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Efficient targeted control of splicing is a major goal of functional genomics and therapeutic applications. Guide (g)RNA-directed, deactivated (d)Cas CRISPR enzymes fused to splicing effectors represent a promising strategy due to the flexibility of these systems. However, efficient, specific, and generalizable activation of endogenous exons using this approach has not been previously reported. By screening over 300 dCasRx-splicing factor fusion proteins tethered to splicing reporters, we identify dCasRx-RBM25 as a potent activator of exons. Moreover, dCasRx-RBM25 efficiently activates the splicing of â¼90% of targeted endogenous alternative exons and displays high on-target specificity. Using gRNA arrays for combinatorial targeting, we demonstrate that dCasRx-RBM25 enables multiplexed activation and repression of exons. Using this feature, the targeting of neural-regulated exons in Ptpb1 and Puf60 in embryonic stem cells reveals combinatorial effects on downstream alternative splicing events controlled by these factors. Collectively, our results enable versatile, combinatorial exon-resolution functional assays and splicing-directed therapeutic applications.
Assuntos
Processamento Alternativo , Sistemas CRISPR-Cas , Éxons , Fatores de Processamento de RNA , Proteínas de Ligação a RNA , Humanos , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Células HEK293 , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , RNA Guia de Sistemas CRISPR-Cas/genética , RNA Guia de Sistemas CRISPR-Cas/metabolismo , Animais , CamundongosRESUMO
RNA sequencing (RNA-seq) has recently been used in translational research settings to facilitate diagnoses of Mendelian disorders. A significant obstacle for clinical laboratories in adopting RNA-seq is the low or absent expression of a significant number of disease-associated genes/transcripts in clinically accessible samples. As this is especially problematic in neurological diseases, we developed a clinical diagnostic approach that enhanced the detection and evaluation of tissue-specific genes/transcripts through fibroblast-to-neuron cell transdifferentiation. The approach is designed specifically to suit clinical implementation, emphasizing simplicity, cost effectiveness, turnaround time, and reproducibility. For clinical validation, we generated induced neurons (iNeurons) from 71 individuals with primary neurological phenotypes recruited to the Undiagnosed Diseases Network. The overall diagnostic yield was 25.4%. Over a quarter of the diagnostic findings benefited from transdifferentiation and could not be achieved by fibroblast RNA-seq alone. This iNeuron transcriptomic approach can be effectively integrated into diagnostic whole-transcriptome evaluation of individuals with genetic disorders.
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Transdiferenciação Celular , Fibroblastos , Neurônios , Análise de Sequência de RNA , Humanos , Transdiferenciação Celular/genética , Fibroblastos/metabolismo , Fibroblastos/citologia , Análise de Sequência de RNA/métodos , Neurônios/metabolismo , Neurônios/citologia , Transcriptoma , Reprodutibilidade dos Testes , Doenças do Sistema Nervoso/genética , Doenças do Sistema Nervoso/diagnóstico , RNA-Seq/métodos , Feminino , MasculinoRESUMO
A major fraction of loci identified by genome-wide association studies (GWASs) mediate alternative splicing, but mechanistic interpretation is hindered by the technical limitations of short-read RNA sequencing (RNA-seq), which cannot directly link splicing events to full-length protein isoforms. Long-read RNA-seq represents a powerful tool to characterize transcript isoforms, and recently, infer protein isoform existence. Here, we present an approach that integrates information from GWASs, splicing quantitative trait loci (sQTLs), and PacBio long-read RNA-seq in a disease-relevant model to infer the effects of sQTLs on the ultimate protein isoform products they encode. We demonstrate the utility of our approach using bone mineral density (BMD) GWAS data. We identified 1,863 sQTLs from the Genotype-Tissue Expression (GTEx) project in 732 protein-coding genes that colocalized with BMD associations (H4PP ≥ 0.75). We generated PacBio Iso-Seq data (N = â¼22 million full-length reads) on human osteoblasts, identifying 68,326 protein-coding isoforms, of which 17,375 (25%) were unannotated. By casting the sQTLs onto protein isoforms, we connected 809 sQTLs to 2,029 protein isoforms from 441 genes expressed in osteoblasts. Overall, we found that 74 sQTLs influenced isoforms likely impacted by nonsense-mediated decay and 190 that potentially resulted in the expression of unannotated protein isoforms. Finally, we functionally validated colocalizing sQTLs in TPM2, in which siRNA-mediated knockdown in osteoblasts showed two TPM2 isoforms with opposing effects on mineralization but exhibited no effect upon knockdown of the entire gene. Our approach should be to generalize across diverse clinical traits and to provide insights into protein isoform activities modulated by GWAS loci.
