RESUMEN
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.
Asunto(s)
Regulación de la Expresión Génica de las Plantas , Gossypium , Plantones , Gossypium/genética , Gossypium/crecimiento & desarrollo , Gossypium/metabolismo , Plantones/genética , Plantones/crecimiento & desarrollo , Plantones/metabolismo , Transcriptoma , Redes Reguladoras de Genes , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Empalme del ARN , Empalme Alternativo , Análisis de Secuencia de ARNRESUMEN
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.
Asunto(s)
Empalme Alternativo , Benzamidas , Piridinas , Precursores del ARN , Factor de Transcripción STAT3 , Factor de Transcripción STAT3/metabolismo , Factor de Transcripción STAT3/genética , Empalme Alternativo/efectos de los fármacos , Humanos , Precursores del ARN/genética , Precursores del ARN/metabolismo , Piridinas/farmacología , Benzamidas/farmacología , Inhibidores de Histona Desacetilasas/farmacología , Células HEK293 , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMEN
Alzheimer's disease is the most common form of dementia, characterized by the pathological accumulation of amyloid-beta (Aß) plaques and tau neurofibrillary tangles. Triggering receptor expressed on myeloid cells 2 (TREM2) is increasingly recognized as playing a central role in Aß clearance and microglia activation in AD. The TREM2 gene transcriptional product is alternatively spliced to produce three different protein isoforms. The canonical TREM2 isoform binds to DAP12 to activate downstream pathways. However, little is known about the function or interaction partners of the alternative TREM2 isoforms. The present study utilized a computational approach in a systematic search for new interaction partners of the TREM2 isoforms by integrating several state-of-the-art structural bioinformatics tools from initial large-scale screening to one-on-one corroborative modeling and eventual all-atom visualization. CD9, a cell surface glycoprotein involved in cell-cell adhesion and migration, was identified as a new interaction partner for two TREM2 isoforms, and CALM, a calcium-binding protein involved in calcium signaling, was identified as an interaction partner for a third TREM2 isoform, highlighting the potential role of cell adhesion and calcium regulation in AD.
Asunto(s)
Empalme Alternativo , Enfermedad de Alzheimer , Glicoproteínas de Membrana , Unión Proteica , Isoformas de Proteínas , Receptores Inmunológicos , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/genética , Humanos , Receptores Inmunológicos/metabolismo , Receptores Inmunológicos/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/genética , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/genética , Biología Computacional/métodosRESUMEN
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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Cromatina , Organoides , Retina , Análisis de la Célula Individual , Humanos , Organoides/metabolismo , Organoides/citología , Cromatina/metabolismo , Cromatina/genética , Retina/metabolismo , Retina/citología , Análisis de la Célula Individual/métodos , Empalme Alternativo , ARN/metabolismo , ARN/genética , Análisis de Secuencia de ARN/métodos , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/genéticaRESUMEN
Insulin-like Growth Factor-1 (IGF-1) is a crucial mitogenic factor with important functions in the mammary gland, mainly through its interaction with the IGF-1 receptor (IGF-1R). This interaction activates a complex signaling network that promotes cell proliferation, epithelial to mesenchymal transition (EMT) and inhibits apoptosis. Despite extensive research, the precise molecular pathways and intracellular mechanisms activated by IGF-1, in cancer, remain poorly understood. Recent evidence highlights the essential roles of IGF-1 and its isoforms in breast cancer (BC) development, progression, and metastasis. The peptides that define the IGF-1 isoforms-IGF-1Ea, IGF-1Eb, and IGF-1Ec-act as key points of convergence for various signaling pathways that influence the growth, metastasis and survival of BC cells. The aim of this review is to provide a detailed exami-nation of the role of the mature IGF-1 and its isoforms in BC biology and their potential use as possible therapeutical targets.
