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The polyploid genome of cotton has significantly increased the transcript complexity. Recent advances in full-length transcript sequencing are now widely used to characterize the complete landscape of transcriptional events. Such studies in cotton can help us to explore the genetic mechanisms of the cotton seedling growth. Through long-read single-molecule RNA sequencing, this study compared the transcriptomes of three yield contrasting genotypes of upland cotton. Our analysis identified different numbers of spliced isoforms from 31,166, 28,716, and 28,713 genes in SJ48, Z98, and DT8 cotton genotypes, respectively, most of which were novel compared to previous cotton reference transcriptomes, and showed significant differences in the number of exon structures and coding sequence length due to intron retention. Quantification of isoform expression revealed significant differences in expression in the root and leaf of each genotype. An array of key isoform target genes showed protein kinase or phosphorylation functions, and their protein interaction network contained most of the circadian oscillator proteins. Spliced isoforms from the GIGANTEA (GI) protien were differentially regulated in each genotype and might be expected to regulate translational activities, including the sequence and function of target proteins. In addition, these spliced isoforms generate diurnal expression profiles in cotton leaves, which may alter the transcriptional regulatory network of seedling growth. Silencing of the novel spliced GI isoform Gh_A02G0645_N17 significantly affected biomass traits, contributed to variable growth, and increased transcription of the early flowering pathway gene ELF in cotton. Our high-throughput hybrid sequencing results will be useful to dissect functional differences among spliced isoforms in the polyploid cotton genome.

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Gustavus, a positive regulator in arthropod reproduction, features a conserved SPRY and a C-terminal SOCS box domain and belongs to the SPSB protein family. The SPSB family, encompassing SPSB1 to SPSB4, plays pivotal roles in higher animals, including immune response, apoptosis, growth, and stress responses. In Neocaridina denticulata sinensis, alternative splicing yielded two NdGustavus isoforms, NdGusX1 and NdGusX2, with distinct expression patterns-high in ovaries and muscles, respectively, and across all ovarian germ cells. These isoforms showed similar expression dynamics during embryogenesis and significant upregulation post-copper ion exposure (P < 0.05). The in situ hybridization result elucidated that NdGusX1 and NdGusX2 were expressed across the germ cell spectrum in the ovary, with NdGusX1 showing enhanced expression in oogonia and primary oocytes. In addition, RNA interference revealed functional complementation in ovaries and potential functional differentiation in muscles. Knockdown of NdGusX1 and NdGusX2 potentially disrupted endogenous vitellogenin synthesis, regulating vitellogenesis and reducing mature oocyte volume, affecting follicular cavity occupation. This study provides a theoretical framework for understanding the biological functions of the SPSB family in crustacean ovarian maturation.

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CD1 isoforms are MHC class I-like molecules that present lipid-antigens to T cells and have been associated with a variety of immune responses. The lipid repertoire bound and presented by the four CD1 isoforms may be influenced by factors such as the cellular lipidome, subcellular microenvironment, and the properties of the binding pocket. In this study, by shotgun mass spectrometry, we performed a comprehensive lipidomic analysis of soluble CD1 molecules. We identified 1040 lipids, of which 293 were present in all isoforms. Comparative analysis revealed that the isoforms bind almost any cellular lipid.CD1a and CD1c closely mirrored the cellular lipidome, while CD1b and CD1d showed a preference for sphingolipids. Each CD1 isoform was found to have unique lipid species, suggesting some distinct roles in lipid presentation and immune responses. These findings contribute to our understanding of the role of CD1 system in immunity and could have implications for the development of lipid-based therapeutics.

