Exon 1B Research Articles

Rescue of Scn5a mis-splicing does not improve the structural and functional heart defects of a DM1 heart mouse model.

Myotonic Dystrophy Type 1 (DM1) is an autosomal dominant multisystemic disorder for which cardiac features, including conduction delays and arrhythmias, are the second leading cause of disease mortality. DM1 is caused by expanded CTG repeats in the 3' untranslated region of the DMPK gene. Transcription of the expanded DMPK allele produces mRNAs containing long tracts of CUG repeats, which sequester the Muscleblind-Like family of RNA binding proteins, leading to their loss-of-function and the dysregulation of alternative splicing. A well-characterized mis-regulated splicing event in the DM1 heart is the increased inclusion of SCN5A exon 6A rather than the mutually exclusive exon 6B that normally predominates in adult heart. As previous work showed that forced inclusion of Scn5a exon 6A in mice recapitulates cardiac DM1 phenotypes, we tested whether rescue of Scn5a mis-splicing would improve the cardiac phenotypes in a DM1 heart mouse model. We generated mice lacking Scn5a exon 6A to force the expression of the adult SCN5A isoform including exon 6B and crossed these mice to our previously established CUG960 DM1 heart mouse model. We showed that correction Scn5a mis-splicing does not improve the DM1 heart conduction delays and structural changes induced by CUG repeat RNA expression. Interestingly, we found that in addition to Scn5a mis-splicing, Scn5a expression is reduced in heart tissues of CUG960 mice and DM1-affected individuals. These data indicate that Scn5a mis-splicing is not the sole driver of DM1 heart deficits and suggest a potential role for reduced Scn5a expression in DM1 cardiac disease.

BRD2 and BRD3 genes independently evolved RNA structures to control unproductive splicing.

The mammalian BRD2 and BRD3 genes encode structurally related proteins from the bromodomain and extraterminal domain protein family. The expression of BRD2 is regulated by unproductive splicing upon inclusion of exon 3b, which is located in the region encoding a bromodomain. Bioinformatic analysis indicated that BRD2 exon 3b inclusion is controlled by a pair of conserved complementary regions (PCCR) located in the flanking introns. Furthermore, we identified a highly conserved element encoding a cryptic poison exon 5b and a previously unknown PCCR in the intron between exons 5 and 6 of BRD3, however, outside of the homologous bromodomain. Minigene mutagenesis and blockage of RNA structure by antisense oligonucleotides demonstrated that RNA structure controls the rate of inclusion of poison exons. The patterns of BRD2 and BRD3 expression and splicing show downregulation upon inclusion of poison exons, which become skipped in response to transcription elongation slowdown, further confirming a role of PCCRs in unproductive splicing regulation. We conclude that BRD2 and BRD3 independently acquired poison exons and RNA structures to dynamically control unproductive splicing. This study describes a convergent evolution of regulatory unproductive splicing mechanisms in these genes, providing implications for selective modulation of their expression in therapeutic applications.

PSEN2 poison exon inclusion is common across brain regions in late‐onset Alzheimer’s disease

AbstractBackgroundPathogenic variants in APP, PSEN1 and PSEN2 can cause early‐onset familial Alzheimer’s disease (AD). The molecular events leading to AD in individuals with late‐onset sporadic AD are still unclear even though they share the same clinical and pathological features of AD.MethodUsing targeted PacBio long read isoform sequencing (Iso‐Seq), we recently reported the characterization of tens of thousands of complete PSEN1 and PSEN2 transcripts in the prefrontal cortex of individuals with sporadic AD, familial AD (carrying PSEN1 and PSEN2 pathogenic variants), and controls.ResultWe found several alternative splicing differences in PSEN2 that are significantly elevated in sporadic AD including inclusion of a human‐specific cryptic exon present in intron 9 of PSEN2 (termed exon 9B) present in 11.8% of full‐length reads in sporadic AD samples relative to 1.9% of controls (p = 0.002) and 3.4% of PSEN variant carriers (p = 0.005) as well as a 77bp intron retention product prior to exon 6. These PSEN2 splice products are predicted to generate a prematurely truncated PSEN2 protein and were confirmed in an independent RNA‐seq dataset of ∼80 AD cases and ∼80 controls from cerebellum tissue (Course et al., Brain 2022). To extend these findings we analyzed PSEN2 exon 9B isoform levels in parietal and temporal lobe tissue samples. We found exon 9B was significantly elevated in sporadic AD relative to controls in the parietal lobe (p = 0.02) as well as the temporal lobe (p = <0.001). However, levels of exon 9B were independent of TDP‐43 pathology or MSUT2 protein levels, which is intriguing given their roles as RNA binding proteins implicated in AD. We are working to establish the consequence of exon 9B inclusion in APP processing, Aβ and PSEN2 protein products in addition to possible cell autonomous roles for PSEN2 splice variants in microglia by leveraging single cell sequencing data.ConclusionCollectively, our findings expand our understanding of PSEN2 splice variants in the development of late‐onset sporadic AD, which can point to novel therapeutic strategies for AD.

