Exon 1A Research Articles

Illumina TruSight Cardiomyopathy Sequencing Panel: Application to hypertrophic cardiomyopathy

exon 8 mutations has been described, to date. In contrast to multiorgan disease caused by G406R either in exon 8A or 8, the G402S carrier manifested only an isolated cardiac phenotype with LQT and cardiac arrest. This isolated phenotype was suggested to be a cause of somatic mosaicism. We describe a teenage patient resuscitated from ventricular fibrillation and treated with ICD. She has no other organ manifestations, no syndactyly, her QTc is <480ms, and she has normal neurodevelopment. There is no family history of arrhythmias or sudden deaths. Targeted oligonucleotideselective sequencing of channelopathy genes revealed a de novo G402S in the exon 8 of CACNA1C. Direct sequencing of blood and oral mucosa derived DNA revealed an identical mutation peak suggesting the mutation is not expressed as mosaicism. The phenotype of our patient and the previously described patient show an isolated arrhythmia disease with no clinical manifestations of TS. Based on current evidence, we suggest the G402S mutation in CACNA1C should not be considered as causative for TS.

CD36 gene variants and their potential in determining T2DM susceptibility

Type 2 diabetes (T2DM) is a non-communicable disease affecting huge populations in India. Several groups all over the world are in search of prognostic genetic markers for the early detection of T2DM. Single nucleotide polymorphisms (SNPs) in CD36, a macrophage scavenger receptor were implicated in the pathogenesis of T2DM and its complications. This molecule is responsible for the uptake of free fatty acids specially oxidized low density lipoprotein (Ox-LDL) during diseased conditions. Aim of the present study was to find out the allelic and genotypic frequencies of 11 SNPs spanning the entire CD36 gene in north Indian population and find their association with T2DM. In addition, haplotypic analysis was undertaken to find out the risk haplotype in individuals of families with a history of T2DM. Material and Methods All the 11 SNPs were genotyped in at least 100 each of controls and T2DM subjects using polymerase chain reaction-restriction fragment length polymorphism (PCRRFLP) and single-strand conformation polymorphism (SSCP) methods. Haplotype analysis was done by SHEsis software. Ten families with diabetic history were identified and blood samples were collected from available family members. DNA was extracted by salting out method and three SNPs viz. rs1761667 (G>A) in exon 1A, rs3211938 (T>G) in exon 10 and rs3212018 (16 bp del) in exon 14 were genotyped in all individuals. Anthropometric characteristics viz. systolic/diastolic blood pressures, body mass index (BMI), waist hip ratio (WHR) and biochemical parameters were measured in all individuals. Results Out of 11 SNPs, six were polymorphic (rs1984112, A>G; rs1761667, G>A; rs1527479, C>T; rs3211938, T>G; rs1527483, C>T; rs3212018, 16bp del) in the study population. Individuals having a haplotypic combination ‘AACGC1’ showed highest significant association with T2DM (P G) or ‘G’ allele of rs1984112 (T>G) had an increased BMI and relatively higher glucose levels. Conclusions Therefore, we conclude that haplotypes/genotypes/alleles of the CD36 gene variants can be potential markers in determining diabetes risk and such analyses may be useful for early identification of individuals susceptible to T2DM and its complications in the north Indian population.

Specific BRCA1 gene variations amongst young Moroccan breast cancer patients

Germline mutations in the BRCA1 gene are known predictive markers for the development of hereditary breast cancer. Nevertheless, no comprehensive study has been performed targeting the presence and relevance of BRCA1 mutations in Moroccan breast cancer patients. We here present an analysis of BRCA1 gene regions (exon 2 and exon 11a/b) of 50 female Moroccan breast cancer patients with early disease onset (≤ 40 years) or familial disease backgrounds. Results showed that no mutation was present in either exon 2 or exon 11a of the BRCA1 gene in any of the 50 patients analysed. However, in exon 11b, a mutation generated by a nucleotide exchange was detected in 8% of patients, most of whom were young women (≤ 40). This mutation leads to substitution of the amino acid glutamine by an arginine at position 356 of the polypeptide sequence (Q356R). Although this mutation was previously characterised at a lower frequency in western populations, our study is the first to describe it in a young Moroccan population. Furthermore, another mutation was detected with a high frequency (4%) on exon 11b of the BRCA1 gene in exclusively young patients (≤ 40). This mutation was silent, encoding the same threonine residue at position 327 (T327T) as the wild type. The present study is the first to describe this mutation as well, particularly in a young Moroccan population. Analysis of a larger population is required in order to highlight the relevance of the Q356R and T327T mutations in young Moroccan breast cancer patients.