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PRLΔE1, a retina-specific isoform of prolactin, is expressed in multiple and diverse forms of canine inherited retinal degeneration (IRD). We find that while PRLΔE1 expression in rods is not associated with the initial phase of disease characterized by acute photoreceptor cell death, it is associated with the protracted phase of slow cell loss. Restoration of photoreceptors to a healthy state by gene-specific replacement therapy of individual IRDs successfully suppresses PRLΔE1 expression. Moreover, short-term PRLΔE1 silencing using shRNA results in preservation of outer nuclear layer thickness, suggesting PRLΔE1 drives retinal disease. However, longer-term observations reveal off-target toxic effects of the PRLΔE1 shRNA, precluding determination of its full therapeutic potential. Future research efforts aimed at enhancing the safety and specificity of PRLΔE1-targeting strategies may identify a potential universal intervention strategy for sustaining photoreceptors during the prolonged phase of multiple IRDs.

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The 24 claudin (CLDN) genes in the human genome encode 26 representative CLDN family proteins. CLDNs are tetraspan­transmembrane proteins at tight junctions. Because several CLDN isoforms, such as CLDN6 and CLDN18.2, are specifically upregulated in human cancer, CLDN­targeting monoclonal antibodies (mAbs), antibody­drug conjugates (ADCs), bispecific antibodies (bsAbs) and chimeric antigen receptor (CAR) T cells have been developed. In the present review, CLDN1­, 4­, 6­ and 18.2­targeting investigational drugs in clinical trials are discussed. CLDN18.2­directed therapy for patients with gastric and other types of cancer is the most advanced area in this field. The mouse/human chimeric anti­CLDN18.2 mAb zolbetuximab has a single­agent objective response rate (ORR) of 9%, and increases progression­free and overall survival in combination with chemotherapy. The human/humanized anti­CLDN18.2 mAb osemitamab, and ADCs AZD0901, IBI343 and LM­302, with single­agent ORRs of 28­60%, have been tested in phase III clinical trials. In addition, bsAbs, CAR T cells and their derivatives targeting CLDN4, 6 or 18.2 are in phase I and/or II clinical trials. AZD0901, IBI343, zolbetuximab and the anti­CLDN1 mAb ALE.C04 have been granted fast track designation or priority review designation by the US Food and Drug Administration.

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Transcripts from the human WDR33 gene, which encodes a central component of the mRNA polyadenylation (PA) machinery, are subject to alternative polyadenylation (APA) within promoter-proximal introns/exons. This APA, which itself involves usage of multiple PA sites, results in the production of two non-canonical protein isoforms, V2 and V3, that are functionally completely unrelated to the full-length protein, with roles in innate immunity. The mechanism and regulation of WDR33 APA are unclear. Here, we report that levels of the PA factor CFIm25 modulate V2 and V3 expression, and that PA site usage of both V2 and V3 varies in distinct immune responses. Using newly developed assays to measure splicing and PA site strength, we show that splicing of V2-associated intron 6 is inefficient, allowing V2 to be produced using weak PA sites. Usage of V3's strong PA sites, on the other hand, is relatively low, reflecting the high efficiency of intron 7 splicing coupled with dependency on usage of an alternative 3' splice site within the intron. Overall, our findings demonstrate that usage of WDR33 alternative PA sites is stochastic, dependent on a complex interplay between splicing and PA, and thus provide new insights into mechanisms underlying APA.

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Germline variants in the NSD1 gene are responsible for Sotos syndrome, while somatic variants promote neoplastic cell transformation. Our previous studies revealed three alternative RNA isoforms of NSD1 present in fibroblast cell lines (FBs): the canonical full transcript and 2 alternative transcripts, termed AT2 (NSD1 Δ5Δ7) and AT3 (NSD1 Δ19-23 at the 5' end). The precise molecular pathways affected by each specific isoform of NSD1 are uncharacterized to date. To elucidate the role of these isoforms, their expression was suppressed by siRNA knockdown in FBs and protein expression and transcriptome data was explored. We demonstrate that one gene target of NSD1 isoform AT2 is ARP3 actin-related protein 3 homolog B (ACTR3B). We show that loss of both canonical NSD1 and AT2 isoforms impaired the ability of fibroblasts to regulate the actin cytoskeleton, and we observed that this caused selective loss of stress fibers. Our findings provide novel insights into NSD1 function by distinguishing isoform function and demonstrating an essential role of NSD1 in regulating the actin cytoskeleton and stress fiber formation in fibroblasts.

