Premature Translation Termination Codons Research Articles

Novel compound heterozygous variants in EMC1 associated with global developmental delay: a lesson from a non-silent synonymous exonic mutation.

The endoplasmic reticulum-membrane protein complex (EMC) as a molecular chaperone is required for the proper synthesis, folding and traffic of several transmembrane proteins. Variants in the subunit 1 of EMC (EMC1) have been implicated in neurodevelopmental disorders. Whole exome sequencing (WES) with Sanger sequencing validation was performed for a Chinese family, including the proband (a 4-year-old girl who displayed global developmental delay, severe hypotonia and visual impairment), her affected younger sister and her non-consanguineous parents. RT-PCR assay and Sanger sequencing were used to detect abnormal RNA splicing. Novel compound heterozygous variants in EMC1, including the maternally inherited chr1: 19566812_1956800delinsATTCTACTT[hg19];NM_015047.3:c.765_777delins ATTCTACTT;p.(Leu256fsTer10) and the paternally inherited chr1:19549890G> A[hg19];NM_015047.3:c.2376G>A;p.(Val792=) are identified in the proband and her affected sister. RT-PCR assay followed by Sanger sequencing reveals that the c.2376G>A variant leads to aberrant splicing, with retention of intron 19 (561bp) in the mature mRNA, which is presumed to introduce a premature translational termination codon (p.(Val792fsTer31)). Novel compound heterozygous variants in EMC1 have been identified in individuals with global developmental delay. Non-silent synonymous mutations should be kept in mind in genetic analysis.

Nonsense-Mediated mRNA Decay as a Mediator of Tumorigenesis.

Nonsense-mediated mRNA decay (NMD) is an evolutionarily conserved and well-characterized biological mechanism that ensures the fidelity and regulation of gene expression. Initially, NMD was described as a cellular surveillance or quality control process to promote selective recognition and rapid degradation of erroneous transcripts harboring a premature translation-termination codon (PTC). As estimated, one-third of mutated and disease-causing mRNAs were reported to be targeted and degraded by NMD, suggesting the significance of this intricate mechanism in maintaining cellular integrity. It was later revealed that NMD also elicits down-regulation of many endogenous mRNAs without mutations (~10% of the human transcriptome). Therefore, NMD modulates gene expression to evade the generation of aberrant truncated proteins with detrimental functions, compromised activities, or dominant-negative effects, as well as by controlling the abundance of endogenous mRNAs. By regulating gene expression, NMD promotes diverse biological functions during development and differentiation, and facilitates cellular responses to adaptation, physiological changes, stresses, environmental insults, etc. Mutations or alterations (such as abnormal expression, degradation, post-translational modification, etc.) that impair the function or expression of proteins associated with the NMD pathway can be deleterious to cells and may cause pathological consequences, as implicated in developmental and intellectual disabilities, genetic defects, and cancer. Growing evidence in past decades has highlighted NMD as a critical driver of tumorigenesis. Advances in sequencing technologies provided the opportunity to identify many NMD substrate mRNAs in tumor samples compared to matched normal tissues. Interestingly, many of these changes are tumor-specific and are often fine-tuned in a tumor-specific manner, suggesting the complex regulation of NMD in cancer. Tumor cells differentially exploit NMD for survival benefits. Some tumors promote NMD to degrade a subset of mRNAs, such as those encoding tumor suppressors, stress response proteins, signaling proteins, RNA binding proteins, splicing factors, and immunogenic neoantigens. In contrast, some tumors suppress NMD to facilitate the expression of oncoproteins or other proteins beneficial for tumor growth and progression. In this review, we discuss how NMD is regulated as a critical mediator of oncogenesis to promote the development and progression of tumor cells. Understanding how NMD affects tumorigenesis differentially will pave the way for the development of more effective and less toxic, targeted therapeutic opportunities in the era of personalized medicine.

