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PubMed2006

Proteasome-mediated degradation of integral inner nuclear membrane protein emerin in fibroblasts lacking A-type lamins.

We previously identified and characterized a homozygous LMNA nonsense mutation leading to the absence of A-type lamins in a premature neonate who died at birth. We show here that the absence of A-type lamins is due to degradation of the aberrant mRNA transcript with a premature termination codon. In cultured fibroblasts from the subject with the homozygous LMNA nonsense mutation, there was a decreased steady-state expression of the integral inner nuclear membrane proteins emerin and nesprin-1alpha associated with their mislocalization to the bulk endoplasmic reticulum and a hyperphosphorylation of emerin. To determine if decreased emerin expression occurred post-translationally, we treated cells with a selective proteasome inhibitor and observed an increase in expression. Our results show that mislocalization of integral inner nuclear membrane proteins to the endoplasmic reticulum in human cells lacking A-type lamins leads to their degradation and provides the first evidence that their degradation is mediated by the proteasome.

Cells, Cultured↗

PubMed2026

A recurrent CCDC82 frameshift variant associated with syndromic neurodevelopmental disorder in a consanguineous Pakistani family.

BACKGROUND: Intellectual disabilities (IDs) are part of neurodevelopmental disorders (NDDs) and are genetically heterogeneous conditions characterized by impairments in cognition, learning, and adaptive functioning. Despite advances in gene discovery, many individuals, particularly those from understudied populations, remain without a molecular diagnosis. Recent reports implicate CCDC82 (HGNC: 26282) as an autosomal recessive ID gene, although the phenotypic spectrum and biological context remain incompletely defined. METHODS: Exome sequencing (ES) was performed in a consanguineous Pakistani family (PKMR06A) with four affected individuals presenting with moderate to severe ID. Variant segregation was confirmed by Sanger sequencing. In silico analyses, including pathogenicity prediction, protein structural modeling, and domain intolerance assessment, were used to evaluate the functional consequences of the identified variant. Spatiotemporal gene expression patterns were examined using bulk and single-cell human brain transcriptomic datasets. RESULTS: Clinically, affected individuals of family PKMR06A presented with early childhood global developmental delay, speech delay, hypotonia, gait abnormalities, spasticity, and mild facial dysmorphism. Genetic screening revealed a recurrent rare homozygous frameshift variant in CCDC82 (NM_024725.4): c.373del; p.(Asp125Ilefs*6), segregating with disease in all available affected individuals of the family. The identified c.373del variant was absent from the gnomAD database and was classified as pathogenic (PVS1, PM2, and PP1) based on ACMG/AMP criteria. The c.373del variant is predicted to introduce a premature termination codon, p.(Asp125Ilefs*6), leading to deletion of essential coiled-coil domains from the encoded protein, supporting a loss-of-function mechanism. In silico, transcriptomic analyses demonstrated preferential CCDC82 expression during prenatal human brain development, providing developmental context for the neurodevelopmental phenotype associated with the identified truncating variant. CONCLUSIONS: This study expands the mutational landscape of CCDC82 and provides additional clinical and molecular evidence supporting its role in autosomal recessive NDD. The findings reinforce the importance of CCDC82 in human neurodevelopment and highlight the value of genomic investigation in underrepresented populations.

Autosomal recessive↗

PubMed2005

Analysis of nonsense-mediated mRNA decay in mutant alleles identified in Spanish Gaucher disease patients.

Most of the mutations described in the GBA gene as responsible for Gaucher disease are missense mutations. Nevertheless, other alterations, including nonsense and frameshift mutations, have been reported. These mutations generate premature termination codons (PTC) that could trigger the degradation of mRNA through a mechanism known as nonsense-mediated decay (NMD). It has been established that NMD requires the presence of at least one intron downstream of the PTC, and that this PTC should be at least 50-55 nucleotides upstream of the 3'-most exon-exon junction. In this study, we analyse four GBA truncating mutations - c.108G > A (W(-4)X; HGVS recommended nomenclature: p.W36X), c.886C > T (R257X; HGVS: p.R296X), c.1098_1099insA and c.1451_1452delAC - found in Spanish Gaucher disease patients in order to determine whether they undergo mRNA decay and, if so, whether this occurs via the NMD pathway. RT-PCR showed a clear reduction of RNA for three of the alleles: W(-4)X, R257X and c.1098_1099insA. After treatment with cycloheximide (CHX), a known inhibitor of both protein synthesis and NMD, two of the mutant alleles, R257X and c.1098_1099insA, showed a partial recovery of the amount of mRNA. The third mutation, W(-4)X, did not show any significant CHX-induced recovery, while allele c.1451_1452delAC did not show mRNA decay at all. Real-time PCR confirmed these results and allowed the decay and recovery to be quantified. Finally, the protein truncation test was performed to detect the corresponding proteins. Expected products for alleles R257X, c.1451_1452delAC and c.1098_1099insA, but not for W(-4)X, were observed.