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First described in the milkweed bug Oncopeltus fasciatus, planar cell polarity (PCP) is a developmental process essential for embryogenesis and development of polarized structures in Metazoans. This signaling pathway involves a set of evolutionarily conserved genes encoding transmembrane (Vangl, Frizzled, Celsr) and cytoplasmic (Prickle, Dishevelled) molecules. Vangl2 is of major importance in embryonic development as illustrated by its pivotal role during neural tube closure in human, mouse, Xenopus, and zebrafish embryos. Here, we report on the molecular and functional characterization of a Vangl2 isoform, Vangl2-Long, containing an N-terminal extension of about 50 aa, which arises from an alternative near-cognate AUA translation initiation site, lying upstream of the conventional start codon. While missing in Vangl1 paralogs and in all invertebrates, including Drosophila, this N-terminal extension is conserved in all vertebrate Vangl2 sequences. We show that Vangl2-Long belongs to a multimeric complex with Vangl1 and Vangl2. Using morpholino oligonucleotides to specifically knockdown Vangl2-Long in Xenopus, we found that this isoform is functional and required for embryo extension and neural tube closure. Furthermore, both Vangl2 and Vangl2-Long must be correctly expressed for the polarized distribution of the PCP molecules Pk2 and Dvl1 and for centriole rotational polarity in ciliated epidermal cells. Altogether, our study suggests that Vangl2-Long significantly contributes to the pool of Vangl2 molecules present at the plasma membrane to maintain PCP in vertebrate tissues.
Assuntos
Polaridade Celular , Proteínas Desgrenhadas , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana , Animais , Humanos , Camundongos , Proteínas de Transporte , Proteínas Desgrenhadas/metabolismo , Proteínas Desgrenhadas/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genética , Biossíntese de Proteínas , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/genética , Xenopus laevis , Proteínas de Xenopus/metabolismo , Proteínas de Xenopus/genética , Peixe-Zebra/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genéticaRESUMO
Force generation and motility by actomyosin in nonmuscle cells are spatially regulated by â¼40 tropomyosin (Tpm) isoforms. The means by which Tpms are targeted to specific cellular regions and the mechanisms that result in differential activity of myosin paralogs are unknown. We show that Tpm3.1 and Tpm1.7 inhibit Myosin-IC (Myo1C), with Tpm1.7 more effectively reducing the number of gliding filaments than Tpm3.1. Strikingly, cosedimentation and fluorescence microscopy assays revealed that Tpm3.1 is displaced from actin by Myo1C and not by myosin-II. In contrast, Tpm1.7 is only weakly displaced by Myo1C. Unlike other characterized myosins, Myo1C motility is inhibited by Tpm when the Tpm-actin filament is activated by myosin-II. These results point to a mechanism for the exclusion of myosin-I paralogs from cellular Tpm-decorated actin filaments that are activated by other myosins. Additionally, our results suggest a potential mechanism for myosin-induced Tpm sorting in cells.
