Exon 1A Research Articles

In vitro identification of CACN1h 9 axonal SNP in epileptic patients

Childhood absence epilepsy (CAE) is an autosomal dominant disorder and a heterogeneous familial condition in which family members express absence seizures initially and then show multiple phenotypes of myoclonic epilepsy, including partial or absence seizures and generalized tonic conic seizures. Multiple types of genetic mutations are involved in epilespy. This study was aimed at investigating the coding regions of CACNA1H gene for analyzing the mutations involved in epilepsy. Blood samples of mutually unrelated true representatives of CAE were collected from the psychiatry department of various hospitals in Lahore. DNA was extracted using the standard protocol and the amplification of the CACNA1H region was achieved with specially designed primers. Later on, the analysis of the results was carried out via the sequencing of target fragments. Sequences were analyzed using the BioEdit software and then aligned with the help of the ClustalW2 software. A series of 12 unrelated patients with CAE were screened for mutations in the CACNA1H gene. No mutation was found in exon 9a. As already reported, mutations in the exonic sequence of CACNA1H gene were found in 5 out of 14 CAE patients. These changes were observed in a PCR fragment amplified by primer 2 in the region of the 9th exon. Subsequent analysis of these fragments identified transition mutations (2025G>A) in exon 9. To conclude, there is a need to explore the site of the gene along with other gene mutations causing epilepsy in the local population of Punjab, Pakistan. It will help to develop genetic counseling strategies, gene therapies, and prenatal diagnostic procedures for the said population.

Developmental dynamics of voltage-gated sodium channel isoform expression in the human and mouse brain

BackgroundGenetic variants in the voltage-gated sodium channels SCN1A, SCN2A, SCN3A, and SCN8A are leading causes of epilepsy, developmental delay, and autism spectrum disorder. The mRNA splicing patterns of all four genes vary across development in the rodent brain, including mutually exclusive copies of the fifth protein-coding exon detected in the neonate (5N) and adult (5A). A second pair of mutually exclusive exons is reported in SCN8A only (18N and 18A). We aimed to quantify the expression of individual exons in the developing human brain.MethodsRNA-seq data from 783 human brain samples across development were analyzed to estimate exon-level expression. Developmental changes in exon utilization were validated by assessing intron splicing. Exon expression was also estimated in RNA-seq data from 58 developing mouse neocortical samples.ResultsIn the mature human neocortex, exon 5A is consistently expressed at least 4-fold higher than exon 5N in all four genes. For SCN2A, SCN3A, and SCN8A, a brain-wide synchronized 5N to 5A transition occurs between 24 post-conceptual weeks (2nd trimester) and 6 years of age. In mice, the equivalent 5N to 5A transition begins at or before embryonic day 15.5. In SCN8A, over 90% of transcripts in the mature human cortex include exon 18A. Early in fetal development, most transcripts include 18N or skip both 18N and 18A, with a transition to 18A inclusion occurring from 13 post-conceptual weeks to 6 months of age. No other protein-coding exons showed comparably dynamic developmental trajectories.ConclusionsExon usage in SCN1A, SCN2A, SCN3A, and SCN8A changes dramatically during human brain development. These splice isoforms, which alter the biophysical properties of the encoded channels, may account for some of the observed phenotypic differences across development and between specific variants. Manipulation of the proportion of splicing isoforms at appropriate stages of development may act as a therapeutic strategy for specific mutations or even epilepsy in general.

Nanopore sequencing reveals full‐length Tropomyosin 1 isoforms and their regulation by RNA‐binding proteins during rat heart development

Alternative splicing (AS) contributes to the diversity of the proteome by producing multiple isoforms from a single gene. Although short‐read RNA‐sequencing methods have been the gold standard for determining AS patterns of genes, they have a difficulty in defining full‐length mRNA isoforms assembled using different exon combinations. Tropomyosin 1 (TPM1) is an actin‐binding protein required for cytoskeletal functions in non‐muscle cells and for contraction in muscle cells. Tpm1 undergoes AS regulation to generate muscle versus non‐muscle TPM1 protein isoforms with distinct physiological functions. It is unclear which full‐length Tpm1 isoforms are produced via AS and how they are regulated during heart development. To address these, we utilized nanopore long‐read cDNA sequencing without gene‐specific PCR amplification. In rat hearts, we identified full‐length Tpm1 isoforms composed of distinct exons with specific exon linkages. We showed that Tpm1 undergoes AS transitions during embryonic heart development such that muscle‐specific exons are connected generating predominantly muscle‐specific Tpm1 isoforms in adult hearts. We found that the RNA‐binding protein RBFOX2 controls AS of rat Tpm1 exon 6a, which is important for cooperative actin binding. Furthermore, RBFOX2 regulates Tpm1 AS of exon 6a antagonistically to the RNA‐binding protein PTBP1. In sum, we defined full‐length Tpm1 isoforms with different exon combinations that are tightly regulated during cardiac development and provided insights into the regulation of Tpm1 AS by RNA‐binding proteins. Our results demonstrate that nanopore sequencing is an excellent tool to determine full‐length AS variants of muscle‐enriched genes.

