Expression Of Isoforms Research Articles

Experimental and Computational Methods for Allelic Imbalance Analysis from Single-Nucleus RNA-seq Data.

Single-cell RNA-seq (scRNA-seq) is emerging as a powerful tool for understanding gene function across diverse cells. Recently, this has included the use of allele-specific expression (ASE) analysis to better understand how variation in the human genome affects RNA expression at the single-cell level. We reasoned that because intronic reads are more prevalent in single-nucleus RNA-Seq (snRNA-Seq), and introns are under lower purifying selection and thus enriched for genetic variants, that snRNA-seq should facilitate single-cell analysis of ASE. Here we demonstrate how experimental and computational choices can improve the results of allelic imbalance analysis. We explore how experimental choices, such as RNA source, read length, sequencing depth, genotyping, etc., impact the power of ASE-based methods. We developed a new suite of computational tools to process and analyze scRNA-seq and snRNA-seq for ASE. As hypothesized, we extracted more ASE information from reads in intronic regions than those in exonic regions and show how read length can be set to increase power. Additionally, hybrid selection improved our power to detect allelic imbalance in genes of interest. We also explored methods to recover allele-specific isoform expression levels from both long- and short-read snRNA-seq. To further investigate ASE in the context of human disease, we applied our methods to a Parkinson's disease cohort of 94 individuals and show that ASE analysis had more power than eQTL analysis to identify significant SNP/gene pairs in our direct comparison of the two methods. Overall, we provide an end-to-end experimental and computational approach for future studies.

Development and tissue specific expression of RAPGEF1 (C3G) transcripts having exons encoding disordered segments with predicted regulatory function.

The ubiquitously expressed Guanine nucleotide exchange factor, RAPGEF1 (C3G), is essential for early development of mouse embryos. It functions to regulate gene expression and cytoskeletal reorganization, thereby controlling cell proliferation and differentiation. While multiple transcripts have been predicted, their expression in mouse tissues has not been investigated in detail. Full length RAPGEF1 isoforms primarily arise due to splicing at two hotspots, one involving exon-3, and the other involving exons 12-14 incorporating amino acids immediately following the Crk binding region of the protein. These isoforms vary in expression across embryonic and adult organs. We detected the presence of unannotated, and unpredicted transcripts with incorporation of cassette exons in various combinations, specifically in the heart, brain, testis and skeletal muscle. Isoform switching was detected as myocytes in culture and mouse embryonic stem cells were differentiated to form myotubes, and embryoid bodies respectively. The cassette exons encode a serine-rich polypeptide chain, which is intrinsically disordered, and undergoes phosphorylation. In silico structural analysis using AlphaFold indicated that the presence of cassette exons alters intra-molecular interactions, important for regulating catalytic activity. LZerD based docking studies predicted that the isoforms with one or more cassette exons differ in interaction with their target GTPase, RAP1A. Our results demonstrate the expression of novel RAPGEF1 isoforms, and predict cassette exon inclusion as an additional means of regulating RAPGEF1 activity in various tissues and during differentiation.

Exploring a Novel Role of Glycerol Kinase 1 in Prostate Cancer PC-3 Cells.

Clinically, prostate cancer is infamous for its histological and molecular heterogeneity, which causes great challenges to pinpoint therapy and pharmaceutical development. To overcome these difficulties, researchers are focusing on modulating tumor microenvironment and immune responses in addition to genetic alteration and epigenetic regulation. Here, we aimed to identify potential biomarkers or modulators of prostate cancer by investigating genes specifically altered in prostate cancer cells treated with established anti-cancer agents. Glycerol kinase 1 (GK1) is phosphotransferase encoded on the X chromosome, is associated with the synthesis of triglycerides and glycerophospholipids, and has been mainly studied for X-linked metabolic disorder GK deficiency (GKD). Interestingly, our DNA microarray analysis showed that several anti-cancer agents highly induced the expression of GK1, especially GK1a and GK1b isoforms, in human prostate cancer PC-3 cells. To elucidate the relationship between GK1 and cancer cell death, a human GK1b-specific expression vector was constructed and transfected into the PC-3 cells. Surprisingly, GK1b overexpression dramatically reduced cell viability and significantly accelerated apoptotic cell death. These findings suggest that GK1b may serve as a promising modulator and biomarker of cell death in prostate cancer, offering potential avenues for therapeutic intervention.