Molecular Effects of Autoimmune-Risk Promoter Polymorphisms on Expression, Exon Choice, and Translational Efficiency of Interferon Regulatory Factor 5

The rs2004640 single nucleotide polymorphism and the CGGGG copy-number variant (rs77571059) are promoter polymorphisms within interferon regulatory factor 5 (IRF5). They have been implicated as susceptibility factors for several autoimmune diseases. IRF5 uses alternative promoter splicing, where any of 4 first exons begin the mRNA. The CGGGG indel is in exon 1A's promoter; the rs2004640 allele creates a splicing recognition site, enabling usage of exon 1B. This study aimed at characterizing alterations in IRF5 mRNA due to these polymorphisms. Cells with risk polymorphisms exhibited ~2-fold higher levels of IRF5 mRNA and protein, but demonstrated no change in mRNA stability. Quantitative PCR demonstrated decreased usage of exons 1C and 1D in cell lines with the risk polymorphisms. RNA folding analysis revealed a hairpin in exon 1B; mutational analysis showed that the hairpin shape decreased translation 5-fold. Although translation of mRNA that uses exon 1B is low due to a hairpin, increased IRF5 mRNA levels in individuals with the rs2004640 risk allele lead to higher overall protein expression. In addition, several new splice variants of IRF5 were sequenced. IRF5's promoter polymorphisms alter first exon usage and increase transcription levels. High levels of IRF5 may bias the immune system toward autoimmunity.

Mouse divalent metal transporter 1 is a copper transporter in HEK293 cells

Divalent Metal Transporter 1 (DMT1) is an apical Fe transporter in the duodenum and is involved in endosomal Fe export. Four protein isoforms have been described for DMT1, two from mRNA with an iron responsive element (IRE) and two from mRNA without it. The sets of two begin in exon 1A or 2. We have characterized copper transport using mouse 2/-IRE DMT1 during regulated ectopic expression. HEK293 cells carrying a TetR:Hyg element were stably transfected with pDEST31 containing a 2/-IRE construct. (64)Cu(1+) incorporation in doxycycline treated cells exhibited 18.6 and 30.0-fold increases in Cu content, respectively when were exposed to 10 and 100 μM of extracellular Cu. Cu content was ~4-fold above that of parent cells or cells carrying just the vector. (64)Cu uptake in transfected cells pre-incubated with 5μM of Cu-His revealed a Vmax and Km of 11.98±0.52pmolmg protein(-1)min(-1) and 2.03±0.03μM, respectively. Doxycycline-stimulated Cu uptake was linear with time. The rates of apical Cu uptake decreased and transepithelial transport increased when intracellular Cu increased. The optimal pH for Cu transport was 6.5; uptake of Cu was temperature dependent. Silver does not inhibit Cu uptake in cells carrying the vector. In conclusion, Cu uptake in HEK293 cells that over-expressed the 2/-IRE isoform of DMT1 transporter supports our earlier contention that DMT1 transports Cu as Cu(1+).

Expression of the Orphan Cytosolic Sulfotransferase SULT1C3 in Human Intestine: Characterization of the Transcript Variant and Implications for Function

The cystolic sulfotransferse 1C3 (SULT1C3) gene was identified by computational analysis of the human genome and suggested to contain duplications of its last two exons (7a/b and 8a/b). Although the SULT1C3 isoform containing the more downstream exons 7b and 8b (SULT1C3d) has been expressed in Escherichia coli, crystallized, and characterized for activity, there is currently no evidence that SULT1C3 is expressed in any human tissue. Using reverse-transcription polymerase chain reaction, we detected SULT1C3 mRNA in the colorectal adenocarcinoma cell line (LS180), colon, and small intestine, but the amplified fragment contained the more upstream exons 7a and 8a. 3'-Rapid amplification of cDNA ends (RACE) confirmed that the SULT1C3 transcript expressed in LS180 cells contained exons 7a/8a, whereas 5'-RACE identified a noncoding exon 1. Full-length SULT1C3 transcript containing exons 7a/8a was amplified from LS180 and intestinal RNA, and in vitro transcription-translation of the cloned cDNA indicated that translation primarily began at the first of three in-frame ATG codons. Since SULT1C3 containing exons 7a/8a (SULT1C3a) would differ by 30 amino acids from SULT1C3d containing exons 7b/8b, we considered the functional implications of expressing one or the other isoform by generating structural models based on the reported crystal structure for SULT1C3d. Comparison of the structures indicated that five of the residues forming the substrate-binding pocket differed between the two isoforms, resulting in a change in both electron density and charge distribution along the inner wall of the substrate-binding pocket. These data indicate that SULT1C3 is expressed in human intestine but suggest that the expressed isoform is likely to differ functionally from the isoform that has been previously characterized.