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The salt-inducible kinases (SIKs) 1 to 3, belonging to the AMPK-related kinase family, serve as master regulators orchestrating a diverse set of physiological processes such as metabolism, bone formation, immune response, oncogenesis, and cardiac rhythm. Owing to its key regulatory role, the SIK kinases have emerged as compelling targets for pharmacological intervention across a diverse set of indications. Therefore, there is interest in developing SIK inhibitors with defined selectivity profiles both to further dissect the downstream biology and for treating disease. However, despite a large pharmaceutical interest in the SIKs, experimental structures of SIK kinases are scarce. This is likely due to the challenges associated with the generation of proteins suitable for structural studies. By adopting a rational approach to construct design and protein purification, we successfully crystallized and subsequently solved the structure of SIK3 in complex with HG-9-91-01, a potent SIK inhibitor. To enable further SIK3-inhibitor complex structures we identified an antibody fragment that facilitated crystallization and enabled a robust protocol suitable for structure-based drug design. The structures reveal SIK3 in an active conformation, where the ubiquitin-associated domain is shown to provide further stabilization to this active conformation. We present four pharmacologically relevant and distinct SIK3-inhibitor complexes. These detail the key interaction for each ligand and reveal how different regions of the ATP site are engaged by the different inhibitors to achieve high affinity. Notably, the structure of SIK3 in complex with a SIK3 specific inhibitor offers insights into isoform selectivity.
Assuntos
Inibidores de Proteínas Quinases , Proteínas Serina-Treonina Quinases , Humanos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Cristalografia por Raios X , Ligação Proteica , Conformação Proteica , Modelos Moleculares , Proteínas QuinasesRESUMO
Gastric cancer is histologically classified into the intestinal subtype, which forms tubular structures, and the aggressive diffuse subtype, characterized by rapid invasion and poor prognosis. The variety and quantity of miRNA isoforms between different histological subtypes of gastric cancer were unknown. Through systematic filtering, we found that more diverse miR-30a-5p isoforms was present in the diffuse subtype of gastric cancer, and was associated with patients' worse survival independent of tumor stage based on the TCGA miRNA-seq data. Among all nine isoforms of miR-30a-5p, miR-30a-5p -1|1 was more abundant than the archetype of miR-30a-5p. Higher expression of miR-30a-5p -1|1 was observed in patients with advanced tumor stage and poor survival. Furthermore, miR-30a-5p -1|1 could promote the metastasis of gastric cancer cells both in vitro and in vivo by down-regulating TMEM66. In clinical samples, decreased expression of TMEM66 was characteristic of gastric cancer, and the low level of TMEM66 correlated with deceased CD8 positive cells in the tumor microenvironment probably due to decreased cytokines production. In conclusion, the variety of miR-30a-5p isoforms correlates with worse survival in gastric cancer patients. Moreover, miR-30a-5p -1|1 could promote gastric cancer metastasis by inhibiting TMEM66 and the infiltration of intratumoral CD8 positive cells.
Assuntos
Regulação Neoplásica da Expressão Gênica , Proteínas de Membrana , MicroRNAs , Neoplasias Gástricas , Linfócitos T Citotóxicos , Animais , Feminino , Humanos , Masculino , Camundongos , Linhagem Celular Tumoral , Proliferação de Células/genética , Progressão da Doença , Regulação Neoplásica da Expressão Gênica/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Nus , MicroRNAs/genética , MicroRNAs/metabolismo , Prognóstico , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Neoplasias Gástricas/mortalidade , Neoplasias Gástricas/metabolismo , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Microambiente Tumoral/genéticaRESUMO
BACKGROUND: Transcriptome assembly from RNA-sequencing data in species without a reliable reference genome has to be performed de novo, but studies have shown that de novo methods often have inadequate ability to reconstruct transcript isoforms. We address this issue by constructing an assembly pipeline whose main purpose is to produce a comprehensive set of transcript isoforms. RESULTS: We present the de novo transcript isoform assembler ClusTrast, which takes short read RNA-seq data as input, assembles a primary assembly, clusters a set of guiding contigs, aligns the short reads to the guiding contigs, assembles each clustered set of short reads individually, and merges the primary and clusterwise assemblies into the final assembly. We tested ClusTrast on real datasets from six eukaryotic species, and showed that ClusTrast reconstructed more expressed known isoforms than any of the other tested de novo assemblers, at a moderate reduction in precision. For recall, ClusTrast was on top in the lower end of expression levels (<15% percentile) for all tested datasets, and over the entire range for almost all datasets. Reference transcripts were often (35-69% for the six datasets) reconstructed to at least 95% of their length by ClusTrast, and more than half of reference transcripts (58-81%) were reconstructed with contigs that exhibited polymorphism, measuring on a subset of reliably predicted contigs. ClusTrast recall increased when using a union of assembled transcripts from more than one assembly tool as primary assembly. CONCLUSION: We suggest that ClusTrast can be a useful tool for studying isoforms in species without a reliable reference genome, in particular when the goal is to produce a comprehensive transcriptome set with polymorphic variants.