Hsa-miR-874-3p Reduces Endogenous Expression of RGS4-1 Isoform In Vitro.

The level of the regulator of G-protein signaling 4-1 (RGS4-1) isoform, the longest RGS4 isoform, is significantly reduced in the dorsolateral prefrontal cortex (DLPFC) of people with schizophrenia. However, the mechanism behind this has not been clarified. The 3'untranslated regions (3'UTRs) are known to regulate the levels of their mRNA splice variants. We constructed recombinant pmir-GLO vectors with a truncated 3' regulatory region of the RGS4 gene (3R1, 3R2, 3R3, 3R4, 3R5, and 3R6). The dual-luciferase reporter assay was conducted to find functional regions in HEK-293, SK-N-SH, and U87cells and then predicted miRNA binding to these regions. We performed a dual-luciferase reporter assay and a Western blot analysis after transiently transfecting the predicted miRNAs. The dual-luciferase reporter assay found that regions +401-+789, +789-+1152, and +1562-+1990 (with the last base of the termination codon being +1) might be functional regions. Hsa-miR-874-3p, associated with many psychiatric disorders, might target the +789-+1152 region in the 3'UTR of the RGS4 gene. In the dual-luciferase reporter assay, the hsa-miR-874-3p mimic, co-transfected with 3R1, down-regulated the relative fluorescence intensities. However, this was reversed when the hsa-miR-874-3p mimic was co-transfected with m3R1 (deletion of +853-+859). The hsa-miR-874-3p mimic significantly decreased the endogenous expression of the RGS4-1 isoform in HEK-293 cells. Hsa-miR-874-3p inhibits the expression of the RGS4-1 isoform by targeting +853-+859.

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.

Deciphering the prognostic value of FGFR2b/2c isoform expression levels in advanced esophagogastric cancer through whole-transcriptome sequencing.

53 Background: Fibroblast growth factor receptor (FGFR) isoform switching from FGFR2b to FGFR2c has been implicated in epithelial-to-mesenchymal transition and enhanced invasive potential in various malignancies and potentially be involved in resistance mechanisms to FGFR2b targeted therapy (e.g., bemarituzumab). This study sought to investigate the potential of respective FGFR2 isoform expression evaluation utilizing whole-transcriptome sequencing (WTS) and its correlation with therapeutic efficacy and clinical outcomes in patients with advanced G/GEJ cancer. Methods: Patients with advanced G/GEJ cancer who received systemic therapy at the National Cancer Center Hospital East between May 2021 and September 2023, and were enrolled in the immunological profiling study (UMIN000019129) and MONSTAR-SCREEN-2 (UMIN000043899) were included. FGFR2 protein and RNA expression were assessed via immunohistochemistry (IHC) and WTS with Caris MI Profile, respectively. FGFR2b and FGFR2c isoforms were distinguished by the detection of their respective splice junctions using WTS data. The FGFR2b/(2b+2c) ratio was stratified into FGFR2b-dominat and -containing subtypes using the median value. The log-rank test was used to discern differences in overall survival (OS). Results: Among the 129 patients, 21 (16.3%) exhibited FGFR2-positivity by IHC (staining intensity 2+ or 3+≥1% membranous staining of tumor cells), and a significant correlation was observed between FGFR2 IHC status and RNA expression levels (TPM: transcript per million) in WTS (FGFR2-positive, median TPM=11.1; FGFR2-negative, median TPM=4.7; P=0.002). The distribution of FGFR2b/(2b+2c) ratio (median, 0.89) was: 10.9% (0≤x<0.2), 5.4% (0.2≤x<0.4), 7.0% (0.4≤x<0.6), 12.4% (0.6≤x<0.8), 43.4% (0.8≤x<1.0), and 20.9% (x=1.0). Among 58 patients treated with 1st line therapy, no significant difference in treatment response was observed between the FGFR2b-dominat (67.9%) and FGFR2c-containing subtypes (53.3%) (P=0.259). Overall, the FGFR2c-containing subtype was associated with significantly shorter OS compared to the FGFR2b-dominat subtype (median OS, 10.1 vs. 17.9 months; hazard ratio, 1.72; 95% confidence interval, 1.03-2.86; log-rank P=0.037). Conclusions: Our findings underscore the substantial concordance between IHC and WTS analysis of FGFR2, the feasibility of distinguishing FGFR2 isoforms using WTS, and the prognostic implications of FGFR2c isoform in patients with advanced G/GEJ cancer. Further analysis is warranted in patients receiving FGFR2b targeted treatment.