Paternal eNOS Deficiency Affects Glucose Homeostasis and Liver Glycogen Content in Male Offspring Without Inheritance of eNOS Deficiency Itself

Background: It was shown that maternal eNOS deficiency causes fatty liver disease and numerically lower fasting glucose in female wild-type offspring suggesting that parental genetic variants may influence the offspring's phenotype via epigenetic modifications in the offspring despite the absence of a primary genetic defect. This, however, was not shown so far for paternal genes. Methods; Heterozygous male eNOS knockout mice or wild-type male mice were bred with female wild type mice. The phenotype of wild-type offspring of heterozygous male eNOS knockout mice was compared to offspring from wild type parents. Findings: Global sperm DNA methylation decreased and sperm miRNAs pattern altered substantially. Fasting glucose and liver glycogen storage were increased when analyzing wild type male and female offspring of +/- eNOS fathers. The endocrine pancreas in wild-type offspring was not affected. Wild-type male but not female offspring of +/- eNOS fathers had increased fasting insulin and increased insulin after glucose load. Analyzing candidate genes for liver fat- and carbohydrate metabolism revealed that the glucocorticoid receptor (GR) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1a) genes were increased while DNA methylation of GR exon 1A and PGC1a promoter were decreased in the liver of male wild-type offspring of +/- eNOS fathers. Interpretation: Our study suggests that paternal genetic defects may alter the epigenome of the sperm without transmission of the defect itself and alter in later life of the offspring epigenetically gene expression and hence the phenotype without inheriting the defect itself and these alterations might be offspring sex dependent. Funding Information: This study was financially supported by the “Deutsche Forschungsgemeinschaft” to Dr. Berthold Hocher. Declaration of Interests: The authors have no conflict of interest to disclose Ethics Approval Statement: Study design and experimental protocols were conducted according to the local institutional guidelines for the care and use of laboratory animals and approved by the animal welfare ethical committee of the state of Berlin.

VEGFA's distal enhancer regulates its alternative splicing in CML.

Enhancer demethylation in leukemia has been shown to lead to overexpression of genes which promote cancer characteristics. The vascular endothelial growth factor A (VEGFA) enhancer, located 157 Kb downstream of its promoter, is demethylated in chronic myeloid leukemia (CML). VEGFA has several alternative splicing isoforms with different roles in cancer progression. Since transcription and splicing are coupled, we wondered whether VEGFA enhancer activity can also regulate the gene’s alternative splicing to contribute to the pathology of CML. Our results show that mutating the VEGFA +157 enhancer promotes exclusion of exons 6a and 7 and activating the enhancer by tethering a chromatin activator has the opposite effect. In line with these results, CML patients present with high expression of +157 eRNA and inclusion of VEGFA exons 6a and 7. In addition, our results show that the positive regulator of RNAPII transcription elongation, CCNT2, binds VEGFA’s promoter and enhancer, and its silencing promotes exclusion of exons 6a and 7 as it slows down RNAPII elongation rate. Thus our results suggest that VEGFA’s +157 enhancer regulates its alternative splicing by increasing RNAPII elongation rate via CCNT2. Our work demonstrates for the first time a connection between an endogenous enhancer and alternative splicing regulation of its target gene.

Abstract 2391: Computation of a user-friendly composite score from three prostate cancer biomarkers

Abstract Summary: Prostate cancer (PCa) is the most commonly diagnosed solid organ cancer in men worldwide. Current diagnosis of PCa includes use of initial prostate specific antigen assay which has a high false positive rate, low specificity, and low sensitivity. Consequently, it results in overdiagnosis and overtreatment, leaving patients with unintended health complications from the side effects of unnecessary prostate biopsies and treatment. PCa biomarkers are being discovered to address this unmet need. Here, we report creation of a composite score based on three PCa biomarkers, plasmacytoma variant translocation 1 exon 4A, exon 4B, and exon 9. Statistical analysis of copy numbers derived from a real-time quantitative polymerase chain reaction - based assay showed these PCa biomarkers to be linearly separable. We trained a supervised learning algorithm using support vector classifiers to generate a classification hyperplane from which a composite score is developed. Our aggregate score when applied to test data from non-cancerous prostate epithelial cells and PCa cells accurately classified 100% PCa cells. Creation of this 3-biomarker based composite score lays the groundwork for clinical trial of its use in PCa diagnosis. Citation Format: Gargi Pal, Emmanuel Asante-Asamani, Leslie Liu, Olorunseun O. Ogunwobi. Computation of a user-friendly composite score from three prostate cancer biomarkers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2391.