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Transcriptoma , Análise de Sequência , RNA-Seq , Análise de Sequência de RNA , Isoformas de Proteínas/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodosRESUMO
In recent years, in the development of emerging immunotherapy, B7-H3 is also termed as CD276 and has become a novel chimeric antigen receptor (CAR)-T target against glioma and other tumours, and aroused extensive attention. However, B7-H3 has three isoforms (2, 3 and 4Ig) with the controversial expression and elusive function in tumour especially glioma. The current study mainly focuses on the regulatory factors and related mechanisms of generation of different B7-H3 isoforms. First, we have determined that 2Ig is dominant in glioma with high malignancy, and 4Ig is widely expressed, whereas 3Ig shows negative expression in all glioma. Next, we have further found that RNA binding protein annexin A2 (ANXA2) is essential for B7-H3 isoform maintenance, but fail to determine the choice of 4Ig or 2Ig. RNA methyltransferase NOP2/Sun RNA methyltransferase 2 (NSUN2) and 5-methylcytosine reader Y-box binding protein 1 (YBX1) facilitate the production of 2Ig. Our findings have uncovered a series of factors (ANXA2/NSUN2/YBX1) that can determine the alternative generation of different isoforms of B7-H3 in glioma. Our result aims to help peers gain a clearer understanding of the expression and regulatory mechanisms of B7H3 in tumour patients, and to provide better strategies for designing B7H3 as a target in immunotherapy.
Assuntos
Anexina A2 , Antígenos B7 , Regulação Neoplásica da Expressão Gênica , Glioma , Isoformas de Proteínas , Humanos , Glioma/genética , Glioma/metabolismo , Glioma/patologia , Antígenos B7/metabolismo , Antígenos B7/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/genética , Anexina A2/metabolismo , Anexina A2/genética , Linhagem Celular Tumoral , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologiaRESUMO
BACKGROUND: Cancers exhibit complex transcriptomes with aberrant splicing that induces isoform-level differential expression compared to non-diseased tissues. Transcriptomic profiling using short-read sequencing has utility in providing a cost-effective approach for evaluating isoform expression, although short-read assembly displays limitations in the accurate inference of full-length transcripts. Long-read RNA sequencing (Iso-Seq), using the Pacific Biosciences (PacBio) platform, can overcome such limitations by providing full-length isoform sequence resolution which requires no read assembly and represents native expressed transcripts. A constraint of the Iso-Seq protocol is due to fewer reads output per instrument run, which, as an example, can consequently affect the detection of lowly expressed transcripts. To address these deficiencies, we developed a concatenation workflow, PacBio Full-Length Isoform Concatemer Sequencing (PB_FLIC-Seq), designed to increase the number of unique, sequenced PacBio long-reads thereby improving overall detection of unique isoforms. In addition, we anticipate that the increase in read depth will help improve the detection of moderate to low-level expressed isoforms. RESULTS: In sequencing a commercial reference (Spike-In RNA Variants; SIRV) with known isoform complexity we demonstrated a 3.4-fold increase in read output per run and improved SIRV recall when using the PB_FLIC-Seq method compared to the same samples processed with the Iso-Seq protocol. We applied this protocol to a translational cancer case, also demonstrating the utility of the PB_FLIC-Seq method for identifying differential full-length isoform expression in a pediatric diffuse midline glioma compared to its adjacent non-malignant tissue. Our data analysis revealed increased expression of extracellular matrix (ECM) genes within the tumor sample, including an isoform of the Secreted Protein Acidic and Cysteine Rich (SPARC) gene that was expressed 11,676-fold higher than in the adjacent non-malignant tissue. Finally, by using the PB_FLIC-Seq method, we detected several cancer-specific novel isoforms. CONCLUSION: This work describes a concatenation-based methodology for increasing the number of sequenced full-length isoform reads on the PacBio platform, yielding improved discovery of expressed isoforms. We applied this workflow to profile the transcriptome of a pediatric diffuse midline glioma and adjacent non-malignant tissue. Our findings of cancer-specific novel isoform expression further highlight the importance of long-read sequencing for characterization of complex tumor transcriptomes.