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Neoplasias de la Mama , Factor I del Crecimiento Similar a la Insulina , Isoformas de Proteínas , Humanos , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Factor I del Crecimiento Similar a la Insulina/metabolismo , Isoformas de Proteínas/metabolismo , Femenino , Receptor IGF Tipo 1/metabolismo , Transducción de Señal , Transición Epitelial-Mesenquimal , Animales , Proliferación Celular , Péptidos Similares a la InsulinaRESUMEN
MDM4 is upregulated in the majority of melanoma cases and has been described as a "key therapeutic target in cutaneous melanoma". Numerous isoforms of MDM4 exist, with few studies examining their specific expression in human tissues. The changes in splicing of MDM4 during human melanomagenesis are critical to p53 activity and represent potential therapeutic targets. Compounding this, studies relying on short reads lose "connectivity" data, so full transcripts are frequently only inferred from the presence of splice junction reads. To address this problem, long-read nanopore sequencing was utilized to read the entire length of transcripts. Here, MDM4 transcripts, both alternative and canonical, are characterized in a pilot cohort of human melanoma specimens. RT-PCR was first used to identify the presence of novel splice junctions in these specimens. RT-qPCR then quantified the expression of major MDM4 isoforms observed during sequencing. The current study both identifies and quantifies MDM4 isoforms present in melanoma tumor samples. In the current study, we observed high expression levels of MDM4-S, MDM4-FL, MDM4-A, and the previously undescribed Ensembl transcript MDM4-209. A novel transcript lacking both exons 6 and 9 is observed and named MDM4-A/S for its resemblance to both MDM4-A and MDM4-S isoforms.
Asunto(s)
Melanoma , Isoformas de Proteínas , Humanos , Melanoma/genética , Melanoma/patología , Melanoma/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/metabolismo , Empalme Alternativo , Regulación Neoplásica de la Expresión Génica , Secuenciación de Nanoporos/métodosRESUMEN
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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Empalme Alternativo , Ovario , Animales , Femenino , Ovario/metabolismo , Ovario/crecimiento & desarrollo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Oocitos/metabolismo , Vitelogénesis/genética , Regulación del Desarrollo de la Expresión GénicaRESUMEN
EWS fusion oncoproteins underlie several human malignancies including Desmoplastic Small Round Cell Tumor (DSRCT), an aggressive cancer driven by EWS-WT1 fusion proteins. Here we combine chromatin occupancy and 3D profiles to identify EWS-WT1-dependent gene regulation networks and target genes. We show that EWS-WT1 is a powerful chromatin activator controlling an oncogenic gene expression program that characterizes primary tumors. Similar to wild type WT1, EWS-WT1 has two isoforms that differ in their DNA binding domain and we find that they have distinct DNA binding profiles and are both required to generate viable tumors that resemble primary DSRCT. Finally, we identify candidate EWS-WT1 target genes with potential therapeutic implications, including CCND1, whose inhibition by the clinically-approved drug Palbociclib leads to marked tumor burden decrease in DSRCT PDXs in vivo. Taken together, our studies identify gene regulation programs and therapeutic vulnerabilities in DSRCT and provide a mechanistic understanding of the complex oncogenic activity of EWS-WT1.