A CRISPR RNA-Binding Protein Screen Reveals Novel Regulators of RUNX1 Alternative Polyadenylation

Alternative polyadenylation (APA) is a crucial post-transcriptional process that regulates the 3' end formation of over 75% of all human genes. Despite its prevalence and importance during development and in disease, there are few studies that focus on APA in the hematology field. Interestingly, APA affects the coding sequence of RUNX1, a pivotal transcription factor for proper hematopoiesis. Usage of the most proximal RUNX1 polyadenylation sequence (PAS) in upstream alternative terminal exon 7a results in the C-terminally truncated RUNX1a isoform. RUNX1a uniquely promotes self-renewal and expansion of hematopoietic stem cells (HSCs). In the endogenous context, usage of this proximal PAS is tightly restricted, resulting in polyadenylation at one of three distal PAS in exon 8 and producing the RUNX1b/c isoforms. RUNX1b/c are much more abundant and promote HSC differentiation and lineage commitment. Importantly, the ratio of RUNX1a to RUNX1b/c is developmentally-regulated. Therefore, it is highly significant to uncover the APA mechanism that controls the RUNX1 isoform ratio and consequently the balance of HSC self-renewal and differentiation. We hypothesize that there are enhancers and suppressors of the proximal RUNX1 PAS, tightly controlling the relative amount of RUNX1a produced during hematopoiesis.To identify RUNX1 APA regulators, we first developed a strategy to monitor RUNX1 proximal PAS usage in vitro. We cloned alternative terminal exon 7a, which contains the proximal PAS, along with 500 bp of up and downstream flanking intron between the two exons of a split GFP reporter. When we introduce this fluorescent minigene reporter into blood cells, two transcripts can be produced: (1) the first GFP exon splices and polyadenylates RUNX1 exon 7a, producing no GFP (RUNX1a formation) or (2) the two GFP exons are spliced together, skipping the proximal PAS in exon 7a, producing GFP (RUNX1b/c formation). Since RUNX1b/c are much more highly expressed than RUNX1a throughout hematopoiesis, we observed a robust GFP signal when this reporter was transfected into MDS-L, K562, and U937 cell lines. RT-PCR analysis confirmed that both transcripts are produced, albeit at dramatically different levels. We can thus use GFP fluorescence as a read-out for RUNX1 proximal PAS usage.Next, we generated a stable, clonal MDS-L cell line expressing this RUNX1 exon 7a split GFP reporter. We used this reporter line to perform a CRISPR screen using an sgRNA library that is enriched for targeting RNA-binding proteins (RBPs). We infected cells with the library, cultured in puromycin to force sgRNA and Cas9 integration, waited three weeks to ensure gene knockout, and then used fluorescence-activated cell sorting (FACS) to isolate GFP low and high cell populations. GFP low cells contained sgRNAs targeting RUNX1a suppressors since these cells exhibited enhanced proximal PAS usage. Conversely, GFP high cells contained sgRNAs targeting RUNX1a enhancers since proximal PAS usage was further diminished. We identified 44 putative RUNX1a suppressors and 41 putative enhancers (p < 0.05).We then sought to validate the genes revealed in our CRISPR screen by using individual sgRNAs targeting a subset of the candidates. One confirmed suppressor is HNRNPA1, an RBP which is upregulated and leukemogenic in BCR/ABL chronic myelogenous leukemia (CML). In HNRNPA1 knockout cells, there was a 5-fold increase in relative RUNX1a and a corresponding decrease in RUNX1b/c levels by RT-qPCR. This effect was dose-dependent as heterozygous HNRNPA1 cells exhibited an intermediate 2.5-fold increase in RUNX1a mRNA. We verified this data by performing shRNA knockdown of HNRNPA1 in MDS-L and K562 cells, again observing a significant increase in relative RUNX1a levels. Interestingly, HNRNPA1 is highly expressed throughout the course of hematopoiesis, indicating that a cooperating protein helps facilitate this suppressive effect. Indeed, numerous HNRNPA1-interacting proteins were also identified in our screen and are the subject of current studies.In summary, we devised a fluorescent minigene reporter to monitor RUNX1 APA in vitro. Using this reporter, we performed a CRISPR RBP screen and identified HNRNPA1 as the founding member of a complex that suppresses RUNX1a production during hematopoiesis. HNRNPA1 and additional regulators represent novel proteins that have the potential to regulate HSC behavior by modifying the RUNX1 isoform ratio. DisclosuresNo relevant conflicts of interest to declare.