Role of Different Variants of Leptin Receptor in Human Adrenal Tumor Types.

The aim of the study was to evaluate the diagnostic and prognostic significance of leptin receptor isoforms in adrenal tumors. In a single-center study, 96 patients (19 with adrenal cortical carcinoma and 77 with benign tumors) underwent an adrenalectomy. A total of 14 unaffected adrenal gland tissues from kidney donors were used as controls. Fasting blood samples were collected for laboratory tests, and mRNA expressions of leptin receptor isoforms were assessed by RT-qPCR. The study analyzed correlations between mRNA expressions and clinical data and measured NCI-H295R cell proliferation via a real-time cell analyzer. All adrenal lesions expressed leptin receptor isoforms. Significantly lower LepR1 expression was observed in carcinoma tissues than in adenomas and controls (p = 0.016). Expressions of LepR3&LepR6 were correlated with overall survival (p = 0.036), while LepR2&LepR4 and LepR5 expressions were inversely related to morning serum cortisol levels (p = 0.041). Leptin reduced NCI-H295R cell proliferation (p < 0.0001). The study highlights the diagnostic and prognostic significance of leptin receptor isoforms in adrenal tumors. Specifically, LepR1 may serve as a diagnostic marker for carcinomas, while LepR3&LepR6 have potential use as prognostic markers.

Increased Homer Activity and NMJ Localization in the Vestibular Lesion het-/- Mouse soleus Muscle.

We investigated the shuttling of Homer protein isoforms identified in soluble (cytosolic) vs. insoluble (membrane-cytoskeletal) fraction and Homer protein-protein interaction/activation in the deep postural calf soleus (SOL) and non-postural gastrocnemius (GAS) muscles of het-/- mice, i.e., mice with an autosomal recessive variant responsible for a vestibular disorder, in order to further elucidate a) the underlying mechanisms of disrupted vestibular system-derived modulation on skeletal muscle, and b) molecular signaling at respective neuromuscular synapses. Heterozygote mice muscles served as the control (CTR). An increase in Homer cross-linking capacity was present in the SOL muscle of het-/- mice as a compensatory mechanism for the altered vestibule system function. Indeed, in both fractions, different Homer immunoreactive bands were detectable, as were Homer monomers (~43-48 kDa), Homer dimers (~100 kDa), and several other Homer multimer bands (>150 kDA). The het-/- GAS particulate fraction showed no Homer dimers vs. SOL. The het-/- SOL soluble fraction showed a twofold increase (+117%, p ≤ 0.0004) in Homer dimers and multimers. Homer monomers were completely absent from the SOL independent of the animals studied, suggesting muscle-specific changes in Homer monomer vs. dimer expression in the postural SOL vs. the non-postural GAS muscles. A morphological assessment showed an increase (+14%, p ≤ 0.0001) in slow/type-I myofiber cross-sectional area in the SOL of het-/- vs. CTR mice. Homer subcellular immuno-localization at the neuromuscular junction (NMJ) showed an altered expression in the SOL of het-/-mice, whereas only not-significant changes were found for all Homer isoforms, as judged by RT-qPCR analysis. Thus, muscle-specific changes, myofiber properties, and neuromuscular signaling mechanisms share causal relationships, as highlighted by the variable subcellular Homer isoform expression at the instable NMJs of vestibular lesioned het-/- mice.