TIN2 Mutations In Dyskeratosis Congenita Cause Telomere Shortening In Induced Pluripotent Stem Cells Through Potent Dominant Negative Inhibition Of Telomerase

Dyskeratosis congenita (DC) is a bone marrow failure syndrome characterized by widespread defects in diverse tissues and a strong predisposition to cancer. DC is caused by germline mutations in genes controlling maintenance of telomeres, nucleoprotein caps that protect chromosome ends. Mutations in components of the telomerase enzyme comprise a large share of cases, including in TERT, TERC, dyskerin, TCAB1, NOP10 and NHP2. These mutations compromise telomerase function leading to telomere shortening, which in turn impairs stem cell function. We previously created patient-derived iPS cells from patients with mutations in TERT, dyskerin or TCAB1 and analyzed these cells to understand the biochemical defects in the telomerase pathway. In each case we found a unique mechanism underlying these telomerase defects, including: reduced catalytic function (TERT mutations), impaired telomerase assembly (dyskerin mutations) and mislocalization of the enzyme to nucleoli (TCAB1 mutations). A six-member protein complex – shelterin - is essential for proper function of telomeres. Despite the critical importance of shelterin proteins in telomere regulation, only a single telomere binding protein – TIN2 – is mutated in DC. However, how these mutations compromise telomere maintenance remains poorly understood. TIN2 mutations occur in a common, autosomal dominant form of DC, presenting in early life, with particularly severe clinical manifestations and poor outcomes. Mutations in the TIN2 gene are clustered in exon 6a, which corresponds to a protein domain of unknown function. To understand how TIN2 mutations impair telomere maintenance and cause DC, we reprogrammed fibroblasts from patients with TIN2 mutations to iPS cells. We succeeded in generating pluripotent iPS cells from a patient with a frame shift mutation at position 284 of the protein. TIN2-mutant iPS cells expressed all the markers of wild-type iPS cells and human ES cells and could be differentiated to all three germ cell layers in culture. With reprogramming from fibroblasts to iPS cells, telomerase is upregulated and causes telomere elongation in wild-type cells. In analyzing telomeres from TIN2-mutant iPS cells, we found that telomere elongation was abrogated. Instead of telomere elongation, TIN2-mutant iPS cells showed telomere shortening with reprogramming and during passage in cell culture. After extended cell passage, TIN2-mutant iPS cells lost the ability to self-renew and differentiated, concomitant with the activation of the telomere surveillance checkpoint p53. To better understand how TIN2 mutant proteins interfere with telomere maintenance, we overexpressed GFP, wild-type TIN2, or TIN2 truncation mutants from DC patients into human, telomerase-positive cancer cells. Genomic DNA was collected from these cells during passage and analyzed for telomere lengths by Southern blot. Expression of GFP or wild-type TIN2 had no effect on telomere lengths, which were stably maintained during the experiment. In marked contrast, expression of the TIN2 truncation mutants from DC patients led to progressive and dramatic telomere shortening with cell passage. Together, these data in patient-derived iPS cells and in human cancer cells suggest that TIN2 mutants inhibit the action of telomerase at telomeres. These results constitute a new molecular mechanism at play in DC and yield new insight into one of the most common forms of DC. Disclosures:No relevant conflicts of interest to declare.