Clinical Relevance of Alternatively Spliced Isoform AML1-ETO9a in Adult Patients with t(8;21)(q22;q22.1) Acute Myeloid Leukemia (AML): A Study of the German-Austrian AML Study Group (AMLSG)

Background: AML with t(8;21)(q22;q22.1) defines a distinct disease entity within the WHO category ‘AML with recurrent genetic abnormalities’ and results in the formation of the AML1-ETO (AE) fusion transcript. Recent data showed that alternative splicing generates a truncated variant of AE, namely AML1-ETO9a (AE9a), containing an additional exon 9a. Co-expression of AE and AE9a was shown to induce a more aggressive leukemic phenotype with a rapid onset of AML in a retrovirally transduced mouse model (Yan et al., Nat Med 2006). However, the clinical relevance of the alternatively spliced AE9a isoform in adult patients (pts) with t(8;21) AML is not well determined yet.Aim: We sought to assess the incidence and prognostic significance of AE9a co-expression in the context of clinical and genetic factors in a large clinically well-annotated cohort of pts with t(8;21) AML.Methods: 129 pts were included based on the availability of a diagnostic bone marrow (BM) or peripheral blood (PB) sample; all pts were either enrolled on an AMLSG clinical trial protocol (n=93) or received standard intensive chemotherapy regimens. AE9a mRNA transcript levels (TL) were determined by quantitative reverse transcription PCR (qRT-PCR) using TaqMan technology. Co-expression of AE9a as a fraction of the full-length AE transcript was reported as AE9a / AE ratio (%). ABL gene expression was used as housekeeping control. Additional gene mutation data were available for KIT, FLT3 -ITD/TKD, NRA S, and ASXL2 .Results: The median age of the pts was 49 years (yrs; range, 18-75 yrs); the alternatively spliced isoform AE9a was identified in all pts; the AE9a / AE ratio (range 3%-77%, median 32%) did not significantly differ between BM (n=116, range 8%-77%, median 31%) and PB (n=13, range 3%-66%, median 52%). Median AE9a / AE ratio was not correlated with clinical features (sex, age, WBC, platelets, BM blasts) or gene mutations affecting KIT (p=0.10), FLT3 -ITD/TKD (p=0.77) or ASXL2 (p=1.00); pts with an additional NRAS mutation showed higher AE9a / AE ratios as compared to wild-type pts (median, 44% vs 31%) without reaching statistical significance (p=0.06). Using Cox regression analysis, AE9a / AE ratios were not associated with the clinical endpoints overall survival (OS), event-free survival (EFS) and cumulative incidence of relapse (CIR). The same remained true when performing univariate analyses comparing AE9a / AE ratios dichotomized at the median (AE9a / AE>median vs AE9a / AE≤median): AE9a / AE had no impact on 4-yr OS (71% vs 79%; p=0.37), 4-yr EFS (59% vs 67%; p=0.83), and 4-yr CIR (32% vs 33%; p=0.35). Correlation of AE9a / AE ratios according to quartiles of distribution did also not affect OS (p=0.14), EFS (p=0.67), and CIR (p=0.70). Finally, we evaluated a possible correlation between AE9a / AE ratio and NRAS or KIT mutations which frequently co-occur in this AML subtype. In a subgroup analysis according to KIT or NRAS mutation status, no significant differences with regard to the clinical endpoints OS, EFS and CIR were found between AE9a / AE>median and AE9a / AE≤median.Conclusion: Alternative splicing of the AML1-ETO fusion transcript is a common feature in pts with t(8;21) AML. This finding is also supported by a recent collaborative transcriptome study where novel high-throughput sequencing (HTS) technologies were applied (Faber et al., Nat Genet 2016). Using different explorative approaches, AE9a has no impact on clinical outcome which precludes its potential as a novel independent prognostic marker in this AML subtype.MA was supported by the Else Kröner-Forschungskolleg. DisclosuresKindler:Novartis: Membership on an entity's Board of Directors or advisory committees. Lübbert:Janssen-Cilag: Other: Travel Funding, Research Funding; Celgene: Other: Travel Funding; Ratiopharm: Other: Study drug valproic acid. Fiedler:Amgen, Pfizer: Research Funding; Amgen, ARIAD/Incyte: Membership on an entity's Board of Directors or advisory committees; Amgen: Patents & Royalties; Amgen, Gilead, GSO, Teva, Jazz Pharmaceuticals: Other: Support for meeting attendance. Götze:Celgene: Honoraria; Novartis: Honoraria; BMS: Honoraria; Abbvie: Honoraria. Griesshammer:Gilead: Consultancy, Honoraria, Speakers Bureau; Baxalta: Consultancy, Honoraria, Speakers Bureau; Sanofi: Honoraria, Speakers Bureau; AOP Orphan: Consultancy, Honoraria, Speakers Bureau; Shire: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau. Greil:Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel support, Research Funding; Novartis, Celgene: Research Funding; Takeda: Honoraria, Research Funding; BMS, Amgen: Honoraria. Döhner:Amgen: Honoraria; Celator: Honoraria; Pfizer: Research Funding; Seattle Genetics: Honoraria; Bristol Myers Squibb: Research Funding; Abbvie: Honoraria; Sunesis: Honoraria; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Agios: Honoraria; Boehringer Ingelheim: Research Funding; Astex Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Arog Pharmaceuticals: Honoraria, Research Funding. Döhner:Novartis: Honoraria, Research Funding.