Hif1α-dependent mitochondrial acute O2 sensing and signaling to myocyte Ca2+ channels mediate arterial hypoxic vasodilation

Vasodilation in response to low oxygen (O2) tension (hypoxic vasodilation) is an essential homeostatic response of systemic arteries that facilitates O2 supply to tissues according to demand. However, how blood vessels react to O2 deficiency is not well understood. A common belief is that arterial myocytes are O2-sensitive. Supporting this concept, it has been shown that the activity of myocyte L-type Ca2+channels, the main ion channels responsible for vascular contractility, is reversibly inhibited by hypoxia, although the underlying molecular mechanisms have remained elusive. Here, we show that genetic or pharmacological disruption of mitochondrial electron transport selectively abolishes O2 modulation of Ca2+ channels and hypoxic vasodilation. Mitochondria function as O2 sensors and effectors that signal myocyte Ca2+ channels due to constitutive Hif1α-mediated expression of specific electron transport subunit isoforms. These findings reveal the acute O2-sensing mechanisms of vascular cells and may guide new developments in vascular pharmacology.

Abstract Mo023: Inhibition of Cyp1a Protects Mice against Anthracycline Cardiomyopathy

Background: Anthracyclines such as doxorubicin (Dox) are highly effective anti-tumor agents, but their use is limited by dose-dependent cardiomyopathy. Our laboratory previously reported that activation of cytochrome P450 family 1 (Cyp1) enzymes contributes to acute doxorubicin (Dox) cardiotoxicity in zebrafish and in mice, and that potent Cyp1 inhibitors prevent cardiotoxicity. However, the role of Cyp1 enzymes in chronic Dox cardiomyopathy, as well as the mechanisms underlying cardioprotection associated with Cyp1 inhibition, have not been fully elucidated. Objective: This study aims to define the role of Cyp1 inhibition in mouse models that more accurately recapitulates the chronic cardiomyopathy seen in patients. Methods: The function of Cyp1 enzymes was evaluated using a small molecule Cyp1 inhibitor in wild-type (WT) mice, or Cyp1-null mice ( Cyp1a1/1a2 -/- , Cyp1b1 -/- , and Cyp1a1/1a2/1b1 -/- ). Low-dose Dox was administered by serial intraperitoneal or intravenous injections, respectively. Expression of Cyp1 isoforms was measured by RT-qPCR, and myocardial tissue was isolated from the left ventricle for RNA sequencing. Cardiac function was evaluated by transthoracic echocardiography. Results: In WT mice, Dox treatment was associated with a decrease in Cyp1a2 and an increase in Cyp1b1 expression in the heart and in the liver. Co-treatment of WT mice with Dox and the potent Cyp1 inhibitor YW-130 protected against cardiac dysfunction compared to Dox treatment alone. Cyp1a1/1a2 -/- and Cyp1a1/1a2/1b1 -/- mice were protected from Dox cardiomyopathy compared to WT mice. Male, but not female, Cyp1b1 -/- mice had increased cardiac dysfunction following Dox treatment compared to WT mice. RNA sequencing of myocardial tissue showed upregulation of Fundc1 and downregulation of Ccl21c in Cyp1a1/1a2 -/- mice treated with Dox, implicating changes in mitophagy and chemokine-mediated inflammation as possible mechanisms of Cyp1a-mediated cardioprotection. Conclusions: Taken together, this study highlights the potential therapeutic value of Cyp1a inhibition in mitigating anthracycline cardiomyopathy.

Abstract Tu023: The VEGF-A splice variants as a mechanism of impaired Inflammatory Angiogenesis in the context of advanced aging.