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Signal transducer and activator of transcription 3 (STAT3) is a pleiotropic factor involved in multiple vital biological processes and a key mediator of gene transcription in response to cytokines, growth factors and aberrant activation of oncogenic signaling. STAT3 has two splicing isoforms, STAT3α and STAT3ß, derived from alternative splicing of exon 23 within pre-mRNA. STAT3ß differs from STAT3α by replacement of 55 amino-acid residues in the C-terminal transactivation domain with 7 specific amino acids. Thus, a shorter STAT3ß was originally regarded as a dominant negative isoform of STAT3α. Recently accumulating evidence from independent studies have shown STAT3 splicing isoforms confer distinct and overlapping functions in many fundamental cellular regulatory steps such as cell differentiation, inflammatory responses, and cancer progression. However, relatively little is known about the mechanisms of STAT3 pre-mRNA splicing, and it remains undiscovered which chemical compounds or bioactive substances can induce the STAT3ß expression. In this study, we generated a potent reporter for detection of alternative splicing of STAT3 pre-mRNA optimized for the screening of function-known chemical library, and successfully identified entinostat, a histone deacetylase inhibitor, as a novel inducer of STAT3ß through modulating mRNA splicing. Our findings demonstrate that alternative splicing of STAT3 can be regulated by a compound, providing an important clue for understanding the regulation mechanisms of the expression balance of STAT3 isoforms in a chemical biology approach. Entinostat is likely to be a promising seed compound for elucidating how the higher ratio of STAT3ß expression impacts on biological responses associated with Janus kinase (JAK)/STAT3 signaling pathway.

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Single-cell multi-omics sequencing is a powerful approach to analyze complex mechanisms underlying neuronal development and regeneration. However, current methods lack the ability to simultaneously profile RNA alternative splicing and chromatin accessibility at the single-cell level. We develop a technique, single-cell RNA isoform and chromatin accessibility sequencing (scRICA-seq), which demonstrates higher sensitivity and cost-effectiveness compared to existing methods. scRICA-seq can profile both isoforms and chromatin accessibility for up to 10,000 single cells in a single run. Applying this method to human retinal organoids, we construct a multi-omic cell atlas and reveal associations between chromatin accessibility, isoform expression of fate-determining factors, and alternative splicing events in their binding sites. This study provides insights into integrating epigenetics, transcription, and RNA splicing to elucidate the mechanisms underlying retinal neuronal development and fate determination.

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Background: Glyoxalase domain containing protein 4 (GLOD4), a protein of an unknown function, is associated with Alzheimer's disease (AD). Three GLOD4 isoforms are known. The mechanism underlying GLOD4's association with AD was unknown. Objective: To assess GLOD4's role in the central nervous system by studying GLOD4 isoforms expression in human frontal cerebral cortical tissues from AD patients and in brains of Blmh-/-5xFAD mouse AD model of AD. Methods: GLOD4 protein and mRNA were quantified in human and mouse brains by western blotting and RT-qPCR, respectively. Mouse brain amyloid-ß (Aß) was quantified by western blotting. Behavioral assessments of mice were performed by cognitive/neuromotor testing. Glod4 gene in mouse neuroblastoma N2a-APPswe cells was silenced by RNA interference and Glod4, Aß precursor protein (Aßpp), Atg5, p62, and Lc3 proteins and mRNAs were quantified. Results: GLOD4 mRNA and protein isoforms were downregulated in cortical tissues from AD patients compared to non-AD controls. Glod4 mRNA was downregulated in brains of Blmh-/-5xFAD mice compared to Blmh+/+5xFAD sibling controls, but not in Blmh-/- mice without the 5xFAD transgene compared to Blmh+/+ sibling controls. The 5xFAD transgene downregulated Glod4 mRNA in Blmh-/- mice of both sexes and in Blmh+/+ males but not females. Attenuated Glod4 was associated with elevated Aß and worsened memory/sensorimotor performance in Blmh-/-5xFAD mice. Glod4 depletion in N2a-APPswe cells upregulated AßPP, and downregulated autophagy-related Atg5, p62, and Lc3 genes. Conclusions: These findings suggest that GLOD4 interacts with AßPP and the autophagy pathway, and that disruption of these interactions leads to Aß accumulation and cognitive/neurosensory deficits.