Truncated Dyrk1A aggravates neuronal apoptosis by inhibiting ASF-mediated Bcl-x exon 2b inclusion.

Aggravated neuronal loss, caused mainly by neuronal apoptosis, is observed in the brain of patients with Alzheimer's disease (AD) and animal models of AD. A truncated form of Dual-specific and tyrosine phosphorylation-regulated protein kinase 1A (Dyrk1A) plays a vital role in AD pathogenesis. Downregulation of anti-apoptotic Bcl-xL is tightly correlated with neuronal loss in AD. However, the molecular regulation of neuronal apoptosis and Bcl-x expression by Dyrk1A in AD remains largely elusive. Here, we aimed to explore the role and molecular mechanism of Dyrk1A in apoptosis. Cell Counting Kit-8 (CCK8), flow cytometry, and TdT-mediated dUTP Nick-End Labeling (TUNEL) were used to check apoptosis. The cells, transfected with Dyrk1A or/and ASF with Bcl-x minigene, were used to assay Bcl-x expression by RT-PCR and Western blots. Co-immunoprecipitation, autoradiography, and immunofluorescence were conducted to check the interaction of ASF and Dyrk1A. Gene set enrichment analysis (GSEA) of apoptosis-related genes was performed in mice overexpressing Dyrk1A (TgDyrk1A) and AD model 5xFAD mice. Dyrk1A promoted Bcl-xS expression and apoptosis. Splicing factor ASF promoted Bcl-x exon 2b inclusion, leading to increased Bcl-xL expression. Dyrk1A suppressed ASF-mediated Bcl-x exon 2b inclusion via phosphorylation. The C-terminus deletion of Dyrk1A facilitated its binding and kinase activity to ASF. Moreover, Dyrk1a1-483 further suppressed the ASF-mediated Bcl-x exon 2b inclusion and aggravated apoptosis. The truncated Dyrk1A, increased Bcl-xS, and enrichment of apoptosis-related genes was observed in the brain of 5xFAD mice. We speculate that increased Dyrk1A and truncated Dyrk1A may aggravate neuronal apoptosis by decreasing the ratio of Bcl-xL/Bcl-xS via phosphorylating ASF in AD.

Identification of a novel mitochondria-localized LKB1 variant required for the regulation of the oxidative stress response

The tumor suppressor Liver Kinase B1 (LKB1) is a multifunctional serine/threonine protein kinase that regulates cell metabolism, polarity, and growth and is associated with Peutz-Jeghers Syndrome and cancer predisposition. The LKB1 gene comprises 10 exons and 9 introns. Three spliced LKB1 variants have been documented, and they reside mainly in the cytoplasm, although two possess a nuclear-localization sequence (NLS) and are able to shuttle into the nucleus. Here, we report the identification of a fourth and novel LKB1 isoform that is, interestingly, targeted to the mitochondria. We show that this mitochondria-localized LKB1 (mLKB1) is generated from alternative splicing in the 5' region of the transcript and translated from an alternative initiation codon encoded by a previously unknown exon 1b (131bp) hidden within the long intron 1 of LKB1 gene. We found by replacing the N-terminal NLS of the canonical LKB1 isoform, the N-terminus of the alternatively spliced mLKB1 variant encodes a mitochondrial transit peptide that allows it to localize to the mitochondria. We further demonstrate that mLKB1 colocalizes histologically with mitochondria-resident ATP Synthase and NAD-dependent deacetylase sirtuin-3, mitochondrial (SIRT3) and that its expression is rapidly and transiently upregulated by oxidative stress. We conclude that this novel LKB1 isoform, mLKB1, plays a critical role in regulating mitochondrial metabolic activity and oxidative stress response.

