Variant Isoforms Research Articles

RNA isoform diversity in human neurodegenerative diseases.

Single-nucleus RNA-sequencing (snRNA-seq) has revealed new levels of cellular organization and diversity within the human brain. However, full-length mRNA isoforms are not resolved in typical snRNA-seq analyses using short-read sequencing that cannot capture full-length transcripts. Here we combine standard 10X Genomics short-read snRNA-seq with targeted PacBio long-read snRNA-seq to examine isoforms of genes associated with neurological diseases at the single-cell level from prefrontal cortex samples of diseased and non-diseased human brain, assessing over 165,000 cells. Samples from 25 post-mortem donors with Alzheimer's disease (AD), dementia with Lewy bodies (DLB), or Parkinson's disease (PD), along with age-matched controls were compared. Analysis of the short-read libraries identified shared and distinct gene expression changes across the diseases. The same libraries were then assayed using enrichment probes to target 50 disease-related genes followed by long-read PacBio sequencing, enabling linkage between cell type and isoform expression. Vast mRNA isoform diversity was observed in all 50 targeted genes, even those that were not differentially expressed in the short-read data. We also developed an informatics method for detection of isoform structural differences in novel isoforms vs. the reference annotation. These data expand available single-cell datasets of the human prefrontal cortical transcriptome with combined short- and long-read sequencing across AD, DLB, and PD, revealing increased mRNA isoform diversity that may contribute to disease features and could potentially represent therapeutic targets for neurodegenerative diseases.Significance Statement Limited comparisons using single-cell transcriptomics analysis have been conducted amongst common neurodegenerative diseases. Here we identify new cell type and disease relationships involving known and novel mRNA isoforms by profiling single nuclei from human prefrontal cortices of Alzheimer's disease, Parkinson's disease, dementia with Lewy bodies and non-diseased controls. Cell-type-specific RNA isoform diversity across the different diseases was examined using the combination of short-read snRNA-seq and targeted long-read single-nucleus isoform sequencing. We identified myriad novel transcripts that highlight an untapped understanding of RNA isoform diversity that exists within the brain and potentially contribute to human neurodegenerative diseases.

537 Full-length transcript sequencing of fibroblasts and keratinocytes reveals isoform diversity in human skin wound healing

537 Full-length transcript sequencing of fibroblasts and keratinocytes reveals isoform diversity in human skin wound healing

Expression of variant isoforms of the tyrosine kinase SYK differentially regulates cervical cancer progression through PI3K/AKT pathway

Invasion and metastasis are the main reasons for the poor prognosis of patients with cervical cancer(CC). SYK is closely related to tumor development. However, the functions of its two isoforms, SYK (L) or SYK (S), are not fully understood to date. In this study, we investigated their biologic functions and possible prognostic values in CC. qRT-PCR was performed to detect the expression of SYK and two variant isoforms in cervical cancer tissues and cells. The association of SYK(L) and SYK(S) with Clinical pathological parameters were evaluated. The migration and invasion was detected by scratch assay and transwell. Western blot was conducted to measure the changes of epithelial mesenchymal transition (EMT)-related markers and PI3K/AKT signaling pathway proteins in cervical cancer cells. LY294002 (inhibitor of PI3K/AKT pathway) and IGF-1 (activator of PI3K/AKT pathway) were applied to evaluate the contribution of PI3K/AKT signaling pathway in cervical cancer cells. The expression of SYK(S) in cervical cancer tissues was significantly higher than that of SYK(L). SYK(L) and SYK(S) were correlated with muscular infiltration, SYK(L) high expression had a better prognosis, whereas SYK(S) high expression predicted a worse disease outcome. Cox multivariate regression analysis demonstrated that SYK(L) expression was an independent prognostic factor. SYK(L) significantly inhibited the proliferation, migration and invasion, while SYK(S) showed the opposite effects. LY294002 blocked SYK (L) knockdown-induced enhancement of migration and invasion as well as the expression EMT-related markers, whereas IGF-1 rescued the decreased migration, invasion and EMT induced by SYK (S) knockdown. The results suggest that SYK(L) and SYK(S) are involved in the progression of cervical cancer through PI3K/AKT signaling pathway, and may serve as potential targets for clinical treatment of advanced cervical cancer.