Novel Fusion Transcripts PVT1-NSMCE2 and BF104016-NSMCE2 Resulting In Depletion Of NSMCE2 Protein In Acute Myeloid Leukemia Harboring 8q24 Amplicons

Little is known about the molecular pathogenesis of acute myeloid leukemia (AML) with high-level DNA amplifications commonly defined as the presence of double minutes (dmins) and/or homogeneously staining regions (hsr), although dmins and hsr are found in nearly 1% of karyotypically abnormal AML and myelodysplastic syndromes (MDS). It is sometimes difficult to precisely assign dmins, hsr, and marker chromosomes (mars) to specific chromosomal bands, while most of them consist of an amplified segment from chromosome band 8q24 or 11q23. In AML, the 8q24 amplicon has been previously identified in a commonly involved segment of about 4 Mb, which contains several genes such as TRIB1 (C8FW), NSMCE2 (non-SMC element 2), PVT1, and MYC. To further define the pathogenetic lesions of 8q24 amplicons in AML, we performed comprehensive molecular studies on leukemic cells from a patient with AML (patient 1) with two mars and leukemic cell lines HL60 and K562, identifying two novel chimeric transcripts, PVT1-NSMCE2 and BF104016-NSMCE2. Regarding PVT1-NSMCE2 fusion transcripts, PVT1 exon 1a fused to NSMCE2 exon 3 in patient 1, PVT1 exon 3a to NSMCE2 exon 4 in patient 2, and PVT1 exon 4b to NSMCE2 exon 4 in K562. Patient 2 was identified from the screening of an additional 50 patients with AML or MDS by RT-PCR. As for BF104016-NSMCE2 fusion transcript, BF104016 exon 1 fused to NSMCE2 exon 6 in HL60 harboring hsr and dmins. BF104016 is located inside the CCDC26 gene, sharing the same exon of CCDC26. In patient 1, fluorescence in situ hybridization identified the amplification of 5'PVT1 on the mars and dmins, and the amplification of NSMCE2 only on the mars. In HL60, PVT1 and NSMCE2 were amplified on dmins. Although real-time quantitative PCR showed the amplification of the aberrant NSMCE2 chimeric transcripts, western blot analysis demonstrated the depletion of NSMCE2 protein. High-resolution oligonucleotide array analysis demonstrated two amplicons at 8q24 commonly found in patient 1 and HL60. Furthermore, the coding directions of these three fusion genes were different at the 8q24; NSMCE2 and PVT1 are transcribed from centromere toward telomere, opposite to the direction of transcription of the CCDC26 gene. These results suggest that the chimeric formation of these genes were caused by chromothripsis. PVT1 and CCDC26 were known as large intervening non-coding RNAs, both of which were considered to be associated with the oncogenesis. NSMCE2 is known as a small ubiquitin-like modifier (SUMO) E3 ligase and is required for DNA repair. Functional analysis of NSMCE2 chimeric transcripts with PVT1 or CCDC26 will contribute to understanding of the leukemogenesis in AML harboring 8q24 amplicons. Disclosures:Taniwaki:Novartis: Honoraria.

Abstract B4: Evaluation of VEGFxxxb isoforms as a predictive biomarker for anti-VEGF cancer therapy.

Abstract Biomarkers are critical tools for measuring efficacy of treatments or predicting which patients are mostly likely to respond to a particular therapy. Recently, the VEGF isoform, VEGF165b, was proposed to be a predictive marker for bevacizumab in metastatic colorectal cancer patients. VEGF165b is an anti-angiogenic splice variant that differs from its pro-angiogenic counterpart, VEGF165, by only six amino acids at the C-terminal end of the protein. Members of the VEGFxxxb isoform family are produced by alternative splicing of the 3’ untranslated region (UTR) to generate exon 8b, instead of the conventional exon 8 which produces exon 8a. The study of VEGF165b in colorectal cancer (Bates et al, Clinical Cancer Research, 2012) reported that patients with low VEGF165b:VEGFtotal ratio had improved PFS following bevacizumab + FOLFOX4 treatment, while individuals with higher VEGF165b:VEGFtotal ratios did not benefit. On the other hand, another recent study (Harris et al, PLoS One, 2012) reported that VEGFxxxb isoforms may be a RT-PCR artifact. In this study, we sought to test the role of VEGF165b in the response to anti-VEGF therapy using preclinical human tumor models, and to evaluate the prevalence of VEGFxxxb isoform in human cancer cells and normal tissues using RNA-Seq, a next generation sequencing (NGS) platform that is independent of RT-PCR. Our results show that xenograft tumors engineered to overexpress VEGF165b had similar growth kinetics, tumor vascular density and were equally sensitive to aflibercept or bevacizumab treatment in humanized VEGF SCID mice compared to their parental counterparts. These results indicate that VEGF165b does not have anti-angiogenic properties, and VEGF165b levels did not correlate with the efficacy of anti-VEGF therapy. Furthermore, although we were able to detect VEGF165b splice variants by NGS profiling in cells engineered to overexpress VEGF165b, no NGS evidence supporting the VEGFxxxb isoforms was found in a panel of 24 human cancer cell lines and 12 different types of human normal tissues. To our knowledge, this is the first report to survey VEGFxxxb isoforms using NGS RNA-seq technology. Overall, these studies suggest that closer inspection of VEGFxxxb isoforms may be required before employing VEGF165b as a predictive biomarker for anti-VEGF therapy. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B4. Citation Format: Wen Zhang, Cristina Abrahams, Ying Huang, Baosheng Li, Asma Parveen, Min Ni, Yi Wei, Yueming Ding, Eric Zhang, Hsin Chieh Lin, Gavin Thurston, Qing Zhang. Evaluation of VEGFxxxb isoforms as a predictive biomarker for anti-VEGF cancer therapy. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B4.