Assuntos
Glioma , Transcriptoma , Humanos , Criança , Perfilação da Expressão Gênica/métodos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Splicing de RNA , Análise de Sequência de RNA , Sequenciamento de Nucleotídeos em Larga Escala/métodosRESUMO
BACKGROUND: The hair follicle development process is regulated by sophisticated genes and signaling networks, and the hair grows from the hair follicle. The Tianzhu white yak population exhibits differences in hair length, especially on the forehead and shoulder region. However, the genetic mechanism is still unclear. Isoform sequencing (Iso-seq) technology with advantages in long reads sequencing. Hence, we combined the Iso-seq and RNA-seq methods to investigate the transcript complexity and difference between long-haired yak (LHY) and normal-haired yak (NHY). RESULTS: The hair length measurement result showed a significant difference between LHY and NHY on the forehead and the shoulder (P-value < 0.001). The skin samples from the forehead and the shoulder of LHY and NHY were pooled for isoform sequencing (Iso-seq). We obtained numerous long transcripts, including novel isoforms, long non-coding RNA, alternative splicing events, and alternative polyadenylation events. Combined with RNA-seq data, we performed differential isoforms (DEIs) analysis between LHY and NHY. We found that some hair follicle and skin development-related DEIs, like BMP4, KRT2, IGF2R, and COL1A2 in the forehead skin; BMP1, KRT1, FGF5, COL2A1, and IGFBP5 in the shoulder skin. Enrichment analysis revealed that DEIs in both two comparable groups significantly participated in skin and hair follicle development-related pathways, like ECM-receptor interaction, focal adhesion, and PI3K-Akt signaling pathways. The results indicated that the hair follicle development of Tianzhu white yak may influence the hair length difference. Besides, the protein-protein interaction (PPI) network of DEIs showed COL2A1 and COL3A1 exhibited a high degree of centrality, and these two genes were suggested as potential candidates for the hair length growth of Tianzhu white yak. CONCLUSIONS: The results provided a comprehensive analysis of the transcriptome complexity and identified differential transcripts that enhance our understanding of the molecular mechanisms underlying the variation in hair length growth in Tianzhu white yak.
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Cabelo , Isoformas de Proteínas , RNA-Seq , Pele , Transcriptoma , Animais , Bovinos/genética , Pele/metabolismo , Cabelo/metabolismo , Cabelo/crescimento & desenvolvimento , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Folículo Piloso/metabolismo , Folículo Piloso/crescimento & desenvolvimento , Perfilação da Expressão Gênica , Processamento Alternativo , Análise de Sequência de RNARESUMO
FARIMPD (Fetal akinesia, respiratory insufficiency, microcephaly, polymicrogyria, and dysmorphic facies) syndrome is a severe condition caused by ATP1A2 gene variants. The syndrome's novelty and rarity have limited its clinical and molecular knowledge. This research tries to provide new insight by investigating the cause of the early deaths due to FARIMPD syndrome in a particular family and reviewing previous studies. DNA and RNA were extracted from the blood samples of newborns and their parents, followed by whole exome sequencing and segregation analysis. A pathogenic homozygous nonsense variant (c.1234C > T: p.Arg412*) in the ATP1A2 gene was found in newborns. This variant is reported as homozygous for the first time. The migraine symptoms were the result of the heterozygous state of this particular variant, which supported the dominant inheritance pattern of this disease. Real-time PCR was used to analyze ATP1A2 gene expression in the newborns compared to parents and control subjects. The expression analysis also showed significant mRNA degradation in the newborns compared to heterozygous and healthy individuals, due to Nonsense-mediated mRNA Decay phenomena. Our study describes an ATP1A2 nonsense variant (c.1234C > T) that appears compatible with infant survival in the heterozygous and compound heterozygous states but is lethal in the homozygous state.