Fluorescent Tagging of Endogenous Beta-Globin Gene in S-HUDEP2 Cell Model for Live Cell Monitoring of Beta-Globin Expression and Regulation

Introduction: The translation of β-globin (HBB) gene is crucial for adult hemoglobin (HbA) formation, and naturally occurring mutations in HBB cause disorders of erythropoiesis. Therefore, understanding the mechanisms governing HBB regulation and expression has been an active area of research. We aim to develop a cell model to monitor HBB expression in live cells as a tool to gain better understanding of HBB expression, regulation and for the discovery of novel therapeutic targets. Methods: Sickle Human Umbilical Cord Derived Erythroid Progenitor cells (S-HUDEP2) are immortalized CD34+ hematopoietic stem and progenitor cells with homozygous sickle mutation that exclusively produce sickle hemoglobin upon erythroid differentiation. To generate the cell model in S-HUDEP2, we delivered HiFi SpCas9/gRNA targeting the C-terminus of HBB together with a double-stranded DNA donor template encoding self-cleaving peptide, eGFP, and β-globin polyA signal flanking homology arms for simultaneous expression of β-globin and eGFP under the HBB promoter. We created this clonal cell model and confirmed biallelic precise in-frame targeted integration by PCR, Sanger sequencing and digital droplet PCR copy number assay to ensure simultaneous monitoring of both HBB alleles. Results: To validate this model, we delivered HiFi SpCas9/R-66S sgRNA as ribonucleoprotein (RNP) targeting HBB currently in pre-clinical studies for sickle cell anemia (SCA). With RNP delivery, CRISPR/Cas9 double-strand breaks (DSBs) will lead to insertions and deletions (indels); we hypothesized that varying indel frequencies and patterns will result in change in GFP expression. After RNP delivery, we conducted 7 days of in vitro cellular expansion to allow for completion of editing and stabilization of GFP signal followed by 5 days of in vitro cellular differentiation. We identified 3 distinct populations based on GFP expression: GFP-, GFPlow, and GFPhigh. These cells were bulk sorted, genomic DNA was extracted and we analyzed indel patterns for each population through targeted amplicon Next Generation Sequencing (NGS). Each population had a unique indel pattern: confirming that GFP expression is based on genotype. In addition, an increase in GFP mean fluorescent intensity (MFI) was seen after in vitro differentiation with preservation of these distinct groups; indicative of expected increase in HBB transcription. Based on these observations, through use of ddPCR copy number assay, we determined that the creation of CRISPR/Cas9 mediated large deletion (>200bp) is the underlying mechanism leading to GFP loss seen in GFP- andGFPlow populations. Nonsense mutations occurring early in exon 1 of HBB resulting in premature translation-termination codons are resistant to nonsense mediated mRNA decay (NMD), a well-known cellular surveillance mechanism. We hypothesized that RNP induced small frameshift indels in exon 1 allow for NMD escape thus preserving HBB translation and GFP expression while large deletions do not, leading to GFP loss. To further validate this model and define its potential for broad applicability, we confirmed its ability to undergo normal differentiation based on erythroid specific cell markers when compared to untagged S-HUDEP2. We also confirmed our model's capacity to form Hb tetramer with α-chains using Hb native PAGE which allows for identification of intact Hb tetramers based on reference migration. Conclusion: We have successfully established and validated a clonal cell model with GFP tagged β-globin, which can be used to monitor HBB expression in live cells via GFP expression, eliminating the need for cellular fixation and HbA antibody staining. Our model may provide a useful tool for a broad range of applications, including the examination of cellular mechanisms underlying HBB expression and the screening of potential therapeutic targets for hemoglobinopathies and other disorders of erythropoiesis.

CRISPR/Cas9-mediated targeted mutagenesis of GmTCP19L increasing susceptibility to Phytophthora sojae in soybean.

The TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) transcription factors is one of the superfamilies of plant-specific transcription factors involved in plant growth, development, and biotic and abiotic stress. However, there is no report on the research of the TCP transcription factors in soybean response to Phytophthora sojae. In this study, Agrobacterium-mediated transformation was used to introduce the CRISPR/Cas9 expression vector into soybean cultivar "Williams 82" and generated targeted mutants of GmTCP19L gene, which was previously related to involve in soybean responses to P. sojae. We obtained the tcp19l mutants with 2-bp deletion at GmTCP19L coding region, and the frameshift mutations produced premature translation termination codons and truncated GmTCP19L proteins, increasing susceptibility to P. sojae in the T2-generation. These results suggest that GmTCP19L encodes a TCP transcription factor that affects plant defense in soybean. The new soybean germplasm with homozygous tcp19l mutations but the BAR and Cas9 sequences were undetectable using strip and PCR methods, respectively, suggesting directions for the breeding or genetic engineering of disease-resistant soybean plants.

CRISRP/Cas9-Mediated Targeted Mutagenesis of Tomato Polygalacturonase Gene (SlPG) Delays Fruit Softening.

Polygalacturonase (PG) gene has been documented as a key candidate for the improvement of fruit firmness, which is a target trait for tomato production because it facilitates transportation and storage. To reduce the expression of the PG gene, most of the elite commercial tomato varieties were obtained by RNA interference technology. However, this approach of producing commercialized tomatoes by integration of the exogenous gene is controversial. In this work, CRISPR/Cas9 technology was used to induce the targeted mutagenesis of the SlPG gene to delay the softening of tomato fruit. Results showed that the SlPG gene was frameshift mutated by 4 bp deletion, 10 bp deletion, and 1 bp insertion, which generated premature translation termination codons. Compared with wild-type (WT), homozygous T1-generation tomato plants exhibited late fruit softening under natural conditions. Consistent with this phenomenon, the firmness value of WT fruit was lower in slpg mutant fruit, and the physiological loss of water was higher. Collectively, these data demonstrate that the mutation of the SlPG gene delays tomato fruit softening. More importantly, 8 out of 20 transgene-free tomato plants, which were homozygous for null alleles of SlPG, were separated in the T3-generation of line slpgT2-#2. This transgene-free slpg may provide materials for more in-depth research of SlPG functions and the molecular mechanism of fruit softening in tomatoes.

Nonsense-Mediated RNA Decay Is a Unique Vulnerability of Cancer Cells Harboring SF3B1 or U2AF1 Mutations.

Nonsense-mediated RNA decay (NMD) is recognized as an RNA surveillance pathway that targets aberrant mRNAs with premature translation termination codons (PTC) for degradation, however, its molecular mechanisms and roles in health and disease remain incompletely understood. In this study, we developed a novel reporter system to accurately measure NMD activity in individual cells. A genome-wide CRISPR-Cas9 knockout screen using this reporter system identified novel NMD-promoting factors, including multiple components of the SF3B complex and other U2 spliceosome factors. Interestingly, cells with mutations in the spliceosome genes SF3B1 and U2AF1, which are commonly found in myelodysplastic syndrome (MDS) and cancers, have overall attenuated NMD activity. Compared with wild-type (WT) cells, SF3B1- and U2AF1-mutant cells were more sensitive to NMD inhibition, a phenotype that is accompanied by elevated DNA replication obstruction, DNA damage, and chromosomal instability. Remarkably, the sensitivity of spliceosome mutant cells to NMD inhibition was rescued by overexpression of RNase H1, which removes R-loops in the genome. Together, these findings shed new light on the functional interplay between NMD and RNA splicing and suggest a novel synthetic lethal strategy for the treatment of MDS and cancers with spliceosome mutations. SIGNIFICANCE: This study has developed a novel NMD reporter system and identified a potential therapeutic approach of targeting the NMD pathway to treat cancer with spliceosome gene mutations.

Nonsense suppression therapies in human genetic diseases.