A structural analysis of the splice-specific functional impact of the pathogenic familial hemiplegic migraine type 1 S218L mutation on Cav2.1 P/Q-type channel gating

P/Q-type (Cav2.1) calcium channels mediate Ca2+ influx essential for neuronal excitability and synaptic transmission. The CACNA1A gene, encoding the Cav2.1 pore forming subunit, is highly expressed throughout the mammalian central nervous system. Alternative splicing of Cav2.1 pre-mRNA generates diverse channel isoforms with distinct biophysical properties and drug affinities, which are differentially expressed in nerve tissues. Splicing variants can also affect channel function under pathological conditions although their phenotypic implication concerning inherited neurological disorders linked to CACNA1A mutations remains unknown. Here, we quantified the expression of Cav2.1 exon 24 (e24) spliced transcripts in human nervous system samples, finding different levels of expression within discrete regions. The corresponding Cav2.1 variants, differing by the presence (+) or absence (Δ) of Ser-Ser-Thr-Arg residues (SSTR) in the domain III S3-S4 linker, were functionally characterized using patch clamp recordings. Further, the + /ΔSSTR isoforms were used to demonstrate the differential impact of the Familial Hemiplegic Migraine Type 1 (FHM-1) S218L mutation, located in the domain I S4-S5 linker, on the molecular structure and electrophysiological properties of Cav2.1 isoforms. S218L has a prominent effect on the voltage-dependence of activation of +SSTR channels when compared to ΔSSTR, indicating a differential effect of the mutation depending on splice-variant context. Structural modeling based upon Cav2.1 cryo-EM data provided further insight reflecting independent contributions of amino acids in distant regions of the channel on gating properties. Our modelling indicates that by increasing hydrophobicity the Leu218 mutation contributes to stabilizing a structural conformation in which the domain I S4-S5 linker is oriented alongside the inner plasma membrane, similar to that occurring when S4 is translocated upon activation.The SSTR insertion appears to exert an influence in the local electric field of domain III due to an change in the distribution of positively charged regions surrounding the voltage sensing domain, which we hypothesize impacts its movement during the transition to the open state. In summary, we reveal molecular changes correlated with distinct functional effects provoked by S218L FHM-1 mutation in hCav2.1 splice isoforms whose differential expression could impact the manifestation of the neurological disorder.

Understanding isoform expression by pairing long-read sequencing with single-cell and spatial transcriptomics.

RNA isoform diversity, produced via alternative splicing, and alternative usage of transcription start and poly(A) sites, results in varied transcripts being derived from the same gene. Distinct isoforms can play important biological roles, including by changing the sequences or expression levels of protein products. The first single-cell approaches to RNA sequencing-and later, spatial approaches-which are now widely used for the identification of differentially expressed genes, rely on short reads and offer the ability to transcriptomically compare different cell types but are limited in their ability to measure differential isoform expression. More recently, long-read sequencing methods have been combined with single-cell and spatial technologies in order to characterize isoform expression. In this review, we provide an overview of the emergence of single-cell and spatial long-read sequencing and discuss the challenges associated with the implementation of these technologies and interpretation of these data. We discuss the opportunities they offer for understanding the relationships between the distinct variable elements of transcript molecules and highlight some of the ways in which they have been used to characterize isoforms' roles in development and pathology. Single-nucleus long-read sequencing, a special case of the single-cell approach, is also discussed. We attempt to cover both the limitations of these technologies and their significant potential for expanding our still-limited understanding of the biological roles of RNA isoforms.