Background and Objectives: Aging and age-related diseases like peripheral artery disease (PAD) is associated with impairment of angiogenesis responses to injury. Critical ischemic limb and wound injury animal models have shown impact of monocytes/macrophages as a major source of angiogenic mediators leading to new arterial growth. Aging has also been associated with impairment of blood flow recovery and reduced VEGF-A expression in animals. However, recombinant VEGF-A administration or clinical trials involving VEGF-A have not fully overcome the foot perfusion and prevent limb/leg amputation. We sought to understand molecular mechanisms of reduced VEGF-A expression which remain incomplete in the context of advanced aging. Results: Firstly, in hindlimb ischemia mouse model, our data from liposomes containing-clodronate treated mice (12-weeks C57Bl6 with macrophage depletion model ) ( Fig. 1a ) demonstrate impairment of blood flow recovery compared to Control group. On day 3 post-surgery, VEGF-A and VEGF-A isoforms expression (VEGF-A 165 a and VEGF-A 165 b, respectively) were reduced in ischemic muscle. Fluorescence staining showed a reduction of macrophages (CD68 + ) was also associated with reduced endothelial cells (CD31 + ). Using a myeloid-specific and inducible deleted VEGF fl/fl mice, we then have shown a key role of early inflammatory macrophages stage as a major source of VEGF-A required for angiogenesis in young mice (Fig. 1b). Secondly, bone marrow-derived-macrophages (BMDMs) from C57Bl6 104-weeks mice also demonstrate reduced VEGF-A expression. Aged BMDMs demonstrate reduced VEGF-A 165 a while VEGF-A 165 b was highly increased. Polarized BMDMs from 12-weeks demonstrate increased VEGFR1 in “M2" macrophages while VEGFR2 was increased in “M1” macrophages. On the other hand, BMDMs from 104-weeks demonstrate reduced VEGFR2 expression while VEGFR1 was unaffected compared to young. Lastly, 104-weeks mice also demonstrate severe impairment of blood flow recovery ( Fig. 2 ) after surgery and reduced VEGF-A and VEGF-A 165 a in ischemic muscle tissue. VEGF-A 165 b was increased and both receptors, VEGF-R1/R2 levels ( Fig. 3 ) were also reduced. Fluorescence staining on day 3 post ligation demonstrates reduced endothelial cells recruitment in muscle tissue while macrophage (CD68+) number was similar. Conclusion: Our investigations will contribute to define mechanisms whereby vascular injury is associated with VEGF-A isoforms specific switch in the context of advanced aging.

Abstract Tu116: Neurofibromin 2 regulates metabolic gene expression and cardiac responses to pressure overload stress

Background: Neurofibromin 2 (NF2) is a tumor suppressor that can engage multiple signaling pathways to modulate cell proliferation and survival. We previously demonstrated that NF2 mediates cardiomyocyte apoptosis and cardiac injury caused by acute myocardial infarction. Research Aim: The role of NF2 in heart failure remains uncharacterized. This study sought to determine whether NF2 modulates heart failure due to chronic stress. Approach: We generated cardiomyocyte-specific NF2 knockout (cKO) mice, and used transverse aortic constriction (TAC) to generate chronic pressure overload (PO) stress, which elicits cardiac remodeling and failure. Complementary cell-based experiments were performed in neonatal rat ventricular myocytes (NRVMs). We analyzed cardiac function by echocardiographic and hemodynamic analysis. We used RNAseq, ChIP, Seahorse metabolic profiling, and biochemical analyses to investigate underlying mechanisms. Results: We found that NF2 is transiently upregulated in wild-type mouse myocardium in response to early phase of PO, but is downregulated during heart failure. Following TAC, NF2 cKO hearts unexpectedly showed significantly worsened cardiac function, compared to controls. RNAseq analysis indicated downregulation of several metabolic pathways and impaired ERR activity in NF2 cKO hearts. qPCR confirmed significantly reduced ERRβ and ERRγ transcripts and decreased metabolic gene expression. Experiments employing NRMVs confirmed that NF2 promoted expression of ERRβ and ERRγ, and DNA pulldown assays demonstrated NF2 association with ERRβ and ERRγ proximal promoters. Analysis of NRVM bioenergetics demonstrated that NF2 depletion impaired mitochondrial respiration, which was reversed by concomitant expression of ERRγ. Moreover, AAV9-mediated restoration of ERRγ in NF2 cKO mice normalized cardiac function in response to PO. Because NF2 does not directly bind DNA, ongoing studies are leveraging a proteomics-based approach to identify the mediator linking NF2 to ERR isoform expression. Conclusion: Based on these findings, we conclude that transient upregulation of myocardial NF2 expression during PO stress is compensatory and regulates metabolic gene expression according to energy demand.