A novel indel in exon 9 of APC upregulates a ‘skip exon 9’ isoform and causes very severe familial adenomatous polyposis

Germline mutation in the adenomatous polyposis coli (APC) gene causes the majority (80%) of familial adenomatous polyposis (FAP), an autosomal dominantly inherited form of colorectal cancer (CRC). Mutation in 5'end of exon 9 of APC usually results in an attenuated form of FAP (aFAP), characterized by later age of onset and fewer polyps. The presence of exon 9a, an in-frame isoform with exon 8 spliced to 3'end of exon 9, modulates any deleterious effect of the mutation. A third lowly expressed isoform that completely skips exon 9 is present in both healthy individuals and FAP patients. We report here an interesting case of a proband with an APC mutation in 5'end of exon 9 that presented with six synchronous advanced CRCs at age 37. The novel insertion-deletion (indel) at codon 409, c.1226-1229delTTTTinsAAA, caused upregulation of the 'skip exon 9' isoform, r934-1312del, resulting in a premature stop codon at exon 10 and a truncated protein that removed all of the β-catenin (CTNNB1) binding motifs, thus activating the downstream T-cell transcription factor (Tcf) pathway. Exon 9a isoform was concomitantly downregulated. This finding emphasizes the necessity of examining the various isoforms of exon 9 to avoid clinical mismanagement and counseling based on just the mutation site by genomic DNA sequencing alone.

Brugada syndrome and abnormal splicing of SCN5A in myotonic dystrophy type 1

In patients with myotonic dystrophy type 1 (DM1), the mechanisms underlying sudden cardiac death, which occurs in up to 1/3 of patients, are unclear. To study the potential role of Brugada syndrome in ventricular tachyarrhythmias and sudden death in DM1 patients. We screened 914 adult patients included in the DM1 Heart Registry during 2000-2009 for the presence of type 1 Brugada pattern on electrocardiogram (ECG). We also performed direct sequencing of SCN5A in patients with Brugada pattern. Further, we analysed SCN5A splicing on ventricular myocardial specimens harvested during cardiac transplantation in a 45-year-old patient with DM1 and three controls with inherited dilated cardiomyopathy. A type 1 Brugada pattern was present on the ECG of seven of 914 patients (0.8%), including five with a history of sustained ventricular tachyarrhythmia or sudden death, who fulfilled the criteria for Brugada syndrome. SCN5A sequencing was normal in all patients. Ventricular myocardial specimen analysis displayed abnormal splicing of SCN5A exon 6, characterized by over-expression of the 'neonatal' isoform, called exon 6A, in the patient with DM1, but not from the controls. Our findings suggest a potential implication of Brugada syndrome in sudden death in DM1, which may be related to missplicing of SCN5A. Our findings provide a new insight into the pathophysiology of heart disease in DM1.