Structural and Functional Peculiarities of Cytoplasmic Tropomyosin Isoforms, the Products of TPM1 and TPM4 Genes

Tropomyosin (Tpm) is one of the major protein partners of actin. Tpm molecules are α-helical coiled-coil protein dimers forming a continuous head-to-tail polymer along the actin filament. Human cells produce a large number of Tpm isoforms that are thought to play a significant role in determining actin cytoskeletal functions. Even though the role of these Tpm isoforms in different non-muscle cells is more or less studied in many laboratories, little is known about their structural and functional properties. In the present work, we have applied various methods to investigate the properties of five cytoplasmic Tpm isoforms (Tpm1.5, Tpm 1.6, Tpm1.7, Tpm1.12, and Tpm 4.2), which are the products of two different genes, TPM1 and TPM4, and also significantly differ by alternatively spliced exons: N-terminal exons 1a2b or 1b, internal exons 6a or 6b, and C-terminal exons 9a, 9c or 9d. Our results demonstrate that structural and functional properties of these Tpm isoforms are quite different depending on sequence variations in alternatively spliced regions of their molecules. The revealed differences can be important in further studies to explain why various Tpm isoforms interact uniquely with actin filaments, thus playing an important role in the organization and dynamics of the cytoskeleton.

Novel Mutations in FGFR1 Causing Idiopathic Hypogonadotropic Hypogonadism: A Cohort Study in Eastern Indian Population

Introduction: IHH is a clinically and genetically heterogeneous condition with more than 80 different mutations described in literature, some of the important being KAL-1 (anosmin), fibroblast growth factor receptor-1 (FGFR1) and fibroblast growth factor-8 (FGF8). Objective: To characterize the clinical findings and molecular analysis of FGFR1 (KAL2) genes in 10 unrelated patients of idiopathic hypogonadotropic hypogonadism with and without hyposmia/anosmia. Methods: After confirming the diagnosis of IHH from detailed history, clinical and biochemical examination the patients were subjected to MRI of pituitary hypothalamic area and olfactory bulb and tract. For genetic analysis PCR of 18 exons was performed on the genomic DNA. The PCR products were purified, sequenced and analysed using BLAST search from NCBI GenBank. Results: We diagnosed ten unrelated sporadic cases of IHH (9 male and 1 female). Out of 10 cases 6 (60%) had anosmia (KS) 4 had normosmia. A variable degree of pubertal development was observed, including various clinical abnormalities, such as micropenis, dental agenesis, skeletal deformity, seizure disorder and bimanual synkinesis. All the patients who had anosmia (KS) had either hypoplasia or aplasia of olfactory bulb and tract on MRI. Two KS patients had novel sporadic FGFR1 mutation, one had a mutation n.2370 C>A detected at exon 10 of FGFR1 gene and another had a mutation n.1875 GA>CA at exon 8C of FGFR1 gene. Discussion: Loss-of-function sequence variants in FGFR1, encoding the fibroblast growth factor receptor-1, account for one autosomal dominant form of KS. Pathogenic changes in KAL1 or FGFR1 have been detected in approximately 20% of the KS patients, which indicates that other responsible genes are still to be discovered. FGFR1 comprises an extracellular region consisting of three immunoglobulin-like domains (D1-D3), a single transmembrane helix, and an intracellular region containing the tyrosine kinase domain. D2, D3 and the short linker between them house all the determinants of ligand (FGF) binding and specificity. In addition, alternative splicing of exon 8, encoding the D3 C-terminal half, leads to two FGFR1 isoforms, FGFR1b (exon 8A) and FGFR1c (exon 8B), with different specificities towards FGF ligands. All the pathogenic changes in FGFR1 that have been reported in KS so far disrupt both isoforms of FGFR1 i.e. FGF1b and FGF1c, therefore leaving open the question of which isoform(s) is (are) required in the olfactory bulb morphogenetic process. We identified a new mutation in exon 8C (patient no 2). Conclusions: IHH and KS patients present with broad spectrum of clinical manifestations. We identified two novel mutations in FGFR1 gene in two unrelated sporadic cases of KS in the Indian population.