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(1) Background: Periostin (Pn) is a secreted protein found in the extracellular matrix, and it plays a variety of roles in the human body. Physiologically, Pn has a variety of functions, including bone formation and wound healing. However, it has been implicated in the pathogenesis of various malignant tumors and chronic inflammatory diseases. Pn has alternative splicing variants (ASVs), and our previous research revealed that aberrant ASVs contribute to the pathogenesis of breast cancer and heart failure. However, the difference in expression pattern between physiologically expressed Pn-ASVs and those expressed during pathogenesis is not clear. (2) Methods and results: We examined normal and breast cancer tissues, focusing on the Pn-ASVs expression pattern to assess the significance of pathologically expressed Pn-ASVs as potential diagnostic and therapeutic targets. We found that most physiologically expressed Pn isoforms lacked exon 17 and 21. Next, we used human breast cancer and normal adjacent tissue (NAT) to investigate the expression pattern of Pn-ASVs under pathological conditions. Pn-ASVs with exon 21 were significantly increased in tumor tissues compared with NAT. In situ hybridization identified the synthesis of Pn-ASVs with exon 21 in peri-tumoral stromal cells. Additionally, the in vivo bio-distribution of 89Zr-labeled Pn antibody against exon 21 (Pn-21Ab) in mice bearing breast cancer demonstrated selective and specific accumulation in tumors, while Pn-21Ab significantly suppressed tumor growth in the mouse breast cancer model. (3) Conclusions: Together, these data indicate that Pn-ASVs might have potential for use as diagnostic and therapeutic targets for breast cancer.

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Vairimorpha (Nosema) ceranae is a single-cellular fungus that obligately infects the midgut epithelial cells of adult honeybees, causing bee microsporidiosis and jeopardizing bee health and production. This work aims to construct the full-length transcriptome of V. ceranae and conduct a relevant investigation using PacBio single-molecule real-time (SMRT) sequencing technology. Following PacBio SMRT sequencing, 41,950 circular consensus (CCS) were generated, and 25,068 full-length non-chimeric (FLNC) reads were then detected. After polishing, 4387 high-quality, full-length transcripts were gained. There are 778, 2083, 1202, 1559, 1457, 1232, 1702, and 3896 full-length transcripts that could be annotated to COG, GO, KEGG, KOG, Pfam, Swiss-Prot, eggNOG, and Nr databases, respectively. Additionally, 11 alternative splicing (AS) events occurred in 6 genes were identified, including 1 alternative 5' splice-site and 10 intron retention. The structures of 225 annotated genes in the V. ceranae reference genome were optimized, of which 29 genes were extended at both 5' UTR and 3' UTR, while 90 and 106 genes were, respectively, extended at the 5' UTR as well as 3' UTR. Furthermore, a total of 29 high-confidence lncRNAs were obtained, including 12 sense-lncRNAs, 10 lincRNAs, and 7 antisense-lncRNAs. Taken together, the high-quality, full-length transcriptome of V. ceranae was constructed and annotated, the structures of annotated genes in the V. ceranae reference genome were improved, and abundant new genes, transcripts, and lncRNAs were discovered. Findings from this current work offer a valuable resource and a crucial foundation for molecular and omics research on V. ceranae.