Target gene variations of PPAR isoforms may contribute to MODY heterogeneity: A preliminary comparative study with type 2 diabetes

AimsThe objective of this study was to evaluate the associations of several genetic variants of peroxisome proliferator-activated receptors (PPARs) on clinical and laboratory parameters in patients with maturity-onset diabetes of the young (MODY), and possible contribution to heterogeneity of the disease. MethodsThe study groups comprised patients with MODY (genetically confirmed (n = 28), clinically relevant but genetically unconfirmed; MODYX (n = 56)), type 2 diabetes mellitus (T2DM; n = 94) and healthy controls (n = 153). PPARA-L162V-(rs1800206), PPARG-C161T-(rs3856806), P12A-(rs1801282), and PPARB/D + 294 T/C-(rs2016520) polymorphisms were genotyped by real-time-PCR. ResultsThe results demonstrated that the frequencies of PPARA-LL162 (p = 0.002), PPARG-CC161 (p = 0.002), and PPARG-ProPro (p = 0.012) genotypes were significantly higher in the MODY group compared to the controls. Furthermore, total-MODY and MODYX groups had a higher frequency of PPARA-LL162 genotype than T2DM (p = 0.005 and p = 0.006, respectively). The frequency of the PPARB/D + 294 T allele was significantly higher in individuals with T2DM than in genetically-determined MODY group (p = 0.019). The PPARA-LL162 genotype was associated with early-onset diabetes in total-MODY (p = 0.022) and T2DM (p < 0.05) groups. ConclusionsThe association of PPARA-L162V polymorphism with early-onset diabetes in both T2DM and MODY is a noteworthy finding. Considering these results, we suggested that genetic polymorphisms in PPAR isoforms may contribute to the clinical and metabolic heterogeneity of MODY.

The Spatial-Temporal Alternative Splicing Profile Reveals the Functional Diversity of FXR1 Isoforms in Myogenesis.

Alternative splicing (AS) is a fundamental mechanism contributing to proteome diversity, yet its comprehensive landscape and regulatory dynamics during skeletal muscle development remain largely unexplored. Here, the temporal AS profiles are investigated during myogenesis in five vertebrates, conducting comprehensive profiling across 27 developmental stages in skeletal muscle and encompassing ten tissues in adult pigs. The analysis reveals a pervasive and evolutionarily conserved pattern of alternative exon usage throughout myogenic differentiation, with hundreds of skipped exons (SEs) showing developmental regulation, particularly within skeletal muscle. Notably, this study identifies a muscle-specific SE (exon 15) within the Fxr1 gene, whose AS generates two dynamically expressed isoforms with distinct functions: the isoform without exon 15 (Fxr1E15 -) regulates myoblasts proliferation, while the isoform incorporating exon 15 (Fxr1E15+) promotes myogenic differentiation and fusion. Transcriptome analysis suggests that specifically knocking-down Fxr1E15+ isoform in myoblasts modulates differentiation by influencing gene expression and splicing of specific targets. The increased inclusion of exon 15 during differentiation is mediated by the binding of Rbm24 to the intron. Furthermore, in vivo experiments indicate that the Fxr1E15+ isoform facilitates muscle regeneration. Collectively, these findings provide a comprehensive resource for AS studies in skeletal muscle development, underscoring the diverse functions and regulatory mechanisms governing distinct Fxr1 isoforms in myogenesis.

Antitumor activities of anti‑CD44 monoclonal antibodies in mouse xenograft models of esophageal cancer.

CD44 is a type I transmembrane glycoprotein associated with poor prognosis in various solid tumors. Since CD44 plays a critical role in tumor development by regulating cell adhesion, survival, proliferation and stemness, it has been considered a target for tumor therapy. Anti‑CD44 monoclonal antibodies (mAbs) have been developed and applied to antibody‑drug conjugates and chimeric antigen receptor‑T cell therapy. Anti-pan‑CD44 mAbs, C44Mab‑5 and C44Mab‑46, which recognize both CD44 standard (CD44s) and variant isoforms were previously developed. The present study generated a mouse IgG2a version of the anti‑pan‑CD44 mAbs (5‑mG2a and C44Mab‑46‑mG2a) to evaluate the antitumor activities against CD44‑positive cells. Both 5‑mG2a and C44Mab‑46‑mG2a recognized CD44s‑overexpressed CHO‑K1 (CHO/CD44s) cells and esophageal tumor cell line (KYSE770) in flow cytometry. Furthermore, both 5‑mG2a and C44Mab‑46‑mG2a could activate effector cells in the presence of CHO/CD44s cells and exhibited complement-dependent cytotoxicity against both CHO/CD44s and KYSE770 cells. Furthermore, the administration of 5‑mG2a and C44Mab‑46‑mG2a significantly suppressed CHO/CD44s and KYSE770 xenograft tumor development compared with the control mouse IgG2a. These results indicate that 5‑mG2a and C44Mab‑46‑mG2a could exert antitumor activities against CD44‑positive cancers and be a promising therapeutic regimen for tumors.