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Genomic imprinting is an epigenetic regulation mechanism in mammals resulting in the parentally dependent monoallelic expression of genes. Imprinting disorders in humans are associated with several congenital syndromes and cancers and remain the focus of many medical studies. Cattle is a better model organism for investigating human embryo development than mice. Imprinted genes usually cluster on chromosomes and are regulated by different methylation regions (DMRs) located in imprinting control regions that control gene expression in cis. There is an imprinted locus on human chromosome 16q24.1 associated with congenital lethal developmental lung disease in newborns. However, genomic imprinting on bovine chromosome 18, which is homologous with human chromosome 16 has not been systematically studied. The aim of this study was to analyze the allelic expressions of eight genes (CDH13, ATP2C2, TLDC1, COTL1, CRISPLD2, ZDHHC7, KIAA0513, and GSE1) on bovine chromosome 18 and to search the DMRs associated gene allelic expression. Three transcript variants of the ZDHHC7 gene (X1, X2, and X5) showed maternal imprinting in bovine placentas. In addition, the monoallelic expression of X2 and X5 was tissue-specific. Five transcripts of the KIAA0513 gene showed tissue- and isoform-specific monoallelic expression. The CDH13, ATP2C2, and TLDC1 genes exhibited tissue-specific imprinting, however, COTL1, CRISLPLD2, and GSE1 escaped imprinting. Four DMRs, established after fertilization, were found in this region. Two DMRs were located between the ZDHHC7 and KIAA0513 genes, and two were in exon 1 of the CDH13 and ATP2C2 genes, respectively. The results from this study support future studies on the molecular mechanism to regulate the imprinting of candidate genes on bovine chromosome 18.
Assuntos
Metilação de DNA , Epigênese Genética , Recém-Nascido , Gravidez , Feminino , Humanos , Bovinos/genética , Animais , Camundongos , Metilação de DNA/genética , Cromossomos Humanos Par 18 , Impressão Genômica/genética , Cromossomos , Mamíferos/genética , Proteínas do Tecido Nervoso/genéticaRESUMO
Microglial Na/H exchanger-1 (NHE1) protein, encoded by Slc9a1, plays a role in white matter demyelination of ischemic stroke brains. To explore underlying mechanisms, we conducted single cell RNA-seq transcriptome analysis in conditional Slc9a1 knockout (cKO) and wild-type (WT) mouse white matter tissues at 3 days post-stroke. Compared to WT, Nhe1 cKO brains expanded a microglial subgroup with elevated transcription of white matter myelination genes including Spp1, Lgals3, Gpnmb, and Fabp5. This subgroup also exhibited more acidic pHi and significantly upregulated CREB signaling detected by ingenuity pathway analysis and flow cytometry. Moreover, the Nhe1 cKO white matter tissues showed enrichment of a corresponding oligodendrocyte subgroup, with pro-phagocytosis and lactate shuffling gene expression, where activated CREB signaling is a likely upstream regulator. These findings demonstrate that attenuation of NHE1-mediated H+ extrusion acidifies microglia/macrophage and may underlie the stimulation of CREB1 signaling, giving rise to restorative microglia-oligodendrocyte interactions for remyelination.