Adolescent↗

PubMed2005

Nonsense-mediated mRNA decay: molecular insights and mechanistic variations across species.

Nonsense-mediated mRNA decay (NMD) is an mRNA surveillance pathway that ensures the rapid degradation of mRNAs containing premature translation termination codons (PTCs), thereby preventing the synthesis of truncated and potentially harmful proteins. In addition, this pathway regulates the expression of approximately 10% of the transcriptome and is essential in mice. Although NMD is conserved in eukaryotes, recent studies in several organisms have revealed that different mechanisms have evolved to discriminate natural from premature stop codons and to degrade the targeted mRNAs. With the elucidation of the first crystal structures of components of the NMD machinery, the way is paved towards a molecular understanding of the protein interaction network underlying this process.

Animals↗

PubMed2004

Initiating cellular stress responses.

The phosphoinositide 3-kinase related kinases (PIKKs) mediate responses to diverse stresses, including DNA double-strand breaks (DSBs), abnormal replication fork progression, the recognition of premature termination codons in mRNAs, and inadequate nutrient availability. Rigorous control of these kinases limits cellular damage and promotes cell viability in the presence of stress. Control mechanisms include the localization of PIKKs into multiprotein complexes at the sites of damage and mediation of protein-protein contacts such that substrates are allowed access to the PIKK catalytic domains.

Animals↗

PubMed2005

mRNA surveillance of expressed pseudogenes in C. elegans.

Messenger RNAs (mRNAs) that contain premature translation termination codons (PTCs) are targeted for rapid degradation in all eukaryotes tested. The mechanisms of nonsense-mediated mRNA decay (NMD) have been described in considerable detail, but the biological roles of NMD in wild-type organisms are poorly understood. mRNAs of wild-type organisms known to be degraded by NMD ("natural targets" of NMD) include by-products of regulated alternative splicing, out-of-frame mRNAs derived from unproductive gene rearrangements, cytoplasmic pre-mRNAs, endogenous retroviral and transposon RNAs, and mRNAs having upstream open reading frames or other unusual sequence features. NMD may function to eliminate aberrant PTC-containing mRNAs in order to protect cells from expression of potentially deleterious truncated proteins. Pseudogenes are nonfunctional genes or gene fragments that accumulate mutations through genetic drift. Such mutations will often introduce shifts of reading frame and/or PTCs, and mRNAs of expressed pseudogenes may thus be substrates of NMD. We demonstrate that mRNAs expressed from C. elegans pseudogenes are degraded by NMD and discuss possible implications for both mRNA surveillance and protein evolution. We describe an expressed pseudogene that encodes a small nucleolar RNA (snoRNA) within an intron and suggest this represents an evolutionary intermediate between snoRNA-encoding host genes that do or do not encode proteins.

Animals↗

PubMedDate not supplied

The ATM-related kinase, hSMG-1, bridges genome and RNA surveillance pathways.

Recent studies have identified, hSMG-1 as the newest member of the phosphoinositide 3-kinase(PI3-kinase)-related kinase (PIKK) family. The protein kinase activity of hSMG-1 resembles that of the related PIKK, ATM, both in terms of substrate specificity and its sensitivity to inhibition by the fungal metabolite wortmannin. hSMG-1 is the ortholog of a Caenorhabditis elegans protein, CeSMG-1, which has been genetically linked to a critical mRNA surveillance pathway termed nonsense-mediated decay (NMD). The function of NMD is to mark for rapid degradation mRNAs that bear a premature termination codon. Compelling evidence now indicates that hSMG-1 is also a central player in the NMD pathway in human cells. In addition, hSMG-1, like ATM, appears to be involved in the recognition and/or repair of damaged DNA in these cells. In this review, we introduce a model in which hSMG-1 teams with ATM and ATR to insure the overall quality of the transcriptome in human cells.

Animals↗

PubMed2005

Mutations in the NDUFS4 gene of mitochondrial complex I alter stability of the splice variants.