A Novel Actin mRNA Splice Variant Regulates ACTG1 Expression

Cytoplasmic actins are abundant, ubiquitous proteins in nucleated cells. However, actin expression is regulated in a tissue- and development-specific manner. We identified a novel cytoplasmic-γ-actin (Actg1) transcript that includes a previously unidentified exon (3a). Inclusion of this exon introduces an in-frame termination codon. We hypothesized this alternatively-spliced transcript down-regulates γ-actin production by targeting these transcripts for nonsense-mediated decay (NMD). To address this, we investigated conservation between mammals, tissue-specificity in mice, and developmental regulation using C2C12 cell culture. Exon 3a is 80% similar among mammals and varies in length from 41 nucleotides in humans to 45 in mice. Though the predicted amino acid sequences are not similar between all species, inclusion of exon 3a consistently results in the in the introduction of a premature termination codon within the alternative Actg1 transcript. Of twelve tissues examined, exon 3a is predominantly expressed in skeletal muscle, cardiac muscle, and diaphragm. Splicing to include exon 3a is concomitant with previously described down-regulation of Actg1 in differentiating C2C12 cells. Treatment of differentiated C2C12 cells with an inhibitor of NMD results in a 7-fold increase in exon 3a-containing transcripts. Therefore, splicing to generate exon 3a-containing transcripts may be one component of Actg1 regulation. We propose that this post-transcriptional regulation occurs via NMD, in a process previously described as “regulated unproductive splicing and translation” (RUST).

Alternative 5’ Untranslated Regions Are Involved in Expression Regulation of Human Heme Oxygenase-1

The single nucleotide polymorphism rs2071746 and a (GT)n microsatellite within the human gene encoding heme oxygenase-1 (HMOX1) are associated with incidence or outcome in a variety of diseases. Most of these associations involve either release of heme or oxidative stress. Both polymorphisms are localized in the promoter region, but previously reported correlations with heme oxygenase-1 expression remain not coherent. This ambiguity suggests a more complex organization of the 5’ gene region which we sought to investigate more fully.We evaluated the 5‘ end of HMOX1 and found a novel first exon 1a placing the two previously reported polymorphisms in intronic or exonic positions within the 5’ untranslated region respectively. Expression of exon 1a can be induced in HepG2 hepatoma cells by hemin and is a repressor of heme oxygenase-1 translation as shown by luciferase reporter assays. Moreover, minigene approaches revealed that the quantitative outcome of alternative splicing within the 5’ untranslated region is affected by the (GT)n microsatellite.This data supporting an extended HMOX1 gene model and provide further insights into expression regulation of heme oxygenase-1. Alternative splicing within the HMOX1 5' untranslated region contributes to translational regulation and is a mechanistic feature involved in the interplay between genetic variations, heme oxygenase-1 expression and disease outcome.

Molecular Diagnosis of Friedreich Ataxia Using Analysis of GAA Repeats and FXN Gene Exons in Population from Western India

The diagnosis of Friedreich ataxia is based on the clinical symptoms and GAA repeats expansions. In our experience, checking FXN gene exons for mutations along with GAA repeat analysis may give better clue for its diagnosis. In the present study, total 49 suspected Friedreich ataxia patients were analyzed for GAA repeat expansion. Eleven patients have normal number of GAA repeats, thereby termed as FRDA negative patients. Thirty-eight patients showed no amplification using GAA repeat analysis. Since no conclusion was possible based on these results, these patients were designated as uninformative. We have analyzed 5 exons of the FXN gene in FRDA negative and uninformative patients to check for possible mutations. It was observed that there were no mutations found in any of FRDA negative and most uninformative patients. We further used long range PCR to check for deletion of exon 5a. It was found that 18 patients showed expression for exon 5a PCR but none in long range PCR. Five patients showed no expression for exon 5a PCR as well as long range PCR indicating that these 5 patients may be positive FRDA patients. These findings need to be correlated with clinical history of these patients for confirmation.

Quaking, an RNA-Binding Protein, Is a Critical Regulator of Vascular Smooth Muscle Cell Phenotype