Abstract A065: Serum PVT1 exons 4A and 4B copy numbers are better predictors of high-grade prostate cancer in Black and Hispanic men than serum prostate specific antigen

Abstract Background Men of African ancestry (moAA) experience the highest incidence and mortality rates of prostate Cancer (PCa) in the United States, where 1 in 8 men are expected to be diagnosed with the disease in their lifetime. Yet, most biomarkers used in PCa screening today, which are based on prostate specific antigen (PSA), are developed and validated in predominantly White populations with weak or nonexistent validation in moAA, making moAA more likely to be subjected to unnecessary prostate biopsies. To reduce this disparity in PCa detection and management, there is a critical need for new biomarkers with demonstrated usefulness in moAA. We recently showed that plasmacytoma variant translocation 1 (PVT1), exons 4A, 4B, and 9 are overexpressed in the prostate tissues and detected in higher copy numbers in serum of moAA with PCa. In this study, we investigated the predictive power of these potential biomarkers in detecting high-grade PCa across different racial populations. Methodology Forty serum samples from White, Hispanic, and Black men, 50% of whom had PCa with 25% showing high-grade PCa, were analyzed to obtain copy numbers for PVT1 exons 4A, 4B, and 9. Seven logistic regression models were developed to predict PCa using individual PTV1 biomarkers, all pairwise combinations of biomarkers and all three biomarkers. The median area under the receiver operator characteristic curve (AUC) was used to measure the predictive accuracy of each model after a repeated, stratified k-fold cross validation. Model accuracy was first evaluated using data from all races and then for just the Black and Hispanic subpopulation. Our best performing models were compared with PSA as a predictor of PCa. We also evaluated the predictive accuracy of composite models that combined our biomarkers with PSA. Results In predicting high-grade PCa for all races, PSA had a median AUC of 0.58. All our PVT1 biomarkers had an AUC greater than 0.64, which was better than PSA. When PSA and PVT1 exon 4B were combined, the median AUC of PSA increased to 0.67, other PTV1 biomarkers did not improve the AUC of PSA. For the Black and Hispanic subpopulation, PVT1 exon 4A achieved the highest median AUC of 0.92 which was significantly higher than the median AUC of 0.33 achieved with PSA. The model with Exon 4A and Exon 4B as well as all three biomarkers achieved perfect discrimination (AUC of 1.0). We were also able to obtain perfect discrimination when PVT1 exon 4A was combined with PSA (AUC: 1.0). Conclusion Our results show that PVT1 exon 4A is more accurate than PSA in predicting high-grade PCa in Black and Hispanic subpopulations, achieving perfect discrimination when combined with PSA. Significant improvement in PSA’s predictability is observed in all races when combined with PTV1 exon 4B. Thus, PVT1 exon 4A and PVT1 exon 4B may hold the key to reducing disparity in high-grade PCa detection among moAA while improving the predictive accuracy of PSA for the general population. Citation Format: Emmanuel Owusu Asante-Asamani, Dinuka S.H. Sewandi De Silva, Gargi Pal, Michael Liss, Robin Leach, Olorunseun O. Ogunwobi. Serum PVT1 exons 4A and 4B copy numbers are better predictors of high-grade prostate cancer in Black and Hispanic men than serum prostate specific antigen [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A065.

Retraction Note: Hypermethylation of the 5′ CpG island of the p14ARF flanking exon 1β in human colorectal cancer displaying a restricted pattern of p53 overexpression concomitant with increased MDM2 expression

Retraction Note: Hypermethylation of the 5′ CpG island of the p14ARF flanking exon 1β in human colorectal cancer displaying a restricted pattern of p53 overexpression concomitant with increased MDM2 expression

TLN1 contains a cancer-associated cassette exon that alters talin-1 mechanosensitivity.

Talin-1 is the core mechanosensitive adapter protein linking integrins to the cytoskeleton. The TLN1 gene is comprised of 57 exons that encode the 2,541 amino acid TLN1 protein. TLN1 was previously considered to be expressed as a single isoform. However, through differential pre-mRNA splicing analysis, we discovered a cancer-enriched, non-annotated 51-nucleotide exon in TLN1 between exons 17 and 18, which we refer to as exon 17b. TLN1 is comprised of an N-terminal FERM domain, linked to 13 force-dependent switch domains, R1-R13. Inclusion of exon 17b introduces an in-frame insertion of 17 amino acids immediately after Gln665 in the region between R1 and R2 which lowers the force required to open the R1-R2 switches potentially altering downstream mechanotransduction. Biochemical analysis of this isoform revealed enhanced vinculin binding, and cells expressing this variant show altered adhesion dynamics and motility. Finally, we showed that the TGF-β/SMAD3 signaling pathway regulates this isoform switch. Future studies will need to consider the balance of these two TLN1 isoforms.