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Omecamtiv mecarbil (OM) is a small molecule that has been shown to improve the function of the slow human ventricular myosin (MyHC) motor through a complex perturbation of the thin/thick filament regulatory state of the sarcomere mediated by binding to myosin allosteric sites coupled to inorganic phosphate (Pi) release. Here, myofibrils from samples of human left ventricle (ß-slow MyHC-7) and left atrium (α-fast MyHC-6) from healthy donors were used to study the differential effects of µmolar [OM] on isometric force in relaxing conditions (pCa 9.0) and at maximal (pCa 4.5) or half-maximal (pCa 5.75) calcium activation, both under control conditions (15 °C; equimolar DMSO; contaminant inorganic phosphate [Pi] ~170 µM) and in the presence of 5 mM [Pi]. The activation state and OM concentration within the contractile lattice were rapidly altered by fast solution switching, demonstrating that the effect of OM was rapid and fully reversible with dose-dependent and myosin isoform-dependent features. In MyHC-7 ventricular myofibrils, OM increased submaximal and maximal Ca2+-activated isometric force with a complex dose-dependent effect peaking (40% increase) at 0.5 µM, whereas in MyHC-6 atrial myofibrils, it had no effect or-at concentrations above 5 µM-decreased the maximum Ca2+-activated force. In both ventricular and atrial myofibrils, OM strongly depressed the kinetics of force development and relaxation up to 90% at 10 µM [OM] and reduced the inhibition of force by inorganic phosphate. Interestingly, in the ventricle, but not in the atrium, OM induced a large dose-dependent Ca2+-independent force development and an increase in basal ATPase that were abolished by the presence of millimolar inorganic phosphate, consistent with the hypothesis that the widely reported Ca2+-sensitising effect of OM may be coupled to a change in the state of the thick filaments that resembles the on-off regulation of thin filaments by Ca2+. The complexity of this scenario may help to understand the disappointing results of clinical trials testing OM as inotropic support in systolic heart failure compared with currently available inotropic drugs that alter the calcium signalling cascade.

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Thyroid hormone receptor alpha (THRα) is a nuclear hormone receptor that binds triiodothyronine (T3) and acts as an important transcription factor in development, metabolism, and reproduction. In mammals, THRα has two major splicing isoforms, THRα1 and THRα2. The better-characterized isoform, THRα1, is a transcriptional stimulator of genes involved in cell metabolism and growth. The less-well-characterized isoform, THRα2, lacks the ligand-binding domain (LBD) and is thought to act as an inhibitor of THRα1 activity. The ratio of THRα1 to THRα2 splicing isoforms is therefore critical for transcriptional regulation in different tissues and during development. However, the expression patterns of both isoforms have not been studied in healthy human tissues or in the developing brain. Given the lack of commercially available isoform-specific antibodies, we addressed this question by analyzing four bulk RNA-sequencing datasets and two scRNA-sequencing datasets to determine the RNA expression levels of human THRA1 and THRA2 transcripts in healthy adult tissues and in the developing brain. We demonstrate how 10X Chromium scRNA-seq datasets can be used to perform splicing-sensitive analyses of isoforms that differ at the 3'-end. In all datasets, we found a strong predominance of THRA2 transcripts at all examined stages of human brain development and in the central nervous system of healthy human adults.

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Most protein-coding genes produce multiple protein isoforms; however, these isoforms are commonly neglected in drug discovery. The expression of protein isoforms can be specific to a disease, tissue and/or developmental stage, and this specific expression can be harnessed to achieve greater drug specificity than pan-targeting of all gene products and to enable improved treatments for diseases caused by aberrant protein isoform production. In recent years, several protein isoform-centric therapeutics have been developed. Here, we collate these studies and clinical trials to highlight three distinct but overlapping modes of action for protein isoform-centric drugs: isoform switching, isoform introduction or depletion, and modulation of isoform activity. In addition, we discuss how protein isoforms can be used clinically as targets for cell type-specific drug delivery and immunotherapy, diagnostic biomarkers and sources of cancer neoantigens. Collectively, we emphasize the value of a focus on isoforms as a route to discovering drugs with greater specificity and fewer adverse effects. This approach could enable the targeting of proteins for which pan-inhibition of all isoforms is toxic and poorly tolerated.