Asunto(s)
Ciclina D1 , Tumor Desmoplásico de Células Pequeñas Redondas , Regulación Neoplásica de la Expresión Génica , Proteínas de Fusión Oncogénica , Isoformas de Proteínas , Piridinas , Proteína EWS de Unión a ARN , Humanos , Tumor Desmoplásico de Células Pequeñas Redondas/genética , Tumor Desmoplásico de Células Pequeñas Redondas/metabolismo , Tumor Desmoplásico de Células Pequeñas Redondas/tratamiento farmacológico , Tumor Desmoplásico de Células Pequeñas Redondas/patología , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Animales , Proteína EWS de Unión a ARN/genética , Proteína EWS de Unión a ARN/metabolismo , Piridinas/farmacología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ciclina D1/metabolismo , Ciclina D1/genética , Ratones , Línea Celular Tumoral , Piperazinas/farmacología , Proteínas WT1/genética , Proteínas WT1/metabolismo , Cromatina/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Redes Reguladoras de Genes , FemeninoRESUMEN
Hematological and oncological diseases are still among the leading causes of childhood mortality. Expression of growth hormone-releasing hormone (GHRH) and its receptors (GHRH-R) has been previously demonstrated in various human tumors, but very limited findings are available about the presence and potential function of GHRH-Rs in oncological and hematological disorders of children. In this study, we aimed to investigate the expression of mRNA for GHRH and splice variant 1 (SV) of GHRH-R in 15 pediatric hematological/oncological specimens by RT-PCR. The presence and binding characteristics of GHRH-R protein were also studied by Western blot and ligand competition assays. Of the fifteen specimens studied, eleven pediatric samples (73%) showed the expression of mRNA for GHRH. These eleven samples also expressed mRNA for GHRH receptor SV1. GHRH-R protein was found to be expressed in two benign tumor samples and five malignant tumors examined by Western blot. The presence of specific, high affinity binding sites on GHRH-R was demonstrated in all of the seven human pediatric solid tumor samples investigated. Our results show that the expression of GHRH and SV1 of GHRH-R in hemato-oncological diseases in children can pave the way for further investigation of GHRH-Rs as potential molecular targets for diagnosis and therapy.
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Hormona Liberadora de Hormona del Crecimiento , Neoplasias , Receptores de Neuropéptido , Receptores de Hormona Reguladora de Hormona Hipofisaria , Humanos , Receptores de Hormona Reguladora de Hormona Hipofisaria/genética , Receptores de Hormona Reguladora de Hormona Hipofisaria/metabolismo , Niño , Masculino , Femenino , Receptores de Neuropéptido/genética , Receptores de Neuropéptido/metabolismo , Proyectos Piloto , Hormona Liberadora de Hormona del Crecimiento/genética , Hormona Liberadora de Hormona del Crecimiento/metabolismo , Preescolar , Adolescente , Neoplasias/genética , Neoplasias/metabolismo , Hungría , Lactante , ARN Mensajero/genética , ARN Mensajero/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Enfermedades Hematológicas/genética , Enfermedades Hematológicas/metabolismo , Estudios de CohortesRESUMEN
Angiogenesis, primarily mediated by vascular endothelial growth factor (VEGF), is a fundamental step in the progression and metastasis of head and neck squamous cell carcinoma (HNSCC). Traditional anti-angiogenic therapies that target the VEGF pathway have shown promise but are often associated with significant side effects and variable efficacy due to the complexity of the angiogenic signaling pathway. This review highlights the potential of a specific VEGF splice form, VEGF165b, as an innovative therapeutic target for HNSCC. VEGF165b, unlike standard VEGF, is a natural inhibitor that binds to VEGF receptors without triggering pro-angiogenic signaling. Its distinct molecular structure and behavior suggest ways to modulate angiogenesis. This concept is particularly relevant when studying HNSCC, as introducing VEGF165b's anti-angiogenic properties offers a novel approach to understanding and potentially influencing the disease's dynamics. The review synthesizes experimental evidence suggesting the efficacy of VEGF165b in inhibiting tumor-induced angiogenesis and provides insight into a novel therapeutic strategy that could better manage HNSCC by selectively targeting aberrant vascular growth. This approach not only provides a potential pathway for more targeted and effective treatment options but also opens the door to a new paradigm in anti-angiogenic therapy with the possibility of reduced systemic toxicity. Our investigation is reshaping the future of HNSCC treatment by setting the stage for future research on VEGF splice variants as a tool for personalized medicine.