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Encéfalo , Microglia , Trocador 1 de Sódio-Hidrogênio , Animais , Camundongos , Encéfalo/metabolismo , Receptor 1 de Quimiocina CX3C/metabolismo , Macrófagos/metabolismo , Microglia/metabolismo , Oligodendroglia/metabolismo , Transdução de Sinais/genética , Trocador 1 de Sódio-Hidrogênio/metabolismoRESUMO
Increasing evidence has demonstrated that glutaminase (GLS) as a key mitochondrial enzyme plays a pivotal role in glutaminolysis, which widely participates in glutamine metabolism serving as main energy sources and building blocks for tumor growth. However, the roles and molecular mechanisms of GLS in esophageal squamous cell carcinoma (ESCC) remains unknown. Here, we found that GLS was highly expressed in ESCC tissues and cells. GLS inhibitor CB-839 significantly suppressed cell proliferation, colony formation, migration and invasion of ESCC cells, whereas GLS overexpression displayed the opposite effects. In addition, CB-839 markedly suppressed glucose consumption and lactate production, coupled with the downregulation of glycolysis-related proteins HK2, PFKM, PKM2 and LDHA, whereas GLS overexpression exhibited the adverse results. In vivo animal experiment revealed that CB-839 dramatically suppressed tumor growth, whereas GLS overexpression promoted tumor growth in ESCC cells xenografted nude mice. Mechanistically, GLS was localized in mitochondria of ESCC cells, which interacted with PDK1 protein. CB-839 attenuated the interaction of GLS and PDK1 in ESCC cells by suppressing PDK1 expression, which further evoked the downregulation of p-PDHA1 (s293), however, GLS overexpression markedly enhanced the level of p-PDHA1 (s293). These findings suggest that interaction of GLS with PDK1 accelerates the glycolysis of ESCC cells by inactivating PDH enzyme, and thus targeting GLS may be a novel therapeutic approach for ESCC patients.
Assuntos
Benzenoacetamidas , Neoplasias Esofágicas , Carcinoma de Células Escamosas do Esôfago , Glutaminase , Glicólise , Piruvato Desidrogenase Quinase de Transferência de Acetil , Tiadiazóis , Animais , Humanos , Camundongos , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/genética , Carcinoma de Células Escamosas do Esôfago/metabolismo , Carcinoma de Células Escamosas do Esôfago/patologia , Regulação Neoplásica da Expressão Gênica , Glutaminase/genética , Glutaminase/metabolismo , Glicólise/genética , Camundongos Nus , Piruvato Desidrogenase Quinase de Transferência de Acetil/genética , Piruvato Desidrogenase Quinase de Transferência de Acetil/metabolismoRESUMO
Ferroportin (FPN) is a transmembrane protein and is the only known iron exporter that helps in maintaining iron homeostasis in vertebrates. To maintain stable iron equilibrium in the body, ferroportin works in conjunction with a peptide called hepcidin. In this study, we have identified an alternatively spliced novel isoform of the human SLC40A1 gene, which encodes for the FPN protein and is found to be expressed in different tissues. The novel transcript has an alternate last exon and encodes 31-amino acid long peptide sequence that replaces 104 amino acids at C-terminal in the novel transcript. Molecular modelling and molecular dynamics (MD) simulation studies revealed key structural features of the novel isoform (FPN-N). FPN-N was predicted to have 12 transmembrane domains similar to the reported isoform (FPN), despite being much smaller in size. FPN-N was found to interact with hepcidin, a key regulator of ferroportin activity. Also, the iron-binding sites were retained in the novel isoform as revealed by the MD simulation of FPN-N in bilipid membrane. The novel isoform identified in this study may play important role in iron homeostasis. However, further studies are required to characterize the FPN-N isoform and decipher its role inside the cell.
Assuntos
Processamento Alternativo , Proteínas de Transporte de Cátions , Hepcidinas , Ferro , Isoformas de Proteínas , Humanos , Sequência de Aminoácidos , Sítios de Ligação , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Proteínas de Transporte de Cátions/química , Hepcidinas/genética , Hepcidinas/metabolismo , Ferro/metabolismo , Modelos Moleculares , Simulação de Dinâmica Molecular , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMO
Cold stress is one of the major environmental factors that limit growth and yield of plants. However, it is still not fully understood how plants account for daily temperature fluctuations, nor how these temperature changes are integrated with other regulatory systems such as the circadian clock. We demonstrate that REVEILLE2 undergoes alternative splicing after chilling that increases accumulation of a transcript isoform encoding a MYB-like transcription factor. We explore the biological function of REVEILLE2 in Arabidopsis thaliana using a combination of molecular genetics, transcriptomics, and physiology. Disruption of REVEILLE2 alternative splicing alters regulatory gene expression, impairs circadian timing, and improves photosynthetic capacity. Changes in nuclear gene expression are particularly apparent in the initial hours following chilling, with chloroplast gene expression subsequently upregulated. The response of REVEILLE2 to chilling extends our understanding of plants immediate response to cooling. We propose that the circadian component REVEILLE2 restricts plants responses to nocturnal reductions in temperature, thereby enabling appropriate responses to daily environmental changes.