Abstract Mo118: Myofilament proteolysis may underlie contractile remodeling in atrial fibrillation

Atrial fibrillation (AFib) is the most common cardiac rhythm disturbance. Treatment of AFib involves restoration of the atrial electrical rhythm. Following rhythm restoration, a period of depressed mechanical function, including decreased blood flow velocity and reduced atrial contractility, known as atrial stunning, occurs. This suggests that defects in contractility occur in AFib and are revealed upon restoration of rhythm. To assess contractile function, we used a canine atrial tachypacing model of induced AFib. Mass spectrometry analysis showed dysregulation of contractile proteins in samples from AFib compared to sinus rhythm atria. In atrial cardiomyocytes that were useable for skinned single cardiomyocyte force-calcium studies, we found reduced force of contraction. There were no significant differences in myosin heavy chain isoform expression. Resting tension is decreased in the AFib samples correlating with reduced full-length titin in the sarcomere. We measured degradation of other myofilament proteins including cMyBP-C, actinin, MHC, and cTnI, showing significant degradation in the AFib samples compared to sinus rhythm atria. Many of the protein degradation products appeared as discrete cleavage products that are generated by calpain proteolysis. We assessed calpain activity and found it to be significantly increased. Skinned cardiomyocytes from AFib atria showed increased calcium sensitivity that was consistent with increased cTnI degradation and decreased TnI phosphorylation. These results provide an understanding of the contractile remodeling that occurs in AFib.

Long-read transcript sequencing identifies differential isoform expression in the entorhinal cortex in a transgenic model of tau pathology

Increasing evidence suggests that alternative splicing plays an important role in Alzheimer’s disease (AD) pathology. We used long-read sequencing in combination with a novel bioinformatics tool (FICLE) to profile transcript diversity in the entorhinal cortex of female transgenic (TG) mice harboring a mutant form of human tau. Our analyses revealed hundreds of novel isoforms and identified differentially expressed transcripts – including specific isoforms of Apoe, App, Cd33, Clu, Fyn and Trem2 – associated with the development of tau pathology in TG mice. Subsequent profiling of the human cortex from AD individuals and controls revealed similar patterns of transcript diversity, including the upregulation of the dominant TREM2 isoform in AD paralleling the increased expression of the homologous transcript in TG mice. Our results highlight the importance of differential transcript usage, even in the absence of gene-level expression alterations, as a mechanism underpinning gene regulation in the development of AD neuropathology.

Novel Identification of Ankyrin-R in Cardiac Fibroblasts and a Potential Role in Heart Failure.

Altered ankyrin-R (AnkR; encoded by ANK1) expression is associated with diastolic function, left ventricular remodeling, and heart failure with preserved ejection fraction (HFpEF). First identified in erythrocytes, the role of AnkR in other tissues, particularly the heart, is less studied. Here, we identified the expression of both canonical and small isoforms of AnkR in the mouse myocardium. We demonstrate that cardiac myocytes primarily express small AnkR (sAnkR), whereas cardiac fibroblasts predominantly express canonical AnkR. As canonical AnkR expression in cardiac fibroblasts is unstudied, we focused on expression and localization in these cells. AnkR is expressed in both the perinuclear and cytoplasmic regions of fibroblasts with considerable overlap with the trans-Golgi network protein 38, TGN38, suggesting a potential role in trafficking. To study the role of AnkR in fibroblasts, we generated mice lacking AnkR in activated fibroblasts (Ank1-ifKO mice). Notably, Ank1-ifKO mice fibroblasts displayed reduced collagen compaction, supportive of a novel role of AnkR in normal fibroblast function. At the whole animal level, in response to a heart failure model, Ank1-ifKO mice displayed an increase in fibrosis and T-wave inversion compared with littermate controls, while preserving cardiac ejection fraction. Collagen type I fibers were decreased in the Ank1-ifKO mice, suggesting a novel function of AnkR in the maturation of collagen fibers. In summary, our findings illustrate the novel expression of AnkR in cardiac fibroblasts and a potential role in cardiac function in response to stress.