About 11% of all human disease-associated gene lesions are nonsense mutations, resulting in the introduction of an in-frame premature translation-termination codon (PTC) into the protein-coding gene sequence. When translated, PTC-containing mRNAs originate truncated and often dysfunctional proteins that might be non-functional or have gain-of-function or dominant-negative effects. Therapeutic strategies aimed at suppressing PTCs to restore deficient protein function-the so-called nonsense suppression (or PTC readthrough) therapies-have the potential to provide a therapeutic benefit for many patients and in a broad range of genetic disorders, including cancer. These therapeutic approaches comprise the use of translational readthrough-inducing compounds that make the translational machinery recode an in-frame PTC into a sense codon. However, most of the mRNAs carrying a PTC can be rapidly degraded by the surveillance mechanism of nonsense-mediated decay (NMD), thus decreasing the levels of PTC-containing mRNAs in the cell and their availability for PTC readthrough. Accordingly, the use of NMD inhibitors, or readthrough-compound potentiators, may enhance the efficiency of PTC suppression. Here, we review the mechanisms of PTC readthrough and their regulation, as well as the recent advances in the development of novel approaches for PTC suppression, and their role in personalized medicine.

Perspective in Alternative Splicing Coupled to Nonsense-Mediated mRNA Decay.

Alternative splicing (AS) of precursor mRNA (pre-mRNA) is a cellular post-transcriptional process that generates protein isoform diversity. Nonsense-mediated RNA decay (NMD) is an mRNA surveillance pathway that recognizes and selectively degrades transcripts containing premature translation-termination codons (PTCs), thereby preventing the production of truncated proteins. Nevertheless, NMD also fine-tunes the gene expression of physiological mRNAs encoding full-length proteins. Interestingly, around one third of all AS events results in PTC-containing transcripts that undergo NMD. Numerous studies have reported a coordinated action between AS and NMD, in order to regulate the expression of several genes, especially those coding for RNA-binding proteins (RBPs). This coupling of AS to NMD (AS-NMD) is considered a gene expression tool that controls the ratio of productive to unproductive mRNA isoforms, ultimately degrading PTC-containing non-functional mRNAs. In this review, we focus on the mechanisms underlying AS-NMD, and how this regulatory process is able to control the homeostatic expression of numerous RBPs, including splicing factors, through auto- and cross-regulatory feedback loops. Furthermore, we discuss the importance of AS-NMD in the regulation of biological processes, such as cell differentiation. Finally, we analyze interesting recent data on the relevance of AS-NMD to human health, covering its potential roles in cancer and other disorders.

A nonsense variant in FBN1 caused autosomal dominant Marfan syndrome in a Chinese family: a case report

BackgroundMarfan syndrome (MFS) is a common autosomal dominant inherited disease, and the occurrence rate is around 0.1–0.2‰. The causative variant of FNB1 gene accounts for approximately 70–80% of all MFS cases. In this study, we found a heterozygous c.3217G > T (p.Glu1073*) nonsense variant in the FBN1 gene. This finding extended the variant spectrum of the FBN1 gene and will provide a solution for patients to bear healthy offspring by preimplantation genetic testing or prenatal diagnosis.Case presentationThe patient was treated due to tachycardia during excitement in a hospital. Echocardiography showed dilatation of the ascending aorta and main pulmonary artery, mitral regurgitation (mild), tricuspid regurgitation (mild), and abnormal left ventricular filling. Electrocardiograph showed sinus rhythm. In addition, flutters of shadows in front of his eyes and vitreous opacity were present in the patient. Genomic DNA was extracted from peripheral blood samples from members of the family and 100 unrelated controls. Potential variants were screened out by next-generation sequencing and confirmed by MLPA & Sanger sequencing. Real-time fluorescence quantitative PCR (RT-qPCR) was performed to detect the relative mRNA quantitation in the patient. A heterozygous nonsense variant c.3217G > T of the FBN1 gene, which resulted in p. Glu1073Term, was identified in both patients. Only wild type bases were found in the cDNA sequence of the patient. Real-time fluorogenic quantitative PCR results showed that the relative expression level of FBN1 cDNA in the patient was only about 21% compared to that of normal individuals. This variant c.3217G > T of the FBN1 gene introduces a Stop codon in the cb-EGF12 domain. We speculated that a premature translational-termination codon (PTC) was located in the mRNA and the target mRNA was disintegrated through a process known as nonsense-mediated mRNA decay (NMD), which led to a significant decrease of the fibrillin-1 protein, eventually causing clinical symptoms in the patient.ConclusionsIn this study, we found a heterozygous c.3217G > T (p.Glu1073*) nonsense variant in the FBN1 gene, which eventually led to Marfan syndrome in a Chinese family.