Regulation of HTT mRNA Biogenesis: The Norm and Pathology.

Huntington's disease (HD) is a neurodegenerative disorder caused by the expansion of the CAG repeat in exon 1 of the HTT gene, leading to the formation of a toxic variant of the huntingtin protein. It is a rare but severe hereditary disease for which no effective treatment method has been found yet. The primary therapeutic targets include the mutant protein and the mutant mRNA of HTT. Current clinical trial approaches in gene therapy involve the application of splice modulation, siRNA, or antisense oligonucleotides for RNA-targeted knockdown of HTT. However, these approaches do not take into account the diversity of HTT transcript isoforms in the normal conditions and in HD. In this review, we discuss the features of transcriptional regulation and processing that lead to the formation of various HTT mRNA variants, each of which may uniquely contribute to the progression of the disease. Furthermore, understanding the role of known transcription factors of HTT in pathology may aid in the development of potentially new therapeutic tools based on endogenous regulators.

Hepatocyte-specific NR5A2 deficiency induces pyroptosis and exacerbates non-alcoholic steatohepatitis by downregulating ALDH1B1 expression

Nonalcoholic steatohepatitis (NASH) is a prevalent chronic disease, yet its exact mechanisms and effective treatments remain elusive. Nuclear receptor subfamily 5 group A member 2 (NR5A2), a transcription factor closely associated with cholesterol metabolism in the liver, has been hindered from comprehensive investigation due to the lethality of NR5A2 loss in cell lines and animal models. To elucidate the role of NR5A2 in NASH, we generated hepatocyte-specific knockout mice for Nr5a2 (Nr5a2HKO) and examined their liver morphology across different age groups under a regular diet. Furthermore, we established cell lines expressing haploid levels of NR5A2 and subsequently reintroduced various isoforms of NR5A2. In the liver of Nr5a2HKO mice, inflammation and fibrosis spontaneously emerged from an early age, independent of lipid accumulation. Pyroptosis occurred in NR5A2-deficient cell lines, and different isoforms of NR5A2 reversed this form of cell death. Our findings unveiled that inhibition of NR5A2 triggers pyroptosis, a proinflammatory mode of cell death primarily mediated by the activation of the NF-κB pathway induced by reactive oxygen species (ROS). As a transcriptionally regulated molecule of NR5A2, aldehyde dehydrogenase 1 family member B1 (ALDH1B1) participates in pyroptosis through modulation of ROS level. In conclusion, the diverse isoforms of NR5A2 exert hepatoprotective effects against NASH by maintaining a finely tuned balance of ROS, which is contingent upon the activity of ALDH1B1.

Isoform-level expression of the constitutive androstane receptor (CAR or NR1I3) transcription factor better predicts the mRNA expression of the cytochrome P450s in human liver samples.