Abstract C060: Investigating the clinical relevance in prostate cancer of the serum biomarkers PVT1 exons 4A, 4B and 9 across risk levels and ethnicity/race

Abstract Chromosome 8q24 is of particular importance for cancer susceptibility. Located in this region is the Plasmacytoma Variant Translocation 1 (PVT1) gene, a long noncoding RNA that has been implicated in multiple cancers including prostate cancer. Amplification of the PVT1 gene locus is a common event in many malignant diseases and is associated with poor clinical outcomes. The pioneering role of PVT1, and its alternatively spliced transcripts, as a cancer biomarker is progressively becoming established. We have demonstrated that copy numbers of PVT1 exons 4A, 4B and 9 is quantifiable in cancer cells, tissue, and serum from cancer patients. In this study, we assessed clinically annotated serum samples from 40 prostate cancer patients to investigate the clinical relevance of PVT1 exons 4A, 4B, and 9 as a biomarker across cancer risk levels and ethnicity/race. Explorative data analysis for the development of composite score for prostate cancer was performed using Kruskal-Wallis Rank Sum Test. We observed significantly higher copy numbers of PVT1 exons 4B and 9 across all races (White, Black and Hispanic) and Blacks and Hispanics when compared to the control. Additionally, using a 3-level cancer risk rating assessment in which 0 = healthy, 1 = low risk and 2 = high risk, we observed that PVT1 exon 9 may distinguish between cancerous and noncancerous cases across all races, but may not help distinguish between indolent and aggressive cancer cases. Notably, PVT1 exon 4B may help distinguish between indolent and aggressive cancer cases for Blacks and Hispanics. The results of this study suggest that using PVT1 exon 4B or 9 may identify cancer regardless of ethnicity/race, and that utilization of serum PVT1 exon 4B copy number may help distinguish between indolent and aggressive prostate cancer in Blacks and Hispanics. Citation Format: Fayola Levine, Emmanuel Asante-Asamani, Gargi Pal, Michael Liss, Olorunseun Ogunwobi. Investigating the clinical relevance in prostate cancer of the serum biomarkers PVT1 exons 4A, 4B and 9 across risk levels and ethnicity/race [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr C060.

The anterior gradient homologue 2 (AGR2) co-localises with the glucose-regulated protein 78 (GRP78) in cancer stem cells, and is critical for the survival and drug resistance of recurrent glioblastoma: in situ and in vitro analyses.

Glioblastomas (GBs) are characterised as one of the most aggressive primary central nervous system tumours (CNSTs). Single-cell sequencing analysis identified the presence of a highly heterogeneous population of cancer stem cells (CSCs). The proteins anterior gradient homologue 2 (AGR2) and glucose-regulated protein 78 (GRP78) are known to play critical roles in regulating unfolded protein response (UPR) machinery. The UPR machinery influences cell survival, migration, invasion and drug resistance. Hence, we investigated the role of AGR2 in drug-resistant recurrent glioblastoma cells. Immunofluorescence, biological assessments and whole exome sequencing analyses were completed under in situ and in vitro conditions. Cells were treated with CNSTs clinical/preclinical drugs taxol, cisplatin, irinotecan, MCK8866, etoposide, and temozolomide, then resistant cells were analysed for the expression of AGR2. AGR2 was repressed using single and double siRNA transfections and combined with either temozolomide or irinotecan. Genomic and biological characterisations of the AGR2-expressed Jed66_GB and Jed41_GB recurrent glioblastoma tissues and cell lines showed features consistent with glioblastoma. Immunofluorescence data indicated that AGR2 co-localised with the UPR marker GRP78 in both the tissue and their corresponding primary cell lines. AGR2 and GRP78 were highly expressed in glioblastoma CSCs. Following treatment with the aforementioned drugs, all drug-surviving cells showed high expression of AGR2. Prolonged siRNA repression of a particular region in AGR2 exon 2 reduced AGR2 protein expression and led to lower cell densities in both cell lines. Co-treatments using AGR2 exon 2B siRNA in conjunction with temozolomide or irinotecan had partially synergistic effects. The slight reduction of AGR2 expression increased nuclear Caspase-3 activation in both cell lines and caused multinucleation in the Jed66_GB cell line. AGR2 is highly expressed in UPR-active CSCs and drug-resistant GB cells, and its repression leads to apoptosis, via multiple pathways.