RNA-binding proteins are critical post-transcriptional regulators of RNA and can influence pre-mRNA splicing, RNA localization, and stability. The RNA-binding protein Quaking (QKI) is essential for embryonic blood vessel development. However, the role of QKI in the adult vasculature, and in particular in vascular smooth muscle cells (VSMCs), is currently unknown. We sought to determine the role of QKI in regulating adult VSMC function and plasticity. We identified that QKI is highly expressed by neointimal VSMCs of human coronary restenotic lesions, but not in healthy vessels. In a mouse model of vascular injury, we observed reduced neointima hyperplasia in Quaking viable mice, which have decreased QKI expression. Concordantly, abrogation of QKI attenuated fibroproliferative properties of VSMCs, while potently inducing contractile apparatus protein expression, rendering noncontractile VSMCs with the capacity to contract. We identified that QKI localizes to the spliceosome, where it interacts with the myocardin pre-mRNA and regulates the splicing of alternative exon 2a. This post-transcriptional event impacts the Myocd_v3/Myocd_v1 mRNA balance and can be modulated by mutating the quaking response element in exon 2a of myocardin. Furthermore, we identified that arterial damage triggers myocardin alternative splicing and is tightly coupled with changes in the expression levels of distinct QKI isoforms. We propose that QKI is a central regulator of VSMC phenotypic plasticity and that intervention in QKI activity can ameliorate pathogenic, fibroproliferative responses to vascular injury.

The 5′‐untranslated region regulates ATF5 mRNA stability via nonsense‐mediated mRNA decay in response to environmental stress

We previously reported that activating transcription factor 5 (ATF5) mRNA increases in response to amino acid limitation, and that this increase is dependent on mRNA stabilization. The ATF5 gene allows transcription of mRNAs with two alternative 5'-UTRs, 5'-UTRα and 5'-UTRβ, derived from exon 1α and exon 1β. 5'-UTRα contains the upstream open reading frames uORF1 and uORF2. Phosphorylation of eukaryotic initiation factor 2α during the integrated stress response had been previously shown to lead to bypassing of uORF2 translation and production of ATF5 protein. Translation of uORF2 is expected to result in translational termination at a position 125 nucleotides upstream of the exon junction, and this fits the criterion of a nonsense-mediated decay target mRNA. We investigated the potential role of 5'-UTRα in the control of mRNA stabilization, and found that 5'-UTRα reduced the stability of ATF5 mRNA. 5'-UTRα-regulated destabilization of mRNA was suppressed by knockdown of the nonsense-mediated decay factors Upf1 and Upf2. Mutation of the downstream AUG (uAUG2) rendered mRNA refractory to Upf1 and Upf2 knockdown. Moreover, 5'-UTRα-regulated down-regulation was hindered by amino acid limitation and tunicamycin treatment, and stress-induced phosphorylation of eukaryotic initiation factor 2α was involved in stabilization of ATF5 mRNA. These studies show that ATF5 mRNA is a naturally occurring normal mRNA target of nonsense-mediated decay, and provide evidence for linkage between stress-regulated translational regulation and the mRNA decay pathway. This linkage constitutes a mechanism that regulates expression of stress response genes.

A GAA repeat expansion reporter model of Friedreich's ataxia recapitulates the genomic context and allows rapid screening of therapeutic compounds.

Friedreich's ataxia (FRDA) is caused by large GAA expansions in intron 1 of the frataxin gene (FXN), which lead to reduced FXN expression through a mechanism not fully understood. Understanding such mechanism is essential for the identification of novel therapies for FRDA and this can be accelerated by the development of cell models which recapitulate the genomic context of the FXN locus and allow direct comparison of normal and expanded FXN loci with rapid detection of frataxin levels. Here we describe the development of the first GAA-expanded FXN genomic DNA reporter model of FRDA. We modified BAC vectors carrying the whole FXN genomic DNA locus by inserting the luciferase gene in exon 5a of the FXN gene (pBAC-FXN-Luc) and replacing the six GAA repeats present in the vector with an ∼310 GAA repeat expansion (pBAC-FXN-GAA-Luc). We generated human clonal cell lines carrying the two vectors using site-specific integration to allow direct comparison of normal and expanded FXN loci. We demonstrate that the presence of expanded GAA repeats recapitulates the epigenetic modifications and repression of gene expression seen in FRDA. We applied the GAA-expanded reporter model to the screening of a library of novel small molecules and identified one molecule which up-regulates FXN expression in FRDA patient primary cells and restores normal histone acetylation around the GAA repeats. These results suggest the potential use of genomic reporter cell models for the study of FRDA and the identification of novel therapies, combining physiologically relevant expression with the advantages of quantitative reporter gene expression.