Asunto(s)
Neoplasias de Cabeza y Cuello , Neovascularización Patológica , Carcinoma de Células Escamosas de Cabeza y Cuello , Factor A de Crecimiento Endotelial Vascular , Humanos , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Carcinoma de Células Escamosas de Cabeza y Cuello/tratamiento farmacológico , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/genética , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/metabolismo , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/patología , Inhibidores de la Angiogénesis/uso terapéutico , Inhibidores de la Angiogénesis/farmacología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Animales , Transducción de Señal/efectos de los fármacosRESUMEN
The different susceptibility to HIV-1 infection in U937 cells-permissive (Plus) or nonpermissive (Minus)-is linked to the expression in Minus cells of interferon (IFN)-γ inducible antiviral factors such as tripartite motif-containing protein 22 (TRIM22) and class II transactivator (CIITA). CIITA interacts with Cyclin T1, a key component of the Positive-Transcription Elongation Factor b (P-TEFb) complex needed for the efficient transcription of HIV-1 upon interaction with the viral transactivator Tat. TRIM22 interacts with CIITA, recruiting it into nuclear bodies together with Cyclin T1. A 50 kDa Cyclin T1 was found only in Minus cells, alongside the canonical 80 kDa protein. The expression of this truncated form remained unaffected by proteasome inhibitors but was reduced by IFNγ treatment. Unlike the nuclear full-length protein, truncated Cyclin T1 was also present in the cytoplasm, and this subcellular localization correlated with its capacity to inhibit Tat-mediated HIV-1 transcription. The 50 kDa Cyclin T1 in Minus cells likely contributes to their non-permissive phenotype by acting as a dominant negative factor, disrupting P-TEFb complex formation and function. Its reduction upon IFNγ treatment suggests a regulatory loop by which its inhibitory role on HIV-1 replication is then exerted by the IFNγ-induced CIITA, which binds to the canonical Cyclin T1, displacing it from the P-TEFb complex.
Asunto(s)
Ciclina T , VIH-1 , Humanos , Ciclina T/metabolismo , VIH-1/fisiología , Células U937 , Infecciones por VIH/virología , Infecciones por VIH/metabolismo , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/genética , Replicación Viral , Fenotipo , Interferón gamma/farmacología , Interferón gamma/metabolismo , Proteínas de Motivos Tripartitos/metabolismo , Proteínas de Motivos Tripartitos/genética , Proteínas Represoras/metabolismo , Proteínas Represoras/genética , Antígenos de Histocompatibilidad MenorRESUMEN
FGF12 belongs to a subfamily of FGF proteins called FGF homologous factors (FHFs), which until recently were thought to be non-signaling intracellular proteins. Our recent studies have shown that although they lack a conventional signal peptide for secretion, they can reach the extracellular space, especially under stress conditions. Here, we unraveled that the long "a" isoform of FGF12 is secreted in a pathway involving the A1 subunit of Na(+)/K(+) ATPase (ATP1A1), Tec kinase and lipids such as phosphatidylinositol and phosphatidylserine. Further, we showed that the short "b" isoform of FGF12, which binds ATP1A1 and phosphatidylserine less efficiently, is not secreted from cells. We also indicated regions in the FGF12a protein sequence that are crucial for its secretion, including N-terminal fragment and specific residues, and proposed that liquid-liquid phase separation may be important in this process. Our results strongly suggest that the mechanism of this process is very similar for all unconventionally secreted FGF proteins.
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Factores de Crecimiento de Fibroblastos , Humanos , Factores de Crecimiento de Fibroblastos/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/genética , Fosfatidilserinas/metabolismo , Secuencia de AminoácidosRESUMEN
Dopamine (DA) D2 receptors (D2Rs) have 2 isoforms, a long form (D2L) and a short form (D2S). D2L is predominantly postsynaptic in the striatal medium spiny neurons and cholinergic interneurons. D2S is principally presynaptic autoreceptors in the nigrostriatal DA neurons. Recently, we demonstrated that L-3,4-dihydroxyphenylalanine (L-DOPA) augments D2L function through the coupling between D2L and GPR143, a receptor of L-DOPA that was originally identified as the gene product of ocular albinism 1. Here we show that GPR143 modifies the functions of D2L and D2S in an opposite manner. Haloperidol-induced catalepsy was attenuated in DA neuron-specific Gpr143 gene-deficient (Dat-cre;Gpr143flox/y) mice, compared with wild-type (Wt) mice. Haloperidol increased in vivo DA release from the dorsolateral striatum, and this increase was augmented in Gpr143-/y mice compared with Wt mice. A D2R agonist quinpirole-induced increase in the phosphorylation of GSK3ß(pGSK3ß(S9)) was enhanced in Chinese hamster ovary (CHO) cells coexpressing D2L and GPR143 compared with cells expressing D2L alone, while it was suppressed in cells coexpressing D2S and GPR143 compared with D2S alone, suggesting that GPR143 differentially modifies D2R functions depending on its isoforms of D2L and D2S.