Inhibition of post-termination ribosome recycling at premature termination codons in UPF1 ATPase mutants.

Recognition and rapid degradation of mRNA harboring premature translation termination codons (PTCs) serves to protect cells from accumulating non-functional and potentially toxic truncated polypeptides. Targeting of PTC-containing transcripts is mediated by the nonsense-mediated mRNA decay (NMD) pathway and requires a conserved set of proteins including UPF1, an RNA helicase whose ATPase activity is essential for NMD. Previously, we identified a functional interaction between the NMD machinery and terminating ribosomes based on 3' RNA decay fragments that accrue in UPF1 ATPase mutants. Herein, we show that those decay intermediates originate downstream of the PTC and harbor 80S ribosomes that migrate into the mRNA 3' UTR independent of canonical translation. Accumulation of 3' RNA decay fragments is determined by both RNA sequence downstream of the PTC and the inactivating mutation within the active site of UPF1. Our data reveal a failure in post-termination ribosome recycling in UPF1 ATPase mutants.

Creation of Early Flowering Germplasm of Soybean by CRISPR/Cas9 Technology.

Soybean is an important economic crop and a typical short-day crop, sensitive to photoperiod, and has narrow geographical adaptative region, which limit the creation of transgenic materials and reduce the breeding efficiency of new varieties. In addition, the genetic transformation efficiency of soybean is lower than that of many other crops, and the available receptor genotypes are limited. In this study, Agrobacterium-mediated transformation were used to introduce the CRISPR/Cas9 expression vector into soybean cultivar Jack and generated targeted mutants of E1 gene controlling soybean flowering. We obtained two novel types of mutations, 11 bp and 40 bp deletion at E1 coding region, respectively, and frameshift mutations produced premature translation termination codons and truncated E1 proteins, causing obvious early flowering under long day condition. In addition, no off-target effects were observed by predicting and analyzing the potential off-target sites of E1 targets. Significant decreased E1 gene expression of two novel mutants showed that the truncated E1 protein disinhibited GmFT2a/5a and increasing GmFT2a/5a gene expressions resulted obvious early flowering. Homozygous trans-clean mutants without T-DNA elements were also obtained and showed early flowering under long day condition. The photo-insensitive soybean transformation receptor we created laid a foundation for breeding excellent transgenic receptors suitable for high latitudes.

Comprehensive Assessment of BARD1 Messenger Ribonucleic Acid Splicing With Implications for Variant Classification.

Introduction: Case–control analyses have shown BARD1 variants to be associated with up to >2-fold increase in risk of breast cancer, and potentially greater risk of triple negative breast cancer. BARD1 is included in several gene sequencing panels currently marketed for the prediction of risk of cancer, however there are no gene-specific guidelines for the classification of BARD1 variants. We present the most comprehensive assessment of BARD1 messenger RNA splicing, and demonstrate the application of these data for the classification of truncating and splice site variants according to American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) guidelines. Methods: Nanopore sequencing, short-read RNA-seq (whole transcriptome and targeted), and capillary electrophoresis analysis were performed by four laboratories to investigate alternative BARD1 splicing in blood, breast, and fimbriae/ovary related specimens from non-cancer affected tissues. Splicing data were also collated from published studies of nine different tissues. The impact of the findings for PVS1 annotation was assessed for truncating and splice site variants. Results: We identified 62 naturally occurring alternative spliced BARD1 splicing events, including 19 novel events found by next generation sequencing and/or reverse transcription PCR analysis performed for this study. Quantitative analysis showed that naturally occurring splicing events causing loss of clinically relevant domains or nonsense mediated decay can constitute up to 11.9% of overlapping natural junctions, suggesting that aberrant splicing can be tolerated up to this level. Nanopore sequencing of whole BARD1 transcripts characterized 16 alternative isoforms from healthy controls, revealing that the most complex transcripts combined only two alternative splicing events. Bioinformatic analysis of ClinVar submitted variants at or near BARD1 splice sites suggest that all consensus splice site variants in BARD1 should be considered likely pathogenic, with the possible exception of variants at the donor site of exon 5. Conclusions: No BARD1 candidate rescue transcripts were identified in this study, indicating that all premature translation-termination codons variants can be annotated as PVS1. Furthermore, our analysis suggests that all donor and acceptor (IVS+/−1,2) variants can be considered PVS1 or PVS1_strong, with the exception of variants targeting the exon 5 donor site, that we recommend considering as PVS1_moderate.