The effect on the stability of alternative transcripts of different mutations of the NDUFS4 gene in patients with Leigh syndrome with complex I deficiency is presented. Normally, two NDUFS4 splice variants are degraded by nonsense mediated mRNA decay (NMD) while a third form does not trigger NMD degradation. In a patient with a premature termination codon in exon 1, all the three splice variants are up-regulated. The present is the first case of a nonsense mutation leading to the abrogation of NMD, which can represent an additional event to be considered in the evaluation of clinically relevant mutations.

Alternative Splicing↗

PubMed2004

Isolation and characterization of a single-copy actin gene from a sterile mutant of Ulva pertusa (Ulvales, Chlorophyta).

We constructed a cDNA library from sterile Ulva pertusa (Ulvales, Chlorophyta), and isolated and characterized a full-length cDNA clone encoding actin. The actin (ACT) cDNA consisted of 1487 nucleotides (nt) and had an open reading frame (ORF) encoding a polypeptide of 377 amino acid (AA) residues. The ACT gene had one intron in the 5'-untranslated region and three introns in the coding region. Transcription started 26 nt downstream of the putative TATA box. A potential polyadenylation signal, TGTAG, was located 100 nt downstream of the terminator codon, TAG. Amino acid alignment with actins from various algae and land plants showed that sterile U. pertusa actin was more similar to actins from Chlorophyta, Phaeophyta, Euglenophyta, and higher plants (over 76.9%) than to actins from Rhodophyta. Southern blot analysis indicated that the sterile U. pertusa genome has only a single actin-encoding gene. Thalli grown on a 12D/12L photoperiod increased in surface area some two-fold over 24 h regardless of the nutritional conditions. The growth rate of thalli during the light period was significantly higher than that during the dark period. Northern hybridization indicated that the expression of actin mRNA was induced and repressed by the light and dark treatments, respectively. These results suggest that the U. pertusa cell division cycle has a periodicity of 24 h and that the ACT gene is highly transcribed during cell growth and development in the light period.

Actins↗

PubMed2006

Complicated RNA splicing of chicken telomerase reverse transcriptase revealed by profiling cells both positive and negative for telomerase activity.

Telomerase reverse transcriptase (TERT) is an essential component of the telomerase ribonucleoprotein complex which maintains telomeres. The objective of this study was to investigate chicken TERT (cTERT) alternative RNA splicing profiles of samples varying for telomerase activity and immortalization parameters. These included systems both in vivo (gastrula embryo, embryo and adult liver) and in vitro (chicken embryo fibroblasts (CEFs) and DT40 cells). Nineteen cTERT variants were discovered, which were generated through exon skipping, intron retention, and alternative usage of splice donor and acceptor sites. Three variants were predicted to introduce in-frame mutations, whereas the others were predicted to have premature termination codons. The number of cTERT variants detected ranged from 10 in adult liver to 13 in CEFs. One variant (V4) was found in all samples and was predicted to generate a truncated protein lacking telomerase catalytic activity. Interestingly, the standard TERT expected from the full-length transcript was expressed not only in telomerase-positive, but also in telomerase-negative samples. The complicated expression profiles of cTERT in various cell systems suggest that sophisticated regulatory pathways are involved in cTERT pre-mRNA editing. Further, these results support the body of increasing evidence that alternative splicing of TERT, both in human and chicken, contributes to telomerase activity regulation.

Animals↗

PubMed2005

A novel ancestral splicing mutation in the multidrug resistance protein 2 gene causes Dubin-Johnson syndrome in Ashkenazi Jewish patients.

Dubin-Johnson syndrome (DJS) is an inherited disorder characterized by chronic conjugated hyperbilirubinemia due to the absence or dysfunction of the multidrug resistance protein 2 (MRP2). We previously identified two distinct ancestral mutations causing DJS in 22 unrelated Iranian and five unrelated Moroccan Jewish patients, respectively. In this study we identified and characterized the mutation causing DJS in Ashkenazi Jewish patients and assessed a possible founder effect. Sequencing of all 32 exons of the MRP2 gene identified a novel IVS8+4A-->G mutation in three unrelated homozygotes. Haplotype analysis using four intragenic dimorphisms disclosed a founder effect for the mutation. RT-PCR and real time PCR analysis of mRNA from one patient revealed three splice variants all leading to frameshifts and predicting premature termination codons. The main splice variant was a consequence of the use of a cryptic donor splice site inside exon 8. Liver biopsy in one patient revealed complete absence of MRP2 from the canalicular membrane of hepatocytes. In conclusion, our results provide strong evidence that an ancestral IVS8+4A-->G mutation causes DJS in Ashkenazi Jewish patients by abolishing normal splicing of intron 8 leading to aberrantly spliced products that predict truncation of MRP2.