A recurrent de novo splice site variant involving DNM1 exon 10a causes developmental and epileptic encephalopathy through a dominant-negative mechanism

Heterozygous pathogenic variants in DNM1 cause developmental and epileptic encephalopathy (DEE) as a result of a dominant-negative mechanism impeding vesicular fission. Thus far, pathogenic variants in DNM1 have been studied with a canonical transcript that includes the alternatively spliced exon 10b. However, after performing RNA sequencing in 39 pediatric brain samples, we find the primary transcript expressed in the brain includes the downstream exon 10a instead. Using this information, we evaluated genotype-phenotype correlations of variants affecting exon 10a and identified a cohort of eleven previously unreported individuals. Eight individuals harbor a recurrent de novo splice site variant, c.1197-8G>A (GenBank: NM_001288739.1), which affects exon 10a and leads to DEE consistent with the classical DNM1 phenotype. We find this splice site variant leads to disease through an unexpected dominant-negative mechanism. Functional testing reveals an in-frame upstream splice acceptor causing insertion of two amino acids predicted to impair oligomerization-dependent activity. This is supported by neuropathological samples showing accumulation of enlarged synaptic vesicles adherent to the plasma membrane consistent with impaired vesicular fission. Two additional individuals with missense variants affecting exon 10a, p.Arg399Trp and p.Gly401Asp, had a similar DEE phenotype. In contrast, one individual with a missense variant affecting exon 10b, p.Pro405Leu, which is less expressed in the brain, had a correspondingly less severe presentation. Thus, we implicate variants affecting exon 10a as causing the severe DEE typically associated with DNM1-related disorders. We highlight the importance of considering relevant isoforms for disease-causing variants as well as the possibility of splice site variants acting through a dominant-negative mechanism.

A lysine residue from an extracellular turret switches the ion preference in a Cav3 T-Type channel from calcium to sodium ions

Cav3 T-type calcium channels from great pond snail Lymnaea stagnalis have a selectivity-filter ring of five acidic residues, EE(D)DD. Splice variants with exons 12b or 12a spanning the extracellular loop between the outer helix IIS5 and membrane-descending pore helix IIP1 (IIS5-P1) in Domain II of the pore module possess calcium selectivity or dominant sodium permeability, respectively. Here, we use AlphaFold2 neural network software to predict that a lysine residue in exon 12a is salt-bridged to the aspartate residue immediately C terminal to the second-domain glutamate in the selectivity filter. Exon 12b has a similar folding but with an alanine residue in place of lysine in exon 12a. We express LCav3 channels with mutated exons Ala-12b-Lys and Lys-12a-Ala and demonstrate that they switch the ion preference to high sodium permeability and calcium selectivity, respectively. We propose that in the calcium-selective variants, a calcium ion chelated between Domain II selectivity-filter glutamate and aspartate is knocked-out by the incoming calcium ion in the process of calcium permeation, whereas sodium ions are repelled. The aspartate is neutralized by the lysine residue in the sodium-permeant variants, allowing for sodium permeation through the selectivity-filter ring of four negatively charged residues akin to the prokaryotic sodium channels with four glutamates in the selectivity filter. The evolutionary adaptation in invertebrate LCav3 channels highlight the involvement of a key, ubiquitous aspartate, "a calcium beacon" of sorts in the outer pore of Domain II, as determinative for the calcium ion preference over sodium ions through eukaryotic Cav1, Cav2, and Cav3 channels.

Maturation of hiPSC-derived cardiomyocytes in cardiac microtissues promotes adult alternative splicing of sodium channel revealing mutation effects associated with cardiac arrhythmia