Asunto(s)
Cricetulus , Dopamina , Haloperidol , Receptores de Dopamina D2 , Animales , Receptores de Dopamina D2/metabolismo , Receptores de Dopamina D2/genética , Haloperidol/farmacología , Células CHO , Dopamina/metabolismo , Cuerpo Estriado/metabolismo , Masculino , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/genética , Ratones , Levodopa/farmacología , Catalepsia/inducido químicamente , Catalepsia/genética , Catalepsia/metabolismo , Ratones Endogámicos C57BL , Fosforilación , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/fisiología , Quinpirol/farmacología , Neuronas Dopaminérgicas/metabolismo , Glucógeno Sintasa Quinasa 3 beta/metabolismoRESUMEN
Accurate detection and quantification of mRNA isoforms from nanopore long-read sequencing remains challenged by technical noise, particularly in single cells. To address this, we introduce Isosceles, a computational toolkit that outperforms other methods in isoform detection sensitivity and quantification accuracy across single-cell, pseudo-bulk and bulk resolution levels, as demonstrated using synthetic and biologically-derived datasets. Here we show Isosceles improves the fidelity of single-cell transcriptome quantification at the isoform-level, and enables flexible downstream analysis. As a case study, we apply Isosceles, uncovering coordinated splicing within and between neuronal differentiation lineages. Isosceles is suitable to be applied in diverse biological systems, facilitating studies of cellular heterogeneity across biomedical research applications.
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ARN Mensajero , Análisis de la Célula Individual , Análisis de la Célula Individual/métodos , Humanos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transcriptoma/genética , Análisis de Secuencia de ARN/métodos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Animales , Programas Informáticos , Biología Computacional/métodos , Neuronas/metabolismo , Perfilación de la Expresión Génica/métodos , Ratones , Secuenciación de Nanoporos/métodosRESUMEN
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.
Asunto(s)
Enfermedad de Alzheimer , Corteza Entorrinal , Ratones Transgénicos , Isoformas de Proteínas , Proteínas tau , Corteza Entorrinal/metabolismo , Corteza Entorrinal/patología , Animales , Humanos , Proteínas tau/metabolismo , Proteínas tau/genética , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Femenino , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratones , Modelos Animales de Enfermedad , Empalme Alternativo/genética , Regulación de la Expresión GénicaRESUMEN
BACKGROUND: 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. METHODS & RESULTS: 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. CONCLUSIONS: 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.