P1/P2 genotyping of known and novel null alleles in the P1PK and GLOB histo‐blood group systems

The rare but clinically important null phenotypes of the P1PK and GLOB blood group systems are due to alterations in A4GALT and B3GALNT1, respectively. A recently identified single-nucleotide polymorphism in Exon 2a of A4GALT predicts the common P1 and P2 phenotypes but rare variants have not been tested. The aim of this study was to analyze 84 p, P1 (k) , and P2 (k) samples, with special emphasis on unknown alleles and the P(1) /P(2) marker. Of these, 27 samples came from individuals not previously investigated genetically and were therefore subjected to sequencing of A4GALT or B3GALNT1, and a subset was tested by flow cytometry. The P(1) /P(2) genotyping linked 20 p-inducing mutations in A4GALT to P(1) or P(2) allelic background. Eight p alleles remain unlinked due to compound heterozygosity. For 23 of 25 P(k) samples, concordant results were observed: P1 (k) samples had at least one P(1) allele while P2 (k) had P(2) only. The two remaining samples typed as P1+ and P1+(w) but were genetically P(2) /P(2) . A tendency toward higher P(k) antigen expression was observed on P1 (k) cells compared to P2 (k) . In total, six previously unknown null mutations were found and characterized in A4GALT while four new changes were revealed in B3GALNT1. For the first time, p alleles were shown to occur on both P(1) and P(2) allelic backgrounds. Furthermore, P(1) /P(2) genotyping predicted the P1 (k) versus P2 (k) phenotype in more than 90% of globoside-deficient samples. The number of GLOB-null alleles was increased by 50% and several P1PK-null alleles were identified.

C-Terminal Alternative Splicing of CaV1.3 Channels Distinctively Modulates Their Dihydropyridine Sensitivity

The transcripts of L-type voltage-gated calcium channels (CaV) 1.3 undergo extensive alternative splicing. Alternative splicing, particularly in the C terminus, drastically modifies gating properties of the channel. However, little is known about whether alternative splicing could modulate the pharmacologic properties of CaV1.3 in a manner similar to the paralogous CaV1.2. Here we undertook the screening of different channel splice isoforms harboring splice variations in either the IS6 segment or the C terminus. Unexpectedly, while inclusion of exon 8a or 8, which code for IS6, did not alter dihydropyridine (DHP) sensitivity, distinct pharmacologic properties were observed for the various C-terminal splice isoforms. In the presence of external Ca(2+), fast inactivating splice variants including CaV1.342a and CaV1.343s with intact calmodulin-IQ domain interaction showed consistently low DHP sensitivity. Interestingly, attenuation of calcium-dependent inactivation with overexpression of calmodulin34 did not enhance the sensitivity of CaV1.342a, suggesting that the low DHP sensitivity may not be a result of fast channel inactivation. Alternatively, disruption of calmodulin-IQ domain binding in the CaV1.3Δ41 and full-length CaV1.342 channels was associated with heightened DHP sensitivity. In distinct contrast to the well-known modulation of DHP blockade of CaV1.2 channels, this study has therefore uncovered a novel mechanism for modulation of the pharmacologic properties of CaV1.3 channels through posttranscriptional modification of the C terminus.

Overlapping Region of p53/Wrap53 Transcripts: Mutational Analysis and Sequence Similarity with microRNA-4732-5p

Although the majority of investigations concerned with TP53 and its protein have focused on coding regions, recently a set of studies highlighted significant roles of regulatory elements located in p53 mRNA, especially 5 ? UTR. The wrap53α transcript is one of those that acts as a natural antisense agent, forming RNA-RNA hybrids with p53 mRNA and protecting it from degradation. In this study, we focused on the mutation status of exon 1α of the WRAP53 gene (according to exon 1 of p53) in 160 breast tumor tissue samples and conducted a bioinformatics search for probable miRNA binding site in the p53/wrap53 overlapping region. Mutations were detected, using single stranded conformation polymorphism (SSCP) and sequencing. We applied the miRBase database for prediction of miRNAs which target overlapping region of p53/wrap53 transcripts. Our results showed all samples to have wild type alleles in exon 1 of TP53 gene. We could detect a novel and unreported intronic mutation (IVS1+ +56, G>C) outside overlapping regions of p53/wrap53 genes in breast cancer tissues and also predict the presence of a binding site for miR-4732-5p in the 5' UTR of Wrap53 mRNA. From our findings we propose designing further studies focused on overexpression of miRNA-4732-5p and introducing different mutations in the overlapping region of wrap53 and p53 genes in order to study their effects on p53 and its δN isoform (δ40p53) expression. The results may provide new pieces in the p53 targeting puzzle for cancer therapy.