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): Marie Skłodowska-Curie Actions - Individual Fellowship Background The cardiac sodium channel encoded by SCN5A gene undergoes fetal-to-adult isoform switch postnatally, regulated by alternative splicing. Various mutations in SCN5A associated with severe cardiac arrhythmia have been previously studied using cardiomyocytes derived from human induced pluripotent stem-cells (hiPSC-CMs), demonstrating the importance of a human cardiac cellular context for such analysis. Mutations in the adult isoform of SCN5A are however of difficult evaluation in hiPSC-CMs, because of their typical immature/fetal-like characteristics. Combining hiPSC-CMs in 3-dimensional microtissues together with hiPSC-derived cardiac fibroblasts and endothelial cells induced maturation in gene expression and functional properties, including electrophysiology. Purpose We investigated whether the maturation promoted by the microtissue system could reveal the functional phenotype of a mutation in the adult isoform of SCN5A. Methods We derived hiPSC-CMs from a cardiac arrhythmia patient carrying two compound mutations in SCN5A: p.W156X in exon 4 and p.R225W in the adult splicing variant of exon 6 (exon 6B). Using CRISPR/Cas9, we corrected exon 4 mutation to investigate specific effects of exon 6B mutation on sodium current by single-cell patch clamp. HiPSC-CMs were matured in tricell-type cardiac microtissues and analysed after dissociation. The mechanism underlying exon 6B expression was investigated by overexpressing or knocking-out the alternative splicing regulator MBNL1 in hiPSC-CMs. Results ddPCR analysis showed low expression of exon 6B-containing transcripts in hiPSC-CMs, accompanied by no effect of exon 6B mutation on the sodium current. Instead, maturing hiPSC-CMs in cardiac microtissues promoted SCN5A exon 6B expression and revealed the contribution of both SCN5A mutations to the diseased cellular electrophysiology. MBNL1 was upregulated in microtissues and indeed its overexpression was sufficient to increase exon 6B levels in hiPSC-CMs, while lack of MBNL1 prevented the switch of SCN5A splicing isoforms. Conclusions Our data demonstrate that a maturation of hiPSC-CMs is required to express the adult SCN5A isoform. This was achieved in the microtissue system via upregulation of MBNL1, and allowed to reveal SCN5A mutation contributions, by dissecting changes in ionic current that cause adult arrhythmic disease phenotypes in humans.

Dysregulation of Skeletal Muscle Mitochondrial Turnover and Dynamics Occurs During Late Symptomatic Stages of Spinal Muscular Atrophy

Spinal muscular atrophy (SMA) is a debilitating neuromuscular disorder caused by a mutation in the survival motor neuron 1 (SMN1) gene and it is the leading genetic cause of infant mortality. Recently approved genetic therapies designed to augment full length SMN protein in the central nervous system fail to ameliorate abnormal skeletal muscle features, which strongly suggests an important role for SMN protein in skeletal muscle homeostasis. This study aimed to characterize key modifiers of skeletal muscle mitochondrial turnover and dynamics during disease progression in a pre‐clinical murine model of SMA in vivo. Additionally, we investigated the effects of a single dose of exercise on these mitochondrial biology‐regulating pathways in the skeletal muscle of SMA mice. Muscle samples were collected from wild‐type (WT) and Smn2B/‐ (SMA) mice at postnatal day 9 (P9), P13, and P21 to examine skeletal muscle‐specific disease progression. Mitochondrial content did not differ between genotypes at all timepoints as evident by similar levels of mitochondrial oxidative phosphorylation proteins, succinate dehydrogenase staining, and citrate synthase content. However, mRNA content of key genes responsible for regulating mitochondrial quality such as nuclear respiratory factor 2, mitochondrial transcription factor A, and p53 were significantly higher in SMA mice versus WT at P21. Additionally, we observed elevated mitochondrial fission activity as indicated by a 2‐fold increase (p < 0.05) in dynamin related protein 1 (DRP1) activation. Concomitantly, the expression of several mitophagy proteins including BLC2 interacting protein 3, parkin, and PTEN‐induced kinase 1 were significantly increased by 2.9‐, 2.7‐, and 2‐fold, respectively, compared to WT animals at P21. Interestingly, we observed blunted (p < 0.05) inclusion of optic atrophy 1 exon 4b, a key exon in mitochondrial biogenesis, between WT and SMA mice at P21. Acute exercise significantly increased the inhibition of DRP1‐mediated mitochondrial fission activity in the muscles of SMA mice relative to sedentary SMA mice. However, a single exercise dose failed to alter the expression of mitophagy proteins up to three hours after running. Our data demonstrate that skeletal muscle mitochondrial health is compromised in SMA mice due to elevated fission and mitophagy processes. Furthermore, SMA mice display aberrant alternative splicing of Opa1 transcripts that may in turn hinder mitochondrial biogenesis in later disease stages. We also highlight that an acute bout of treadmill running elicits pro‐fusion signaling to potentially improve the mitochondrial reticulum. Collectively, this study is the first to reveal mitochondrial dysfunction in SMA skeletal muscle and outlines signalling associated with mitochondrial plasticity following a single dose of exercise.