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Style penetration by pollen tubes is essential for reproductive success, a process requiring canonical Rab5s in Arabidopsis. However, functional loss of Arabidopsis Vps9a, the gene encoding for guanine nucleotide exchange factor (GEF) of Rab5s, did not affect male transmission, implying the presence of a compensation program or redundancy. By combining genetic, cytological, and molecular approaches, we report that Arabidopsis Vps9b is a pollen-preferential gene, redundantly mediating pollen tube penetration of style with Vps9a. Vps9b is functionally interchangeable with Vps9a, whose functional distinction results from distinct expression profiles. Functional loss of Vps9a and Vps9b results in the mis-targeting of Rab5-dependent tonoplast proteins, defective vacuolar biogenesis, disturbed distribution of post-Golgi vesicles, increased cellular turgor, cytosolic acidification, and disrupted organization of actin microfilaments (MF) in pollen tubes, which collectively lead to the failure of pollen tubes to grow through style.

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The Growth Hormone Receptor (GHR) gene encodes a protein that is essential for mediating the biological effects of growth hormone (GH). A series of molecular events are set off when GH binds to its receptor, resulting in a variety of physiological reactions linked to development, growth, and metabolism. Recently a particular genetic variation, within the GHR gene that is labeled as the "d3GHR," which lacks exon 3 was associated with longevity. This specific deletion isoform was connected to changes in the structure of the GHR protein, which may have an impact on the GHR's function. To test in vitro the advantage of the d3 carrier that may link to longevity, we employed the CRISPR/Cas9 technique to produce two isoforms: the homozygotes isoform (d3/d3) and the heterozygotes isoform (d3/fl) using HEK293 cell line. The CRISPR editing effectiveness was >85 %, indicating that we had successfully built the Cas9-gRNA complex that is appropriate for the GHR gene. The viability of the resulted isoform cells was examined under three environmental stressors that mimic some aging processes. In addition, we examined the GHR signaling pathway by selecting potential downstream genes in the GHR signaling cascade. The results show that heterozygotes cells demonstrated higher survival rates under UV radiation compared with the WT cells (87 % compared with 67 % for the WT cells when exposed to 2 min of UV radiation), and in fasting conditions, the d3GHR cells showed a 15 % greater viability than the WT cells. Moreover, the baseline expression levels (without intervention) of the IGF1 and JAK/STAT genes signaling pathways significantly declined in the homozygotes cells compared with the WT (p < 0.05). This noteworthy finding might offer a practical approach to test illness prevention and give the scientific community critical new insights on mechanism associated with lifespan.

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PURPOSE OF REVIEW: In humans, tissue factor pathway inhibitor (TFPI) exists in two alternatively spliced isoforms, TFPIα and TFPIß. TFPIα consists of three Kunitz domains (K1, K2 and K3) and a highly basic C-terminal tail. K1 inhibits the tissue factor-activated factor VII complex, K2 specifically inhibits activated factor X, K3 is essential for interaction with its cofactor, protein S, and the basic C-terminus is binds factor V-short (FV-short) with high affinity. TFPIß consists of K1 and K2 that is glycosylphosphatidylinositol anchored directly to cell surfaces. This review explores the structure/function of TFPI and its cofactors (protein S and FV-short), and the relative contributions that different TFPI isoforms may play in haemostatic control. RECENT FINDINGS: Recent data have underscored the importance of TFPIα function and its reliance on its cofactors, protein S and FV-short, in influencing haemostatic control as well as bleeding and thrombotic risk. SUMMARY: TFPIα is likely the most important pool of TFPI in modifying the risk of thrombosis and bleeding. TFPIα forms a trimolecular complex with FV-short and protein S in plasma. FV-short expression levels control the circulating levels of TFPIα, whereas protein S exerts essential cofactor mediated augmentation of it anticoagulant function.

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