert specific adenosines (As) to inosines (Is), with the latter recognized as guanosines (Gs) during the translation process. Efforts have been made to engineer the deaminase domain of ADAR1 and the MS2 system to target specific A residues to restore G→A mutations. The target consisted of an ochre (TAA) stop codon, generated from the TGG codon encoding amino acid 58 (Trp) of enhanced green fluorescent protein (EGFP). This system had the ability to convert the stop codon (TAA) to a readable codon (TGG), thereby restoring fluorescence in a cellular system, as shown by JuLi fluorescence and LSM confocal microscopy. The specificity of the editing was confirmed by polymerase chain reaction-restriction fragment length polymorphism, as the restored EGFP mRNA could be cleaved into fragments of 160 and 100 base pairs. Direct sequencing analysis with both sense and antisense primers showed that the restoration rate was higher for the 5′ than for the 3′A. This system may be very useful for treating genetic diseases that result from G→A point mutations. Successful artificial editing of RNA in vivo can accelerate research in this field, and pioneer genetic code restoration therapy, including stop codon read-through therapy, for various genetic diseases.

Bama minipig is a unique miniature swine bred from China. Their favorable characteristics include delicious meat, strong adaptability, tolerance to rough feed, and high levels of stress tolerance. Unfavorable characteristics are their low lean meat percentage, high fat content, slow growth rate, and low feed conversion ratio. Genome-editing technology using CRISPR/Cas9 efficiently knocked out the myostatin gene (MSTN) that has a negative regulatory effect on muscle production, effectively promoting pig muscle growth and increasing lean meat percentage of the pigs. However, CRISPR/Cas9 genome editing technology is based on random mutations implemented by DNA double-strand breaks, which may trigger genomic off-target effects and chromosomal rearrangements. The application of CRISPR/Cas9 to improve economic traits in pigs has raised biosafety concerns. Base editor (BE) developed based on CRISPR/Cas9 such as cytosine base editor (CBE) effectively achieve targeted modification of a single base without relying on DNA double-strand breaks. Hence, the method has greater safety in the genetic improvement of pigs. The aim of the present study is to utilize a modified CBE to generate MSTN-knockout cells of Bama minipigs. Our results showed that the constructed “all-in-one”-modified CBE plasmid achieved directional conversion of a single C·G base pair to a T·A base pair of the MSTN target in Bama miniature pig fibroblast cells. We successfully constructed multiple single-cell colonies of Bama minipigs fibroblast cells carrying the MSTN premature termination and verified that there were no genomic off-target effects detected. This study provides a foundation for further application of somatic cell cloning to construct MSTN-edited Bama minipigs that carry only a single-base mutation and avoids biosafety risks to a large extent, thereby providing experience and a reference for the base editing of other genetic loci in Bama minipigs.

Ribosomes are evolutionary conserved ribonucleoprotein complexes that function as two separate subunits in all kingdoms. During translation initiation, the two subunits assemble to form the mature ribosome, which is responsible for translating the messenger RNA. When the ribosome reaches a stop codon, release factors promote translation termination and peptide release, and recycling factors then dissociate the two subunits, ready for use in a new round of translation. A tethered ribosome, called Ribo-T, in which the two subunits are covalently linked to form a single entity, was recently described in Escherichia coli. A hybrid ribosomal RNA (rRNA) consisting of both the small and large subunit rRNA sequences was engineered. The ribosome with inseparable subunits generated in this way was shown to be functional and to sustain cell growth. Here, we investigated the translational properties of Ribo-T. We analyzed its behavior during amino acid misincorporation, −1 or +1 frameshifting, stop codon readthrough, and internal translation initiation. Our data indicate that covalent attachment of the two subunits modifies the properties of the ribosome, altering its ability to initiate and terminate translation correctly.

Regulation of translation via stop codon readthrough (SC-RT) expands not only tissue-specific but also viral proteomes in humans and, therefore, represents an important subject of study. Understanding this mechanism and all involved players is critical also from a point of view of prospective medical therapies of hereditary diseases caused by a premature termination codon. tRNAs were considered for a long time to be just passive players delivering amino acid residues according to the genetic code to ribosomes without any active regulatory roles. In contrast, our recent yeast work identified several endogenous tRNAs implicated in the regulation of SC-RT. Swiftly emerging studies of human tRNA-ome also advocate that tRNAs have unprecedented regulatory potential. Here, we developed a universal U6 promotor-based system expressing various human endogenous tRNA iso-decoders to study consequences of their increased dosage on SC-RT employing various reporter systems in vivo. This system combined with siRNA-mediated downregulations of selected aminoacyl-tRNA synthetases demonstrated that changing levels of human tryptophan and tyrosine tRNAs do modulate efficiency of SC-RT. Overall, our results suggest that tissue-to-tissue specific levels of selected near-cognate tRNAs may have a vital potential to fine-tune the final landscape of the human proteome, as well as that of its viral pathogens.

In bacteria, selenocysteine (Sec) is incorporated into proteins via the recoding of a particular codon, the UGA stop codon in most cases. Sec-tRNASec is delivered to the ribosome by the Sec-dedicated elongation factor SelB that also recognizes a Sec-insertion sequence element following the codon on the mRNA. Since the excess of SelB may lead to sequestration of Sec-tRNASec under selenium deficiency or oxidative stress, the expression levels of SelB and tRNASec should be regulated. In this bioinformatic study, I analyzed the Rhizobiales SelB species because they were annotated to have a non-canonical C-terminal extension. I found that the open reading frame (ORF) of diverse Alphaproteobacteria selB genes includes an entire tRNASec sequence (selC) and overlaps with the start codon of the downstream ORF. A remnant tRNASec sequence was found in the Sinorhizobium melilotiselB genes whose products have a shorter C-terminal extension. Similar overlapping traits were found in Gammaproteobacteria and Nitrospirae. I hypothesized that once the tRNASec moiety is folded and processed, the expression of the full-length SelB may be repressed. This is the first report on a nested tRNA gene inside a protein ORF in bacteria.

CRISPR-stop converts protein-coding sequences into stop codons, which, in the appropriate location, results in a null allele. CRISPR-stop induction in one-cell-stage zygotes generates Founder 0 (F0) mice that are homozygous mutants; this avoids mouse breeding and serves as a rapid screening approach for nonlethal genes. However, loss of function of 25% of mammalian genes causes early lethality. Here, we induced CRISPR-stop in one of the two blastomeres of the zygote, a method we name mosaic CRISPR-stop, to produce mosaic Atoh1 and Sox10 F0 mice; these mice not only survived longer than regular Atoh1/Sox10 knockout mice but also displayed their recognized cochlear phenotypes. Moreover, by using mosaic CRISPR-stop, we uncovered a previously unknown role of another lethal gene, Rbm24, in the survival of cochlear outer hair cells (OHCs), and we further validated the importance of Rbm24 in OHCs by using our Rbm24 conditional knockout model. Together, our results demonstrated that mosaic CRISPR-stop is reliable and rapid, and we believe this method will facilitate rapid genetic screening of developmentally lethal genes in the mouse inner ear and also in other organs.