RNA Helicases from the DEA(D/H)-Box Family Contribute to Plant NMD Efficiency.

Nonsense-mediated mRNA decay (NMD) is a conserved eukaryotic RNA surveillance mechanism that degrades aberrant mRNAs comprising a premature translation termination codon. The adenosine triphosphate (ATP)-dependent RNA helicase up-frameshift 1 (UPF1) is a major NMD factor in all studied organisms; however, the complexity of this mechanism has not been fully characterized in plants. To identify plant NMD factors, we analyzed UPF1-interacting proteins using tandem affinity purification coupled to mass spectrometry. Canonical members of the NMD pathway were found along with numerous NMD candidate factors, including conserved DEA(D/H)-box RNA helicase homologs of human DDX3, DDX5 and DDX6, translation initiation factors, ribosomal proteins and transport factors. Our functional studies revealed that depletion of DDX3 helicases enhances the accumulation of NMD target reporter mRNAs but does not result in increased protein levels. In contrast, silencing of DDX6 group leads to decreased accumulation of the NMD substrate. The inhibitory effect of DDX6-like helicases on NMD was confirmed by transient overexpression of RH12 helicase. These results indicate that DDX3 and DDX6 helicases in plants have a direct and opposing contribution to NMD and act as functional NMD factors.

A role for DIS3L2 over natural nonsense-mediated mRNA decay targets in human cells

The nonsense-mediated decay (NMD) pathway selectively degrades mRNAs carrying a premature translation-termination codon but also regulates the abundance of a large number of physiological mRNAs that encode full-length proteins. In human cells, NMD-targeted mRNAs are degraded by endonucleolytic cleavage and exonucleolytic degradation from both 5-’ and 3′-ends. This is done by a process not yet completely understood that recruits decapping and 5′-to-3′ exonuclease activities, as well as deadenylating and 3′-to-5′ exonuclease exosome activities. In yeast, DIS3/Rrp44 protein is the catalytic subunit of the exosome, but in humans, there are three known paralogues of this enzyme: DIS3, DIS3L1, and DIS3L2. However, little is known about their role in NMD. Here, we show that some NMD-targets are DIS3L2 substrates in human cells. In addition, we observed that DIS3L2 acts over full-length transcripts, through a process that also involves UPF1. Moreover, DIS3L2-mediated decay is dependent on the activity of the terminal uridylyl transferases Zcchc6/11 (TUT7/4). Together, our findings establish a role for DIS3L2 and uridylation in NMD.

Environmental stresses suppress nonsense-mediated mRNA decay (NMD) and affect cells by stabilizing NMD-targeted gene expression

Nonsense-mediated mRNA decay (NMD) is a cellular mechanism that eliminates mRNAs that harbor premature translation termination codons (PTCs). Here, we investigated the effects of environmental stresses (oxidative stress and endoplasmic reticulum (ER) stress) on NMD activity. Methylmercury (MeHg) was used to cause oxidative stress and thapsigargin to stress the ER. NMD suppression, evidenced by upregulation of NMD-sensitive mRNAs and a decrease in UPF1 phosphorylation, was observed in MeHg-treated myogenic cells, cerebral cortical neuronal cells, and astroglial cells. Mild ER stress amplified NMD suppression caused by MeHg. To elucidate the cause of stress-induced NMD suppression, the role of the phospho-eIF2α/ATF4 pathway was investigated. Knockdown and non-phosphorylatable eIF2α-transfection studies demonstrated the critical role of phospho-eIF2α-mediated repression of translation in mild ER stress-induced NMD suppression. However, NMD suppression was also observed in phospho-eIF2α-deficient cells under mild ER stress. Mechanistic target of rapamycin suppression-induced inhibition of cap-dependent translation, and downregulation of the NMD components UPF1, SMG7, and eIF4A3, were probably involved in stress-induced NMD suppression. Our results indicate that stress-induced NMD suppression has the potential to affect the condition of cells and phenotypes of PTC-related diseases under environmental stresses by stabilizing NMD-targeted gene expression.