Many factors cause inter-person variability in the activity and expression of liver cytochrome P450 (CYP) drug-metabolizing enzymes, leading to variable drug exposure and treatment outcomes. Several liver-enriched transcription factors (TFs) are associated with CYP expression, with estrogen receptor alpha (ESR1) and constitutive androstane receptor (CAR or NR1I3) being the two top factors. ESR1 and NR1I3 undergo extensive alternative splicing that results in numerous splice isoforms, but how these splice isoforms associate with CYP expression is unknown. Here, we quantified 18 NR1I3 splice isoforms and the three most abundant ESR1 isoforms in 260 liver samples derived from African Americans (AA, n=125) and European Americans (EA, n=135). Our results showed variable splice isoform populations in the liver for both NR1I3 and ESR1. Multiple linear regression analyses revealed that, compared to gene-level NR1I3, isoform-level NR1I3 expression better predicted the mRNA expression of most CYPs and three UDP-glucuronosyltransferases (UGTs), while ESR1 isoforms improved predictive models for the UGTs and CYP2D6, but not for most CYPs. Also, different NR1I3 isoforms were associated with different CYPs, and the associations varied depending on sample ancestry. Surprisingly, non-canonical NR1I3 isoforms having retained introns (introns 2 or 6) were abundantly expressed and associated with the expression of most CYPs and UGTs, whereas the reference isoform (NR1I3-205) only associated with CYP2D6. Moreover, NR1I3 isoform diversity increased during the differentiation of induced pluripotent stem cells to hepatocytes, paralleling increasing CYP expression. These results suggest that isoform-level TF expression may help to explain variation in CYP or UGT expression between individuals. Significance Statement We quantified 18 NR1I3 splice isoforms and three ESR1 splice isoforms in 260 liver samples derived from AA and EA donors and found variable NR1I3 and ESR1 splice isoform expression in the liver. Multiple linear regression analysis showed that, compared to gene-level expression, isoform-level expression of NR1I3 and ESR1 better predicted the mRNA expression of some CYPs and UGTs, highlighting the importance of isoform-level analyses to enhance our understanding of gene transcriptional regulatory networks controlling the expression of drug-metabolizing enzymes.

MECOM Locus classical transcript isoforms affect tumor immune microenvironment and different targets in ovarian cancer

The MECOM locus is a gene frequently amplified in high-grade serous ovarian carcinoma (HGSOC). Nevertheless, the body of research examining the associations among MECOM transcripts, patient prognosis, and their role in modulating the tumor immune microenvironment (TIME) remains sparse, particularly in large cohorts. This study assessed the expression of MECOM transcripts in 352 HGSOC patients and 88 normal ovarian tissues from the combined GTEx/TCGA database. Using resources such as the UCSC Genome Browser, Ensembl, and NextProt, two transcripts corresponding to classical protein isoforms from MECOM were identified. Cox proportional hazards regression analysis, Kaplan-Meier survival curves, and a comprehensive TIME evaluation algorithm were employed to elucidate the connections between the expression levels of these transcripts and both patient prognosis and TIME status. Chromatin Immunoprecipitation sequencing (ChIP-seq) data for the two protein isoforms, as well as RNA sequencing data post-targeted silencing, were analyzed to identify potential regulatory targets of the different transcription factors. Elevated expression of the MECOM isoform transcripts was correlated with poorer survival in HGSOC patients, potentially through the modulation of cancer-associated fibroblasts (CAFs) and immunosuppressive cell populations. In contrast, higher levels of EVI1 isoform transcripts were linked to enhanced survival, possibly due to the regulation of CD8+ T cells, macrophages, and a reduction in the expression of JUN protein, or its DNA-binding activity on downstream genes. Diverse protein isoforms derived from MECOM were found to differentially affect the survival and tumor development in ovarian cancer patients through specific mechanisms. Investigating the molecular mechanisms underlying disease pathogenesis and identifying potential drug target proteins at the level of splice variant isoforms were deemed crucial.

The alternative splicing landscape of infarcted mouse heart identifies isoform level therapeutic targets

Alternative splicing is an important process that contributes to highly diverse transcripts and protein products, which can affect the development of disease in various organisms. Cardiovascular disease (CVD) represents one of the greatest global threats to humans, particularly acute myocardial infarction (MI) and subsequent ischemic reperfusion (IR) injury, which involve complex transcriptomic changes in heart tissues associated with metabolic reshaping and immunological response. In this study, we used a newly developed ONT full-length transcriptomic approach and performed transcript-resolved differential expression profiling in murine models of MI and IR. We built an analytical pipeline to reliably identify and quantify alternative splicing products (isoforms), expanding on the currently available catalog of isoforms described in mice. The updated alternative splicing landscape included transcripts, genes, and pathways that were differentially regulated during IR and MI. Our study establishes a pipeline to profile highly diverse isoforms using state-of-the-art long-read sequencing, builds a landscape of alternative splicing in the mouse heart during MI and IR.