A homozygous nonsense variant in the alternatively spliced VLDLR exon 4 causes a neurodevelopmental disorder without features of VLDLR cerebellar hypoplasia.

VLDLR cerebellar hypoplasia is characterized by intellectual disability, non-progressive cerebellar ataxia, and seizures. The characteristic MRI findings include hypoplasia of the inferior portion of the cerebellar vermis and hemispheres, simplified cortical gyration, and a small brain stem. Biallelic VLDLR pathogenic variants cause loss-of-function of the encoded very low-density lipoprotein receptor. VLDLR exons 4 and 16 are alternatively spliced, resulting in the expression of four transcript variants, including two exon 4-lacking mRNAs expressed in the human brain. Previously reported VLDLR pathogenic variants affect all four transcript variants. Here we report on two sisters with facial dysmorphism, microcephaly, intellectual disability, and normal brain imaging. Exome sequencing in one patient identified the homozygous VLDLR nonsense variant c.376C>T; p.(Gln126*) in exon 4; her similarly affected sister also carried the homozygous variant and parents were heterozygous carriers. VLDLR transcript analysis identified mRNAs with and without exon 4 in patient fibroblasts, while exon 4-containing VLDLR mRNAs were predominantly detected in control fibroblasts. We found significantly reduced VLDLR mRNA levels in patient compared to control cells, likely caused by nonsense-mediated mRNA decay of exon 4-containing VLDLR transcripts. Expression of neuronal VLDLR isoforms produced from exon 4-lacking transcripts may have protected both patients from developing the cerebellar hypoplasia phenotype.

Fumonisin B1 induces endoplasmic reticulum damage and inflammation by activating the NXR response and disrupting the normal CYP450 system, leading to liver damage in juvenile quail.

Fumonisin B1 (FB1) is a mycotoxin affecting animal health through the food chain and has been closely associated with several diseases such as pulmonary edema in pigs and diarrhea in poultry. FB1 is mainly metabolized in the liver. Although a few studies have shown that FB1 causes liver damage, the molecular mechanism of liver damage is unclear. This study aimed to evaluate the role of liver damage, nuclear xenobiotic receptor (NXR) response and cytochrome P450 (CYP450)-mediated defense response during FB1 exposure. A total of 120 young quails were equally divided into two groups (control and FB1 groups). The quails in the control group were fed on a normal diet, while those in the FB1 group were fed on a quail diet containing 30mg/kg for 42 days. Histopathological and ultrastructural changes in the liver, biochemical parameters, inflammatory factors, endoplasmic reticulum (ER) factors, NXR response and CYP450 cluster system and other related genes were examined at 14 days, 28 days and 42 days. The results showed that FB1 exposure impaired the metabolic function and caused liver injury. FB1 caused ER stress and decreased adenosine triphosphatease activity, induced the expression of inflammation-related genes such as interleukin 6 and nuclear factor kappa-B, and promoted inflammation. In addition, FB1 disrupted the expression of multiple CYP450 isoforms by activating nuclear xenobiotic receptors (NXRs). The present study confirms that FB1 exposure disturbs the homeostasis of cytochrome P450 systems (CYP450s) in quail liver by activating NXR responses and thereby causing liver damage. This study's findings provide insight into the molecular mechanisms of FB1-induced hepatotoxicity.

High-throughput single-molecule long-read RNA sequencing analysis of tissue-specific genes and isoforms in lettuce (Lactuca sativa L.)