The nonsense-mediated mRNA decay (NMD) pathway differentially regulates COX17, COX19 and COX23 mRNAs

The differential regulation of COX17, COX19 and COX23 mRNAs by the nonsense-mediated mRNA decay (NMD) pathway was investigated. The NMD pathway regulates mRNAs that aberrantly terminate translation. This includes mRNAs harboring premature translation termination codons and natural mRNAs. Most natural mRNAs regulated by NMD encode fully functional proteins involved in various cellular processes. However, the cause and targeting of most of these mRNAs by the pathway is not understood. Analysis of a set of mRNAs involved in copper homeostasis showed that a subset of these mRNAs function in mitochondrial copper homeostasis. Here, we examined the regulation of COX17, COX19 and COX23 mRNAs by NMD. These mRNAs encode homologous mitochondrial proteins involved in metallation of cytochrome c oxidase. We found that COX17, COX19 and COX23 mRNAs are differentially regulated by NMD depending on environmental copper levels. A long 3′-UTR contributes to the direct regulation of COX19 mRNA by the pathway. Alternatively, COX23 mRNA contains a long 3′-UTR, but is indirectly regulated by the pathway under two conditions tested here. Analysis of the functionality of the NMD targeting features in COX23 mRNA showed that the COX23 3′-UTR is sufficient to trigger NMD. The regulation of mRNAs involved in mitochondrial copper metabolism by NMD is physiologically significant because excess copper enhances growth of NMD mutants on a non-fermentable carbon source. These findings suggest that regulation of mRNAs encoding homologous proteins by NMD can be differential depending on environmental copper levels. Furthermore, these findings suggest copper ion homeostatic mechanisms in the mitochondria occur at the mRNA level via the NMD pathway.

Compound C inhibits nonsense-mediated RNA decay independently of AMPK.

The nonsense mediated RNA decay (NMD) pathway safeguards the integrity of the transcriptome by targeting mRNAs with premature translation termination codons (PTCs) for degradation. It also regulates gene expression by degrading a large number of non-mutant RNAs (including mRNAs and noncoding RNAs) that bear NMD-inducing features. Consequently, NMD has been shown to influence development, cellular response to stress, and clinical outcome of many genetic diseases. Small molecules that can modulate NMD activity provide critical tools for understanding the mechanism and physiological functions of NMD, and they also offer potential means for treating certain genetic diseases and cancer. Therefore, there is an intense interest in identifying small-molecule NMD inhibitors or enhancers. It was previously reported that both inhibition of NMD and treatment with the AMPK-selective inhibitor Compound C (CC) induce autophagy in human cells, raising the possibility that CC may be capable of inhibiting NMD. Here we show that CC indeed has a NMD-inhibitory activity. Inhibition of NMD by CC is, however, independent of AMPK activity. As a competitive ATP analog, CC does not affect the kinase activity of SMG1, an essential NMD factor and the only known kinase in the NMD pathway. However, CC treatment down-regulates the protein levels of several NMD factors. The induction of autophagy by CC treatment is independent of ATF4, a NMD target that has been shown to promote autophagy in response to NMD inhibition. Our results reveal a new activity of CC as a NMD inhibitor, which has implications for its use in basic research and drug development.