HIF1α and NFκB Regulate Mineralocorticoid Receptor Gene Expression in Aging Human Vascular Smooth Muscle Cells

BackgroundMineralocorticoid receptor (MR) activation drives aging‐associated cardiovascular diseases including hypertension, vascular stiffness and heart failure. Vascular MR expression increases with age and drives the aging phenotype but the mechanism of MR gene regulation is unknown. The MR has two mRNA isoforms (1α and 1β) that differ in only the 5’‐untranslated region (exon 1α or 1β) and in the promoter sequence (P1 or P2, respectively). Thus, we investigated the transcriptional mechanisms regulating MR gene expression in aging human smooth muscle cells (SMC).MethodsMR mRNA and protein expression were quantified in primary human aortic SMC (HASMC) and human aortic tissue comparing adult (average age 40s) and aged (average age 70s) male and female donors and comparing adult HASMC or Pac1 (rat SMC line) treated with H2O2 to induce aging. MR promoter activity was tested by luciferase reporter assays. In silico analysis of the MR promoters identified putative transcription factor (TF) binding sites and inhibitor studies and chromatin immunoprecipitations (ChIP) were performed to validate the predicted regulatory mechanisms.ResultsIncreased SMC donor age or H2O2 treatment were associated with significantly increased expression of both of the MR mRNA isoforms, MR protein level, and activity of both MR promoters in HASMCs. The hypoxia‐inducible TF, HIF1α, and the inflammatory TF, NFκB, were predicted in silico to bind to the MR promoters and found to increase significantly with age in HASMCs. HIF1α was induced by H2O2 in HASMCs and this was associated with significant enrichment of HIF1α binding specifically at a site in the 1a exon, near the MR P1 promoter. HIF1α inhibition attenuated expression specifically of the MR‐1α mRNA isoform resulting in decreased MR protein in HASMC. NFκB also increased with age in HASMCs and contributes to MR expression. In response to H2O2,NFκB binding is enriched at to two different binding sites, one each in the P1 and P2 promoters and NFκB inhibition decreased expression of both MR mRNA isoforms (1α and 1β) and MR protein in SMC. Finally, HIF1α and NFκB expression each positively correlated with donor age and with MR expression (p<0.0001) in human aortic tissue from males and females.ConclusionThe MR gene is regulated by the inflammatory transcription factor NFkB and by the hypoxia/oxidative stress‐induced TF HIF1α, to increase MR expression in SMC of the aging human vasculature. This molecular insight may explain enhanced MR activity in cardiovascular aging and could potentially contribute to other cardiovascular conditions driven by oxidative stress and inflammation.

Four cases of gastric adenocarcinoma and proximal polyposis of the stomach treated by robotic total gastrectomy

BackgroundGastric adenocarcinoma and proximal polyposis of the stomach (GAPPS) is a rare disease and characterized by a unique point mutation in the promoter 1B region of the adenomatous polyposis coli (APC) gene. There are two aims in surgery for GAPPS; the first is prophylactic gastrectomy, and the second is excising concurrent cancer. We performed robotic total gastrectomy (RTG) for four cases of GAPPS.Case presentationCase 1 was a woman in her 40 s whose sister had died from gastric cancer. Mutational analysis revealed mutation of APC exon 1B. We performed prophylactic gastrectomy. Case 2 was a woman in her 30 s who had a mutation of APC exon 1B, and preoperative biopsy revealed suspected adenocarcinoma. Case 3 was a woman in her 40 s who was diagnosed with gastric cancer with multiple polyps in the stomach and a mutation of APC exon 1B. Case 4 was a woman in her 20 s in whom biopsy revealed low-grade dysplasia of a raised lesion. She had a mutation in APC exon 1B. We performed RTG with D1 + lymphadenectomy in all patients, and there were no intraoperative complications.ConclusionsPatients with GAPPS are mainly followed regularly with repeat biopsy, and tumors are detected in an early stage. As the safety of robotic surgery for the early gastric cancer is reported, RTG is an option for these patients. This is the first report of RTG for GAPPS patients.

Maturation of hiPSC-derived cardiomyocytes promotes adult alternative splicing of SCN5A and reveals changes in sodium current associated with cardiac arrhythmia.