8773 CD44 Variant Isoforms Expressed Within PAX7/SOX2-Positive Pituitary Neuroendocrine Tumors Attribute To Therapy Resistance And Recurrence Of Cushing’s Disease

Abstract Disclosure: J. Chakrabarti: None. J. Eschbacher: None. S. Mallick: None. E. Ferris: None. B. Hermes: None. A. Little: None. Y. Zavros: None. Background: Cushing’s disease (CD) is a serious rare endocrine disease caused by an adrenocorticotropic hormone (ACTH)-secreting pituitary neuroendocrine tumor (PitNET) that subsequently stimulates the adrenal glands to overproduce cortisol. Approximately 56% of patients will experience disease recurrence during the 2 to 10-year follow-up period. Surgical failures and late recurrences of CD are common, and despite multiple treatments, biochemical control is achieved in only 50% of patients. Therefore, there is a need to advance targeted therapies that prevent recurrence and therapy resistance. The Cluster of Differentiation (CD) 44 transmembrane glycoprotein (CD44) is a prominent WNT signaling target, and deregulation of the Wnt pathway is associated with constitutively active βcatenin in SOX2-positive cells. Although a similar mechanism has been proposed in PitNETs, it has not been fully tested. The alternative mRNA splice variant, CD44v9 is expressed on cancer stem cells promotes resistance to ROS through the stabilization of the SLC7A11 (xCT) cystine glutamate transporter. Proteolytic cleavage of CD44 releases the CD44 intracellular domain which translocates to the nucleus activating cell stemness genes including SOX2. Although CD44 is a known cancer stem cell marker that plays multiple key roles including drug resistance and tumor cell invasiveness, the mechanisms underlying human PitNET tumorigenesis are unclear. Hypothesis: CD44 expressed on PAX7/SOX2-positive corticotroph PitNET cells define a cell population that attributes to therapy resistance and disease recurrence. Methods: CD patient PitNET tissues and matched PitNET-generated organoids (PitNETOs) were used for CosMx™ Spatial Molecular Imaging (SMI) and scRNAseq analyses respectively. Resistance to drug- and radiation-induced apoptosis was analyzed using PitNETOs generated. Results: SMI and scRNAseq data revealed a cell population co-expressing PAX7 and SOX2, CD44 and EpCAM that was abundant in invasive and silent corticotroph PitNET subtypes with Knosp grades 3-4. Invasive and silent corticotroph PitNETs also expressed an increased cell population of CD163+ macrophages, myofibroblast cancer associated fibroblasts (myCAFs), inflammatory CAFs. While cells isolated from the dura revealed growth of PitNETOs, adherent cells expressed increased proliferation (Ki67), and expression of somatostatin receptor subtype 2, cancer stem markers (CD44, SOX2, NESTIN, FUT4, PROM1), PAX7 progenitor cell population expressing stem cell markers NESTIN, SOX2, CD44 and EpCAM. Increased CD44/SLCA7A11 expression in CD44/PAX7/SOX2-positive PitNETOs rendered tumor cells resistant to radiation-induced apoptosis. Conclusions: Drugs targeting the CD44 variant isoforms may increase the efficacy of medical therapies and stereotactic radiosurgery to prevent residual and recurrent PitNETs in CD patients. Presentation: 6/3/2024

One-Pot multicomponent synthesis of novel pyrazole-linked thiazolyl-pyrazolines: Molecular docking and cytotoxicity assessment on breast and lung cancer cell-lines