Journal Article↗

PubMed2006

Identification of a novel mutation and a genetic polymorphism of EVER1 gene in two families with epidermodysplasia verruciformis.

BACKGROUND: Epidermodysplasia verruciformis (EV) is a rare autosomal recessive disease. The main clinical features include three kinds of lesions, high risk of skin cancer, and abnormal susceptibility to HPV 5 and 8. Recent studies have shown that mutations in EVER1 and EVER2 genes are responsible for the condition. OBJECTIVE: In the present study, we investigated the molecular basis of EV in two families with EV. METHODS: PCR and direct sequencing of the EVER1 and EVER2 genes were used to identify and confirm the mutations in our probands in the two families. Direct sequencing and SacI digestion were used to detect the polymorphism of exon 6. RESULTS: Sequencing of the EVER1 and EVER2 genes revealed a novel mutation and a genetic polymorphism. The novel mutation by inserting CATGT after nucleotide 916 in exon 9 resulted in a nonsense mutation and a premature termination codon. Direct sequencing and SacI digestion revealed genotype frequencies of C457T, 457T, and 457C alleles in 16 individuals of EV families were of 9, 3, and 4, which were 26, 0, and 24 in 50 unrelated normal controls, respectively. To our knowledge, the novel mutation and genetic polymorphism have not been described in literatures. CONCLUSIONS: The growing number of mutations in EV pedigrees supports the hypothesis that EVER1 and EVER2 are the molecular basis of EV.

Adult↗

PubMed2026

A Conserved 3'UTR Stem-loop Directs UPF1/eIF4AIII-Dependent Regulation of GABARAPL1 mRNA.

RNA-binding proteins (RBP) interact with mRNA untranslated regions containing cis-regulatory elements to govern mRNA localization, stability, and translational efficiency. Among these trans-regulatory factors, RNA helicase UPF1 is a central factor which play a role in multiple mRNA decay pathways, including nonsense-mediated mRNA decay (NMD). NMD is triggered when an exon-junction complex (EJC) is located downstream of a premature termination codon. However, in some cases, NMD can be activated in an EJC-independent manner through mechanisms involving the 3'UTR. In the present study, we focused on the GABARAPL1 3'UTR, as previous studies had shown that this region plays a key role in NMD targeting, although the underlying molecular mechanism had not yet been elucidated. Unlike canonical NMD targets such as SC35, we found that the chemical inhibition of eIF4AIII helicase activity did not affect GABARAPL1 transcript levels, indicating that this transcript is regulated through its 3'UTR via an EJC-independent mechanism. We therefore investigated the potential presence of cis-regulatory element within the 3'UTR of GABARAPL1 which can regulate mRNA and protein levels in a UPF1-dependent manner. Furthermore, we identified a conserved RNA region spanning nucleotides 364-421 involved in GABARAPL1 targeting and used biochemical analysis to demonstrate the direct binding of UPF1 and eIF4AIII to this RNA region, to analyse its secondary structure in solution, and to map the protein-binding sites. By complementing these approaches with molecular modelling, we showed that this stem-loop adopts a stable global fold but a local flexibility and dynamic behaviour properties. Together, our results support the role of UPF1 and eIF4AIII as specific regulators of GABARAPL1 transcript and reveal a novel RNA regulatory element within its 3'UTR, which provides a completely unexpected binding site for these factors.

3' Untranslated Regions↗

PubMed2004

Hyperornithinemia, hyperammonemia, and homocitrullinuria syndrome with evidence of mitochondrial dysfunction due to a novel SLC25A15 (ORNT1) gene mutation in a Palestinian family.