A preferential switch between placental GR exon 1 promoter variants in the presence of maternal asthma or inflammation upregulates GRα D isoforms

IntroductionThe human placenta expresses multiple glucocorticoid receptor (GR) isoforms that may be partially regulated by the untranslated 5’ exon 1 GR gene promoter region which consists of 9 different promoters and 13 splice variants. The objective of this study was to determine which GR exon 1 variants are expressed in the human placenta and relate these findings to GR mRNA and protein expression. MethodsPlacental extracts from pregnancies with or without the complication of maternal asthma and trophoblast cells exposed to an inflammatory challenge in vitro were examined using PCR and Western blot to measure GR exon 1 variants, GR splice variant mRNA and GR protein isoforms, respectively. ResultsAll 9 GR exon 1 variants were detectable in the human placenta and included GR exons 1A, 1B, 1C, 1D, 1E, 1F, 1H, 1I and 1J. In the presence of maternal asthma and a male fetus there was preferential expression of GR exon 1B, 1C, IF and 1J (KW-ANOVA, P < 0.05) which were positively correlated with GRα D3 protein isoform. In female placentae from pregnancies complicated by asthma there was no upregulation of any exon 1 variant (KW-ANOVA, P < 0.05). Exposure of BeWo trophoblast cell line to an inflammatory challenge, lipopolysaccharide, in vitro, resulted in preferential expression of GR exon 1B, 1D, 1E and 1H and associated with GRα-D1 protein upregulation. DiscussionThe preferential expression of different GR exon 1 promoters drive the upregulation of GRα D isoforms and contribute to glucocorticoid resistance observed in male placentae of pregnancies complicated by asthma.

Epstein-Barr virus nuclear antigen 3C (EBNA3C) interacts with the metabolism sensing C-terminal binding protein (CtBP) repressor to upregulate host genes.

Epstein-Barr virus (EBV) infection is associated with the development of specific types of lymphoma and some epithelial cancers. EBV infection of resting B-lymphocytes in vitro drives them to proliferate as lymphoblastoid cell lines (LCLs) and serves as a model for studying EBV lymphomagenesis. EBV nuclear antigen 3C (EBNA3C) is one of the genes required for LCL growth and previous work has suggested that suppression of the CDKN2A encoded tumor suppressor p16INK4A and possibly p14ARF is central to EBNA3C’s role in this growth transformation. To directly assess whether loss of p16 and/or p14 was sufficient to explain EBNA3C growth effects, we used CRISPR/Cas9 to disrupt specific CDKN2A exons in EBV transformed LCLs. Disruption of p16 specific exon 1α and the p16/p14 shared exon 2 were each sufficient to restore growth in the absence of EBNA3C. Using EBNA3C conditional LCLs knocked out for either exon 1α or 2, we identified EBNA3C induced and repressed genes. By trans-complementing with EBNA3C mutants, we determined specific genes that require EBNA3C interaction with RBPJ or CtBP for their regulation. Unexpectedly, interaction with the CtBP repressor was required not only for repression, but also for EBNA3C induction of many host genes. Contrary to previously proposed models, we found that EBNA3C does not recruit CtBP to the promoters of these genes. Instead, our results suggest that CtBP is bound to these promoters in the absence of EBNA3C and that EBNA3C interaction with CtBP interferes with the repressive function of CtBP, leading to EBNA3C mediated upregulation.

Restriction of an intron size en route to endothermy.

Ca2+-insensitive and -sensitive E1 subunits of the 2-oxoglutarate dehydrogenase complex (OGDHC) regulate tissue-specific NADH and ATP supply by mutually exclusive OGDH exons 4a and 4b. Here we show that their splicing is enforced by distant lariat branch points (dBPs) located near the 5′ splice site of the intervening intron. dBPs restrict the intron length and prevent transposon insertions, which can introduce or eliminate dBP competitors. The size restriction was imposed by a single dominant dBP in anamniotes that expanded into a conserved constellation of four dBP adenines in amniotes. The amniote clusters exhibit taxon-specific usage of individual dBPs, reflecting accessibility of their extended motifs within a stable RNA hairpin rather than U2 snRNA:dBP base-pairing. The dBP expansion took place in early terrestrial species and was followed by a uridine enrichment of large downstream polypyrimidine tracts in mammals. The dBP-protected megatracts permit reciprocal regulation of exon 4a and 4b by uridine-binding proteins, including TIA-1/TIAR and PUF60, which promote U1 and U2 snRNP recruitment to the 5′ splice site and BP, respectively, but do not significantly alter the relative dBP usage. We further show that codons for residues critically contributing to protein binding sites for Ca2+ and other divalent metals confer the exon inclusion order that mirrors the Irving-Williams affinity series, linking the evolution of auxiliary splicing motifs in exons to metallome constraints. Finally, we hypothesize that the dBP-driven selection for Ca2+-dependent ATP provision by E1 facilitated evolution of endothermy by optimizing the aerobic scope in target tissues.

Multiple competing RNA structures dynamically control alternative splicing in the human ATE1 gene.