Cancer remains a foremost cause of mortality globally, with approximately 20 million new cases and nearly 10 million deaths reported in 2022. Lung and breast cancers are particularly prevalent and deadly, underscoring the critical need for novel therapeutic strategies. This study utilized a one-pot, multicomponent reaction (MCR) approach to synthesize a novel series of pyrazole-linked thiazolyl-pyrazolines. The optimal reaction conditions were determined by systematically varying bases, temperatures, reaction times, and solvents. The best yield was achieved using NaOH as the base at 50 °C with 2 h of stirring in ethanol. The synthesized compounds underwent thorough characterization through IR, mass spectrometry, and NMR spectroscopy. Molecular docking studies indicated strong binding affinities to the ErbB4 kinase, suggesting potential utility as kinase inhibitors. Compounds 5E, 5F, and 5G exhibited significant cytotoxicity against breast cancer cell lines, with IC50 values ranging from 15.79±1.272 to 28.38±1.520 µM. Meanwhile, compounds 5A, 5B, 5D, and 5G demonstrated potent effects against lung cancer cell lines, with IC50 values between 7.945±0.99 and 9.295±1.074 µM. Remarkably, compound 5G showed potent efficacy against both breast and lung cancers, with IC50 values of 28.38±1.520 µM and 9.02±1.07 µM, respectively. This study illuminates the critical role of pyrazole-linked thiazolyl-pyrazoline scaffolds in crafting powerful cancer therapeutics targeting ErbB4 (HER4) kinase. Unique in its dual functionality, ErbB4 can act as either an oncogene or a tumor suppressor in lung and breast cancers, contingent on specific cellular contexts and isoform variations. The dynamic expression of ErbB4 intricately modulates tumor growth and patient outcomes, marking it as a compelling therapeutic target for innovative treatments in both breast and lung cancer.

NADPH oxidases in healthy white adipose tissue and in obesity: function, regulation, and clinical implications.

Reactive oxygen species, when produced in a controlled manner, are physiological modulators of healthy white adipose tissue (WAT) expansion and metabolic function. By contrast, unbridled production of oxidants is associated with pathological WAT expansion and the establishment of metabolic dysfunctions, most notably insulin resistance and type 2 diabetes mellitus. NADPH oxidases (NOXs) produce oxidants in an orderly fashion and are present in adipocytes and in other diverse WAT-constituent cell types. Recent studies have established several links between aberrant NOX-derived oxidant production, adiposity, and metabolic homeostasis. The objective of this review is to highlight the physiological roles attributed to diverse NOX isoforms in healthy WAT and summarize current knowledge of the metabolic consequences related to perturbations in their adequate oxidant production. We detail WAT-related alterations in preclinical investigations conducted in NOX-deficient murine models. In addition, we review clinical studies that have employed NOX inhibitors and currently available data related to human NOX mutations in metabolic disturbances. Future investigations aimed at understanding the integration of NOX-derived oxidants in the regulation of the WAT cellular redox network are essential for designing successful redox-related precision therapies to curb obesity and attenuate obesity-associated metabolic pathologies.

Fine mapping of RNA isoform diversity using an innovative targeted long-read RNA sequencing protocol with novel dedicated bioinformatics pipeline.

Solving the structure of mRNA transcripts is a major challenge for both research and molecular diagnostic purposes. Current approaches based on short-read RNA sequencing and RT-PCR techniques cannot fully explore the complexity of transcript structure. The emergence of third-generation long-read sequencing addresses this problem by solving this sequence directly. However, genes with low expression levels are difficult to study with the whole transcriptome sequencing approach. To fix this technical limitation, we propose a novel method to capture transcripts of a gene panel using a targeted enrichment approach suitable for Pacific Biosciences and Oxford Nanopore Technologies platforms. We designed a set of probes to capture transcripts of a panel of genes involved in hereditary breast and ovarian cancer syndrome. We present SOSTAR (iSofOrmS annoTAtoR), a versatile pipeline to assemble, quantify and annotate isoforms from long read sequencing using a new tool specially designed for this application. The significant enrichment of transcripts by our capture protocol, together with the SOSTAR annotation, allowed the identification of 1,231 unique transcripts within the gene panel from the eight patients sequenced. The structure of these transcripts was annotated with a resolution of one base relative to a reference transcript. All major alternative splicing events of the BRCA1 and BRCA2 genes described in the literature were found. Complex splicing events such as pseudoexons were correctly annotated. SOSTAR enabled the identification of abnormal transcripts in the positive controls. In addition, a case of unexplained inheritance in a family with a history of breast and ovarian cancer was solved by identifying an SVA retrotransposon in intron 13 of the BRCA1 gene. We have validated a new protocol for the enrichment of transcripts of interest using probes adapted to the ONT and PacBio platforms. This protocol allows a complete description of the alternative structures of transcripts, the estimation of their expression and the identification of aberrant transcripts in a single experiment. This proof-of-concept opens new possibilities for RNA structure exploration in both research and molecular diagnostics.