Hyperornithinemia, hyperammonemia, and homocitrullinuria (HHH) syndrome is caused by mutations in the SLC25A15 (ORNT1) gene encoding the mitochondrial ornithine transporter, but the mechanism of pathogenesis of the encephalopathy, spastic paraparesis and hepatopathy remains undetermined. HHH syndrome was diagnosed in a 2-year-old Palestinian boy with developmental delay and seizures, and subsequently in his 13-year-old brother with developmental delay. Direct sequencing of the PCR products of SLC25A15 exon amplifications revealed that both brothers were homozygous for a novel 446G deletion in exon 3 as well as for a 760A>T (I254L) polymorphism in exon 5, which is downstream of a premature termination codon produced by the frameshift resulting from the 446G deletion. The index patient had elevated liver enzymes as well as hyperalaninemia, lactic acidemia with an elevated lactate to pyruvate ratio, and increased urinary excretion of lactate, glutarate and Krebs cycle intermediates. These findings are indicative of mitochondrial dysfunction and are in accordance with ultrastructural studies showing increased numbers of large and bizarre mitochondria in liver, muscle, leukocytes and fibroblasts of some HHH patients. Neurologic and hepatic manifestations are characteristic of some primary mitochondrial disorders. Secondary mitochondrial dysfunction may contribute to the pathogenesis of these same features in HHH syndrome.

Adolescent↗

PubMed2005

Exon-junction complex components specify distinct routes of nonsense-mediated mRNA decay with differential cofactor requirements.

Messenger RNAs (mRNAs) bearing premature translation termination codons (PTCs) are degraded by nonsense-mediated mRNA decay (NMD). For mammalian NMD, current models propose a linear pathway that involves the splicing-dependent deposition of exon-junction complexes (EJCs) and the sequential action of the NMD factors UPF3, UPF2, and UPF1. We show here that different EJC proteins serve as entry points for the formation of distinguishable NMD-activating mRNPs. Specifically, Y14, MAGOH, and eIF4A3 can activate NMD in an UPF2-independent manner, whereas RNPS1-induced NMD requires UPF2. We identify the relevant regions of RNPS1, eIF4A3, Y14, and MAGOH, which are essential for NMD and provide insights into the formation of complexes, that classify alternative NMD pathways. These results are integrated into a nonlinear model for mammalian NMD involving alternative routes of entry that converge at a common requirement of UPF1.

Animals↗

PubMed2006

Applying nonsense-mediated mRNA decay research to the clinic: progress and challenges.

Premature termination codons (PTCs) are equivalent to nonsense sequences. They encode no amino acid, and their presence precludes the synthesis of full-length proteins. Furthermore, the resulting truncated proteins, if synthesized and stable, are likely to be non-functional or might even be deleterious to cellular metabolism. Approximately one third of genetic and acquired diseases are due to PTCs. In fact, PTCs are apt to cause at least some cases of all diseases that involve protein insufficiency. Cells have evolved a way to eliminate mRNAs that contain PTCs using a mechanism called nonsense-mediated mRNA decay (NMD). Here, we will review how to determine which PTCs elicit NMD, what is currently known about the mechanism of NMD, and additional information that is pertinent to establishing therapies for PTC-associated diseases.

Animals↗

PubMed2006

A new mutation in exon 3 of the SCL39A4 gene in a Tunisian family with severe acrodermatitis enteropathica.

Acrodermatitis enteropathica is a rare autosomal recessive disease that manifests as an inability of the affected individual to absorb intestinal zinc, and therefore patients have nutritional zinc deficiency. Without zinc therapy, this condition is fatal. Mutations in the SLC39A4 gene are responsible for acrodermatitis enteropathica. This gene encodes one member of a human zinc/iron-regulated transporter-like protein, also known as ZIP4, and consists of 12 exons and spans about 4.7 kb. We describe a novel mutation in a Tunisian family in which a chain termination codon in exon 3 yielded a truncated ZIP4 zinc transporter protein.

Acrodermatitis↗

PubMed2005

Expression and purification of exendin-4, a GLP-1 receptor agonist, in Escherichia coli.

Exendin-4 is a 39 amino acid peptide isolated from salivary secretions of Gila monster (Heloderma suspectum). It shows 53% sequence similarity to glucagon-like peptide-1 (GLP-1), which is evaluated for the regulation of plasma glucose in type 2 diabetes. Exendin-4 is a potent and long-acting agonist of GLP-1 receptor. In the present study, the exendin-4 gene obtained by PCR with an enterokinase site at N-terminus and a termination codon at C-terminus was expressed in Escherichia coli strain BL21 (DE3) harboring pET32a(+). The fusion protein was purified by chromatography on Ni-NTA-agarose column. Recombinant exendin-4 was obtained by enterokinase cleavage of the fusion protein and subsequent purification. The yield of recombinant exendin-4 was 3.15mg/10g bacteria. The obtained recombinant exendin-4 shows glucose-lowering action in vivo.

Animals↗