Lettuce is one of the most widely cultivated and consumed dicotyledonous vegetables globally. Despite the availability of its reference genome sequence, lettuce gene annotation remains incomplete, impeding comprehensive research and the broad application of genomic resources. Long-read RNA isoform sequencing (Iso-Seq) offers substantial advantages for analyzing RNA alternative splicing and aiding gene annotation, yet it faces throughput limitations. We present the HIT-ISOseq method tailored for bulk sample analysis, significantly enhancing RNA sequencing throughput on the PacBio platform by concatenating cDNA. Here we show, HIT-ISOseq generates 3-4 cDNA molecules per CCS read in lettuce, yielding 15.7 million long reads per PacBio Sequel II SMRT Cell 8 M. We validate its effectiveness in analyzing six lettuce tissue samples, including roots, stems, and leaves, revealing tissue-specific gene expression patterns and RNA isoforms. Leveraging diverse tissue long-read RNA sequencing, we refine the transcript annotation of the lettuce reference genome, expanding its GO and KEGG annotation repertoire. Collectively, this study serves as a foundational reference for genome annotation and the analysis of multi-sample isoform expression, utilizing high-throughput long-read transcriptome sequencing.

MRNA and circRNA mislocalization to synapses are key features of Alzheimer's disease.

Proper transport of RNAs to synapses is essential for localized translation of proteins in response to synaptic signals and synaptic plasticity. Alzheimer's disease (AD) is a neurodegenerative disease characterized by accumulation of amyloid aggregates and hyperphosphorylated tau neurofibrillary tangles followed by widespread synapse loss. To understand whether RNA synaptic localization is impacted in AD, we performed RNA sequencing on synaptosomes and brain homogenates from AD patients and cognitively healthy controls. This resulted in the discovery of hundreds of mislocalized mRNAs in AD among frontal and temporal brain regions. Similar observations were found in an APPswe/PSEN1dE9 mouse model. Furthermore, major differences were observed among circular RNAs (circRNAs) localized to synapses in AD including two overlapping isoforms of circGSK3β, one upregulated, and one downregulated. Expression of these distinct isoforms affected tau phosphorylation in neuronal cells substantiating the importance of circRNAs in the brain and pointing to a new class of therapeutic targets.

Long-read proteogenomics to connect disease-associated sQTLs to the protein isoform effectors of disease

A major fraction of loci identified by genome-wide association studies (GWASs) mediate alternative splicing, but mechanistic interpretation is hindered by the technical limitations of short-read RNA sequencing (RNA-seq), which cannot directly link splicing events to full-length protein isoforms. Long-read RNA-seq represents a powerful tool to characterize transcript isoforms, and recently, infer protein isoform existence. Here, we present an approach that integrates information from GWASs, splicing quantitative trait loci (sQTLs), and PacBio long-read RNA-seq in a disease-relevant model to infer the effects of sQTLs on the ultimate protein isoform products they encode. We demonstrate the utility of our approach using bone mineral density (BMD) GWAS data. We identified 1,863 sQTLs from the Genotype-Tissue Expression (GTEx) project in 732 protein-coding genes that colocalized with BMD associations (H4PP ≥ 0.75). We generated PacBio Iso-Seq data (N= ∼22 million full-length reads) on human osteoblasts, identifying 68,326 protein-coding isoforms, of which 17,375 (25%) were unannotated. By casting the sQTLs onto protein isoforms, we connected 809 sQTLs to 2,029 protein isoforms from 441 genes expressed in osteoblasts. Overall, we found that 74 sQTLs influenced isoforms likely impacted by nonsense-mediated decay and 190 that potentially resulted in the expression of unannotated protein isoforms. Finally, we functionally validated colocalizing sQTLs in TPM2, in which siRNA-mediated knockdown in osteoblasts showed two TPM2 isoforms with opposing effects on mineralization but exhibited no effect upon knockdown of the entire gene. Our approach should be to generalize across diverse clinical traits and to provide insights into protein isoform activities modulated by GWAS loci.