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Exones , Factores de Intercambio de Guanina Nucleótido , Isoformas de Proteínas , Animales , Exones/genética , Ratones , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Especificidad de Órganos/genética , Diferenciación Celular/genética , Empalme Alternativo/genética , Regulación del Desarrollo de la Expresión Génica/genética , Masculino , Células Madre Embrionarias de Ratones/metabolismoAsunto(s)
Melanoma , Osteopontina , Humanos , Melanoma/genética , Melanoma/diagnóstico , Osteopontina/genética , Osteopontina/metabolismo , Pronóstico , Regulación Neoplásica de la Expresión Génica , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/diagnóstico , Neoplasias Cutáneas/patología , Empalme Alternativo , Femenino , MasculinoRESUMEN
Targeting wild-type epidermal growth factor receptor (EGFR) using tyrosine kinase inhibitors (TKIs) never achieved its purported success in cancers such as head and neck squamous cell carcinoma, which are largely EGFR-dependent. We had previously shown that exceptional responders to TKIs have a genetic aberration that results in overexpression of an EGFR splice variant, isoform D (IsoD). IsoD lacks an integral transmembrane and kinase domain and is secreted in extracellular vesicles (EVs) in TKI-sensitive patient-derived cultures. Remarkably, the exquisite sensitivity to TKIs could be transferred to TKI-resistant tumor cells, and IsoD protein in the EV is necessary and sufficient to transfer the phenotype in vitro and in vivo across multiple models and drugs. This drug response requires an intact endocytic mechanism, binding to full-length EGFR, and signaling through Src-phosphorylation within the endosomal compartment. We propose a therapeutic strategy using EVs containing EGFR IsoD as a co-drug to expand the use of TKI therapy to EGFR-driven cancers.
Asunto(s)
Carcinoma de Células Escamosas , Receptores ErbB , Vesículas Extracelulares , Isoformas de Proteínas , Animales , Humanos , Ratones , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Resistencia a Antineoplásicos/efectos de los fármacos , Receptores ErbB/metabolismo , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Vesículas Extracelulares/metabolismo , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/metabolismo , Neoplasias de Cabeza y Cuello/patología , Neoplasias de Cabeza y Cuello/genética , Fosforilación/efectos de los fármacos , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/genética , Transducción de Señal/efectos de los fármacos , Carcinoma de Células Escamosas de Cabeza y Cuello/tratamiento farmacológico , Carcinoma de Células Escamosas de Cabeza y Cuello/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , /uso terapéuticoRESUMEN
Fibronectin (FN) is a ubiquitous extracellular matrix glycoprotein essential for the development of various tissues. Mutations in FN cause a unique form of spondylometaphyseal dysplasia, emphasizing its importance in cartilage and bone development. However, the relevance and functional role of FN during skeletal development has remained elusive. To address these aspects, we have generated conditional knockout mouse models targeting the cellular FN isoform in cartilage (cFNKO), the plasma FN isoform in hepatocytes (pFNKO), and both isoforms together in a double knockout (FNdKO). We used these mice to determine the relevance of the two principal FN isoforms in skeletal development from postnatal day one to the adult stage at two months. We identified a distinct topological FN deposition pattern in the mouse limb during different gestational and postnatal skeletal development phases, with prominent levels at the resting and hypertrophic chondrocyte zones and in the trabecular bone. Cartilage-specific cFN emerged as the predominant isoform in the growth plate, whereas circulating pFN remained excluded from the growth plate and confined to the primary and secondary ossification centers. Deleting either isoform independently (cFNKO or pFNKO) yielded only relatively subtle changes in the analyzed skeletal parameters. However, the double knockout of cFN in the growth plate and pFN in the circulation of the FNdKO mice significantly reduced postnatal body weight, body length, and bone length. Micro-CT analysis of the adult bone microarchitecture in FNdKO mice exposed substantial reductions in trabecular bone parameters and bone mineral density. The mice also showed elevated bone marrow adiposity. Analysis of chondrogenesis in FNdKO mice demonstrated changes in the resting, proliferating and hypertrophic growth plate zones, consistent alterations in chondrogenic markers such as collagen type II and X, decreased apoptosis of hypertrophic chondrocytes, and downregulation of bone formation markers. Transforming growth factor-ß1 and downstream phospho-AKT levels were significantly lower in the FNdKO than in the control mice, revealing a crucial FN-mediated regulatory pathway in chondrogenesis and bone formation. In conclusion, the data demonstrate that FN is essential for chondrogenesis and bone development. Even though cFN and pFN act in different regions of the bone, both FN isoforms are required for the regulation of chondrogenesis, cartilage maturation, trabecular bone formation, and overall skeletal growth.