Subtranscriptome analysis of phospholipases D in Loxosceles venom glands: Confirmation of predominance, intra-species diversity, and description of novel isoforms

Spiders of Loxosceles genus, or Brown spiders produce a potent venom with minimal volume and protein content. Among its toxins, phospholipases D (PLDs) are notable for causing primary local and systemic manifestations observed following envenomation. They degrade cellular phospholipids, mainly sphingomyelin and lysophosphatidylcholine. We present a robust and detailed analysis of PLD transcripts from venom glands of three major clinically relevant South American species—L. intermedia, L. laeta, and L. gaucho—using next-generation sequencing. Results confirmed that PLDs are the most highly expressed toxins, accounting for 65.4 % of expression in L. intermedia, 71.8 % in L. gaucho, and 50.4 % in L. laeta. These findings further support the idea that these enzymes form a protein family both within and across species. Eighteen contigs for PLDs were found for L. gaucho, 24 for L. intermedia, and 21 for L. laeta. A detailed analysis revealed that, although all contigs display conserved amino acid residues directly involved in catalysis, magnesium coordination, and substrate affinity, they also possess distinct primary sequences with important substitutions. Such data reinforces the hypothesis that these toxins may act synergistically. Furthermore, new PLD sequences were identified within the contigs. For L. intermedia, 14 potential new isoforms were identified; 16 for L gaucho; and 16 novel sequences for L. laeta. This indicates that there is still a wealth of undisclosed information about these toxins. These data will help identify structural and functional differences among these proteins, support future functional studies, and to the comprehensive understanding of the mechanism of action of PLDs.

The intestinal mucin isoform landscape reveals region-specific biomarker panels for inflammatory bowel disease patient stratification.

Mucosal healing is considered as a key therapeutic endpoint in inflammatory bowel diseases (IBD) and comprises endoscopic improvement of inflammation without taking barrier healing into account. Mucins are critical components of the mucosal barrier function that give rise to structurally diverse isoforms. Unraveling disease-associated mucin isoforms that could act as an indication for barrier function would greatly enhance IBD management. We present the intestinal mucin RNA isoform landscape in IBD and control patients using a targeted mucin isoform sequencing approach on a discovery cohort (n = 106). Random Forest modeling (n = 1683 samples) with external validation (n = 130 samples) identified unique mucin RNA isoform panels that accurately stratified IBD patients in multiple subpopulations based on inflammation, IBD subtype (Crohn's disease (CD), ulcerative colitis (UC)), and anatomical location of the intestinal tract (i.e. ileum, proximal colon, distal colon, rectum). Particularly, the mucin RNA isoform panels obtained from the inflamed UC and CD distal colon showed high performance in distinguishing inflamed biopsies from their control counterparts (AUC of 93.3% and 91.1% in the training, 95.0% and 96.0% in the test, and 89.5% and 78.3% in the external validation datasets, respectively). Furthermore, the differentially expressed MUC4 (PB.1238.363), MUC5AC (PB.2811.15), MUC16 (ENST00000397910.8) and MUC1 (ENST00000462317.5, ENST00000620103.4) RNA isoforms frequently occurred throughout the different panels highlighting their role in IBD pathogenesis. We unveiled region-specific mucin RNA isoform panels capturing the heterogeneity of the IBD patient population and showing great potential to indicate barrier function in IBD patients.

RNA in axons, dendrites, synapses and beyond.

In neurons, a diverse range of coding and non-coding RNAs localize to axons, dendrites, and synapses, where they facilitate rapid responses to local needs, such as axon and dendrite extension and branching, synapse formation, and synaptic plasticity. Here, we review the extent of our current understanding of RNA subclass diversity in these functionally demanding subcellular compartments. We discuss the similarities and differences identified between axonal, dendritic and synaptic local transcriptomes, and discuss the reported and hypothesized fates and functions of localized RNAs. Furthermore, we outline the RNA composition of exosomes that bud off from neurites, and their implications for the biology of neighboring cells. Finally, we highlight recent advances in third-generation sequencing technologies that will likely provide transformative insights into splice isoform and RNA modification diversity in local transcriptomes.