The mammalian Ate1 gene encodes an arginyl transferase enzyme with tumor suppressor function that depends on the inclusion of one of the two mutually exclusive exons (MXE), exons 7a and 7b. We report that the molecular mechanism underlying MXE splicing in Ate1 involves five conserved regulatory intronic elements R1–R5, of which R1 and R4 compete for base pairing with R3, while R2 and R5 form an ultra-long-range RNA structure spanning 30 Kb. In minigenes, single and double mutations that disrupt base pairings in R1R3 and R3R4 lead to the loss of MXE splicing, while compensatory triple mutations that restore RNA structure revert splicing to that of the wild type. In the endogenous Ate1 pre-mRNA, blocking the competing base pairings by LNA/DNA mixmers complementary to R3 leads to the loss of MXE splicing, while the disruption of R2R5 interaction changes the ratio of MXE. That is, Ate1 splicing is controlled by two independent, dynamically interacting, and functionally distinct RNA structure modules. Exon 7a becomes more included in response to RNA Pol II slowdown, however it fails to do so when the ultra-long-range R2R5 interaction is disrupted, indicating that exon 7a/7b ratio depends on co-transcriptional RNA folding. In sum, these results demonstrate that splicing is coordinated both in time and in space over very long distances, and that the interaction of these components is mediated by RNA structure.

Crosstalk between vrille transcripts, proteins, and regulatory elements controlling circadian rhythms and development in Drosophila.

SummaryThe vrille (vri) gene encodes a transcriptional repressor required for Drosophila development as well as circadian behavior in adults. Alternate first exons produce vri transcripts predicted to produce a short VRI isoform during development and long VRI in adults. A vri mutant (vriΔ679) lacking long VRI transcripts is viable, confirming that short VRI is sufficient for developmental functions, yet behavioral rhythms in vriΔ679 flies persist, showing that short VRI is sufficient for clock output. E-box regulatory elements that drive rhythmic long VRI transcript expression are required for developmental expression of short VRI transcripts. Surprisingly, long VRI transcripts primarily produce short VRI in adults, apparently due to a poor Kozak sequence context, demonstrating that short VRI drives circadian behavior. Thus, E-box-driven long VRI transcripts primarily control circadian rhythms via short VRI, whereas the same E-boxes drive short VRI transcripts that control developmental functions using short VRI.

The Fuchs corneal dystrophy-associated CTG repeat expansion in the TCF4 gene affects transcription from its alternative promoters

The CTG trinucleotide repeat (TNR) expansion in Transcription factor 4 (TCF4) intron 3 is the main cause of Fuchs’ endothelial corneal dystrophy (FECD) and may confer an increased risk of developing bipolar disorder (BD). Usage of alternative 5′ exons for transcribing the human TCF4 gene results in numerous TCF4 transcripts which encode for at least 18 N-terminally different protein isoforms that vary in their function and transactivation capability. Here we studied the TCF4 region containing the CTG TNR and characterized the transcription initiation sites of the nearby downstream 5′ exons 4a, 4b and 4c. We demonstrate that these exons are linked to alternative promoters and show that the CTG TNR expansion decreases the activity of the nearby downstream TCF4 promoters in primary cultured neurons. We confirm this finding using two RNA sequencing (RNA-seq) datasets of corneal endothelium from FECD patients with expanded CTG TNR in the TCF4 gene. Furthermore, we report an increase in the expression of various other TCF4 transcripts in FECD, possibly indicating a compensatory mechanism. We conclude that the CTG TNR affects TCF4 expression in a transcript-specific manner both in neurons and in the cornea.

Association of mineralocorticoid receptor gene (NR3C2) hypermethylation in adult males with aggressive behavior

Aggressive behavior may have adaptive value under some environmental conditions. However, when it is extreme or improper, it may also lead to maladaptive results, seriously threatening human and social well-being. Aggressive behavior is a multifactorial disease, and the etiology is largely unknown. The stress-related hypothalamic-pituitary-adrenal (HPA) axis is a crucial system in the stress response that has emerged as a potential mechanism of aggressive behavior. The NR3C2 gene is an important regulator of the HPA axis: it is involved in regulating HPA axis activity and behavioral adaptation to stressors. Moreover, the epigenetic mechanism of DNA methylation has been suggested to mediate the development of aggressive behavior. However, the association between NR3C2 methylation and aggressive behavior has not been studied. In the present study, we assessed NR3C2 methylation (including three regions: promoter P1, exon 1α, and the sequence downstream of exon 1α) in peripheral blood DNA of adult males with aggressive behavior (n = 106) and healthy controls (n = 104). We found the NR3C2 gene to be associated with aggressive behavior, with hypermethylation detected in the entire aggressive behavior group as well as in the robbery subgroup compared to controls. In addition, analysis of methylation at 75 CpG sites revealed that some important CpG sites are associated with aggressive behavior. Our results suggest that HPA axis-related gene NR3C2 methylation is associated with aggressive behavior. These results lend support for using NR3C2 DNA methylation as a potential biomarker of aggressive behavior.