Molecular cloning of novel transcripts of the adaptor-related protein complex 2 alpha 1 subunit (AP2A1) gene, using Next-Generation Sequencing

The adaptor-related protein (AP) complexes play important roles in cargo selection and vesicle formation, and hence in intracellular membrane trafficking. Five different AP complexes are currently known, each consisting of four subunits, known as adaptins. AP-2, the most thoroughly characterized of the five AP complexes, facilitates clathrin-mediated endocytosis. In this study, we describe the discovery and molecular cloning of seventy-seven novel alternatively spliced transcripts of the human AP2A1 gene, which encodes the αA adaptin of the AP-2 complex. For this purpose, we have used Next-Generation Sequencing (NGS), a powerful tool for studying alternative splicing. In brief, we subcultured fifty-five established human cell lines, originating from several distinct cancerous and normal tissues, extracted total RNA, and synthesized first-strand cDNA. Next, we used nested touchdown PCR to amplify the whole coding region of the AP2A1 transcripts of each cell line, mixed all PCR products, and proceeded to NGS library construction, template preparation, and semiconductor sequencing. Extensive bioinformatic analysis revealed thirteen novel splice junctions of previously annotated exons, as also verified via nested PCR with primers targeting these splice junctions. Moreover, consecutive nested PCRs led to the determination of the primary structure of seventy-seven novel AP2A1 transcripts, all of which were shown to comprise at least one premature translation termination codon, thus representing nonsense-mediated mRNA decay (NMD) candidates. NMD is a mechanism that cells use to control gene expression. Consequently, alterations in the levels of these potentially non-coding AP2A1 transcripts could lead to a decrease in the number of AP2A1 mRNA molecules, when needed. Undoubtedly, the exact role of these new APA1 splice variants merits elucidation.

Two novel COL6A3 mutations disrupt extracellular matrix formation and lead to myopathy from Ullrich congenital muscular dystrophy and Bethlem myopathy spectrum

Here we present a case report of collagen VI related myopathy in a patient, 8 y.o. boy, with intermediate phenotype between severe Ullrich congenital muscular dystrophy and milder Bethlem myopathy. Whole exome sequencing revealed two novel single nucleotide variants in COL6A3 gene: paternal p.Glu2402Ter, resulting in premature translation termination codon and degradation of mRNA from this allele probably due to nonsense-mediated decay, and maternal p.Arg1660Cys leading to amino-acid substitution in N2-terminal domain. COL6A3 expression analysis of proband's fibroblasts reveals functional homozygosity of the latter variant. Paternal fibroblasts showed only WT allele expression, which could lead to a reduction in mature transcript level, while maternal fibroblasts expressed both alleles. Functional assay of immunofluorescent staining of COL6A3 protein in fibroblasts culture reveals profound changes in COL6A3 localization and reduction of protein level in studied cultures when comparing with the controls. This study not only broadens the allelic spectrum of pathogenic COL6A3 variants in myopathy but also gives an additional support to Ullrich congenital muscular dystrophy and Bethlem myopathy clinical continuum.

Dissecting the functions of SMG5, SMG7, and PNRC2 in nonsense-mediated mRNA decay of human cells.

The term “nonsense-mediated mRNA decay” (NMD) originally described the degradation of mRNAs with premature translation–termination codons (PTCs), but its meaning has recently been extended to be a translation-dependent post-transcriptional regulator of gene expression affecting 3%–10% of all mRNAs. The degradation of NMD target mRNAs involves both exonucleolytic and endonucleolytic pathways in mammalian cells. While the latter is mediated by the endonuclease SMG6, the former pathway has been reported to require a complex of SMG5–SMG7 or SMG5–PNRC2 binding to UPF1. However, the existence, dominance, and mechanistic details of these exonucleolytic pathways are divisive. Therefore, we have investigated the possible exonucleolytic modes of mRNA decay in NMD by examining the roles of UPF1, SMG5, SMG7, and PNRC2 using a combination of functional assays and interaction mapping. Confirming previous work, we detected an interaction between SMG5 and SMG7 and also a functional need for this complex in NMD. In contrast, we found no evidence for the existence of a physical or functional interaction between SMG5 and PNRC2. Instead, we show that UPF1 interacts with PNRC2 and that it triggers 5′–3′ exonucleolytic decay of reporter transcripts in tethering assays. PNRC2 interacts mainly with decapping factors and its knockdown does not affect the RNA levels of NMD reporters. We conclude that PNRC2 is probably an important mRNA decapping factor but that it does not appear to be required for NMD.