Human-induced pluripotent stem cell-cardiomyocytes (hiPSC-CMs) are widely used to study arrhythmia-associated mutations in ion channels. Among these, the cardiac sodium channel SCN5A undergoes foetal-to-adult isoform switching around birth. Conventional hiPSC-CM cultures, which are phenotypically foetal, have thus far been unable to capture mutations in adult gene isoforms. Here, we investigated whether tri-cellular cross-talk in a three-dimensional (3D) cardiac microtissue (MT) promoted post-natal SCN5A maturation in hiPSC-CMs. We derived patient hiPSC-CMs carrying compound mutations in the adult SCN5A exon 6B and exon 4. Electrophysiological properties of patient hiPSC-CMs in monolayer were not altered by the exon 6B mutation compared with isogenic controls since it is not expressed; further, CRISPR/Cas9-mediated excision of the foetal exon 6A did not promote adult SCN5A expression. However, when hiPSC-CMs were matured in 3D cardiac MTs, SCN5A underwent isoform switch and the functional consequences of the mutation located in exon 6B were revealed. Up-regulation of the splicing factor muscleblind-like protein 1 (MBNL1) drove SCN5A post-natal maturation in microtissues since its overexpression in hiPSC-CMs was sufficient to promote exon 6B inclusion, whilst knocking-out MBNL1 failed to foster isoform switch. Our study shows that (i) the tri-cellular cardiac microtissues promote post-natal SCN5A isoform switch in hiPSC-CMs, (ii) adult splicing of SCN5A is driven by MBNL1 in these tissues, and (iii) this model can be used for examining post-natal cardiac arrhythmias due to mutations in the exon 6B. The cardiac sodium channel is essential for conducting the electrical impulse in the heart. Postnatal alternative splicing regulation causes mutual exclusive inclusion of fetal or adult exons of the corresponding gene, SCN5A. Typically, immature hiPSCCMs fall short in studying the effect of mutations located in the adult exon. We describe here that an innovative tri-cellular three-dimensional cardiac microtissue culture promotes hiPSC-CMs maturation through upregulation of MBNL1, thus revealing the effect of a pathogenic genetic variant located in the SCN5A adult exon. These results help advancing the use of hiPSC-CMs in studying adult heart disease and for developing personalized medicine applications.

Altered HIV-1 mRNA Splicing Due to Drug-Resistance-Associated Mutations in Exon 2/2b

The underlying molecular mechanism and their general effect on the replication capacity of HIV 1 drug-resistance-associated mutations is often poorly understood. To elucidate the effect of two such mutations located in a region with a high density of spicing regulatory elements on the HIV-1-splicing outcome, bioinformatic predictions were combined with transfection and infection experiments. Results show that the previously described R263K drug-resistance-associated integrase mutation has additionally a severe effect on the ESE2b splicing regulatory element (SRE) in exon 2b, which causes loss of SD2b recognition. This was confirmed by an R263R silent mutation with a similar predicted effect on the exon 2b SRE. In contrast, a V260I mutation and its silent counterpart with a lower effect on ESS2b did not exhibit any differences in the splicing pattern. Since HIV-1 highly relies on a balanced splicing reaction, changes in the splicing outcome can contribute to changes in viral replication and might add to the effect of escape mutations toward antiviral drugs. Thus, a classification of mutations purely addressing proteins is insufficient.

KRAS4A induces metastatic lung adenocarcinomas in vivo in the absence of the KRAS4B isoform

In mammals, the KRAS locus encodes two protein isoforms, KRAS4A and KRAS4B, which differ only in their C terminus via alternative splicing of distinct fourth exons. Previous studies have shown that whereas KRAS expression is essential for mouse development, the KRAS4A isoform is expendable. Here, we have generated a mouse strain that carries a terminator codon in exon 4B that leads to the expression of an unstable KRAS4B154 truncated polypeptide, hence resulting in a bona fide Kras4B-null allele. In contrast, this terminator codon leaves expression of the KRAS4A isoform unaffected. Mice selectively lacking KRAS4B expression developed to term but died perinatally because of hypertrabeculation of the ventricular wall, a defect reminiscent of that observed in embryos lacking the Kras locus. Mouse embryonic fibroblasts (MEFs) obtained from Kras4B-/- embryos proliferated less than did wild-type MEFs, because of limited expression of KRAS4A, a defect that can be compensated for by ectopic expression of this isoform. Introduction of the same terminator codon into a KrasFSFG12V allele allowed expression of an endogenous KRAS4AG12V oncogenic isoform in the absence of KRAS4B. Exposure of Kras+/FSF4AG12V4B- mice to Adeno-FLPo particles induced lung tumors with complete penetrance, albeit with increased latencies as compared with control Kras+/FSFG12V animals. Moreover, a significant percentage of these mice developed proximal metastasis, a feature seldom observed in mice expressing both mutant isoforms. These results illustrate that expression of the KRAS4AG12V mutant isoform is sufficient to induce lung tumors, thus suggesting that selective targeting of the KRAS4BG12V oncoprotein may not have significant therapeutic consequences.

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.