Alternative N-terminal regions of Drosophila myosin heavy chain II regulate communication of the purine binding loop with the essential light chain

We investigated the biochemical and biophysical properties of one of the four alternative exon-encoded regions within the Drosophila myosin catalytic domain. This region is encoded by alternative exons 3a and 3b and includes part of the N-terminal β-barrel. Chimeric myosin constructs (IFI-3a and EMB-3b) were generated by exchanging the exon 3-encoded areas between native slow embryonic body wall (EMB) and fast indirect flight muscle myosin isoforms (IFI). We found that this exchange alters the kinetic properties of the myosin S1 head. The ADP release rate (k-D ) in the absence of actin is completely reversed for each chimera compared with the native isoforms. Steady-state data also suggest a reciprocal shift, with basal and actin-activated ATPase activity of IFI-3a showing reduced values compared with wild-type (WT) IFI, whereas for EMB-3b these values are increased compared with wild-type (WT) EMB. In the presence of actin, ADP affinity (KAD ) is unchanged for IFI-3a, compared with IFI, but ADP affinity for EMB-3b is increased, compared with EMB, and shifted toward IFI values. ATP-induced dissociation of acto-S1 (K1k+2 ) is reduced for both exon 3 chimeras. Homology modeling, combined with a recently reported crystal structure for Drosophila EMB, indicates that the exon 3-encoded region in the myosin head is part of the communication pathway between the nucleotide binding pocket (purine binding loop) and the essential light chain, emphasizing an important role for this variable N-terminal domain in regulating actomyosin crossbridge kinetics, in particular with respect to the force-sensing properties of myosin isoforms.

Hypoxia-induced TGF-β-RBFOX2-ESRP1 axis regulates human MENA alternative splicing and promotes EMT in breast cancer.

Hypoxic microenvironment heralds epithelial–mesenchymal transition (EMT), invasion and metastasis in solid tumors. Deregulation of alternative splicing (AS) of several cancer-associated genes has been instrumental in hypoxia-induced EMT. Our study in breast cancer unveils a previously unreported mechanism underlying hypoxia-mediated AS of hMENA, a crucial cytoskeleton remodeler during EMT. We report that the hypoxia-driven depletion of splicing regulator ESRP1 leads to skipping of hMENA exon 11a producing a pro-metastatic isoform, hMENAΔ11a. The transcriptional repression of ESRP1 is mediated by SLUG, which gets stimulated via hypoxia-driven TGF-β signaling. Interestingly, RBFOX2, an otherwise RNA-binding protein, is also found to transcriptionally repress ESRP1 while interacting with SLUG. Similar to SLUG, RBFOX2 gets upregulated under hypoxia via TGF-β signaling. Notably, we found that the exosomal delivery of TGF-β contributes to the elevation of TGF-β signaling under hypoxia. Moreover, our results show that in addition to hMENA, hypoxia-induced TGF-β signaling contributes to global changes in AS of genes associated with EMT. Overall, our findings reveal a new paradigm of hypoxia-driven AS regulation of hMENA and insinuate important implications in therapeutics targeting EMT.

Tivantinib Decreases Hepatocyte Growth Factor-Induced BCRP Expression in Hepatocellular Carcinoma HepG2 Cells.

Tivantinib, a mesenchymal-epithelial transition factor (cMET) inhibitor, is a molecular targeting drug that kills hepatocellular carcinoma (HCC) cells. Tivantinib alone does not affect the overall survival of patients with HCC, and combination treatment with tivantinib and other therapies has not been evaluated. This study was conducted to clarify the effect of the tivantinib in regulating breast cancer therapy-resistant protein (BCRP), a key transporter of 5-fluorouracil (5-FU), and dihydropyridine dehydrogenase (DPYD), a major metabolic enzyme of 5-FU. To this end, cMET gene expression was determined by RT-PCR in HepG2 (human hepatoma) cells. The transcriptional start sites of BCRP were determined by 5'-rapid amplification of cDNA ends (5'-RACE). BCRP and DPYD mRNA levels were determined by real-time RT-PCR, and promoter activities were measured by dual-luciferase assays. Results show that hepatocyte growth factor (HGF) upregulated the mRNA level of BCRP, but not DPYD, in HepG2 cells. The upregulation of BCRP expression by HGF was down-regulated by tivantinib. We also identified two transcriptional start sites (E1α, E1β) in BCRP by 5'-RACE. The transcriptional activity of the region -287 to E1α of BCRP was upregulated by HGF, which was decreased by tivantinib, whereas activity of the region -297 to E1βo f BCRP was not affected by tivantinib. Therefore, tivantinib regulates BCRP expression upstream of exon 1α. Combination treatment of tivantinib and 5-FU should be further evaluated for HCC therapy.