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Proteínas de Arabidopsis , Arabidopsis , Relógios Circadianos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Relógios Circadianos/genética , Ritmo Circadiano/genética , Regulação da Expressão Gênica de Plantas , TemperaturaRESUMO
The advent of full-length transcriptome sequencing technologies has accelerated the discovery of novel splicing isoforms. However, existing alternative splicing (AS) tools are either tailored for short-read RNA-Seq data or designed for human and animal studies. The disparities in AS patterns between plants and animals still pose a challenge to the reliable identification and functional exploration of novel isoforms in plants. Here, we developed integrated full-length alternative splicing analysis (iFLAS), a plant-optimized AS toolkit that introduced a semi-supervised machine learning method known as positive-unlabeled (PU) learning to accurately identify novel isoforms. iFLAS also enables the investigation of AS functions from various perspectives, such as differential AS, poly(A) tail length, and allele-specific AS (ASAS) analyses. By applying iFLAS to three full-length transcriptome sequencing datasets, we systematically identified and functionally characterized maize (Zea mays) AS patterns. We found intron retention not only introduces premature termination codons, resulting in lower expression levels of isoforms, but may also regulate the length of 3'UTR and poly(A) tail, thereby affecting the functional differentiation of isoforms. Moreover, we observed distinct ASAS patterns in two genes within heterosis offspring, highlighting their potential value in breeding. These results underscore the broad applicability of iFLAS in plant full-length transcriptome-based AS research.
Assuntos
Processamento Alternativo , Transcriptoma , Humanos , Processamento Alternativo/genética , Transcriptoma/genética , Zea mays/genética , Perfilação da Expressão Gênica/métodos , Melhoramento Vegetal , Isoformas de Proteínas/genética , RNA Mensageiro/genética , Análise de Sequência de RNARESUMO
Human papillomavirus (HPV) is a major causative factor of head and neck squamous cell carcinoma (HNSCC), and the incidence of HPV- associated HNSCC is increasing. The role of tumor microenvironment in viral infection and metastasis needs to be explored further. We studied the molecular characteristics of primary tumors (PTs) and lymph node metastatic tumors (LNMTs) by stratifying them based on their HPV status. Eight samples for single-cell RNA profiling and six samples for spatial transcriptomics (ST), composed of matched primary tumors (PT) and lymph node metastases (LNMT), were collected from both HPV- negative (HPV- ) and HPV-positive (HPV+ ) patients. Using the 10x Genomics Visium platform, integrative analyses with single-cell RNA sequencing were performed. Intracellular and intercellular alterations were analyzed, and the findings were confirmed using experimental validation and publicly available data set. The HPV+ tissues were composed of a substantial amount of lymphoid cells regardless of the presence or absence of metastasis, whereas the HPV- tissue exhibited remarkable changes in the number of macrophages and plasma cells, particularly in the LNMT. From both single-cell RNA and ST data set, we discovered a central gene, pyruvate kinase muscle isoform 1/2 (PKM2), which is closely associated with the stemness of cancer stem cell-like populations in LNMT of HPV- tissue. The consistent expression was observed in HPV- HNSCC cell line and the knockdown of PKM2 weakened spheroid formation ability. Furthermore, we found an ectopic lymphoid structure morphology and clinical effects of the structure in ST slide of the HPV+ patients and verified their presence in tumor tissue using immunohistochemistry. Finally, the ephrin-A (EPHA2) pathway was detected as important signals in angiogenesis for HPV- patients from single-cell RNA and ST profiles, and knockdown of EPHA2 declined the cell migration. Our study described the distinct cellular composition and molecular alterations in primary and metastatic sites in HNSCC patients based on their HPV status. These results provide insights into HNSCC biology in the context of HPV infection and its potential clinical implications.
Assuntos
Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Infecções por Papillomavirus , Humanos , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas/patologia , Papillomavirus Humano , Papillomaviridae/genética , Neoplasias de Cabeça e Pescoço/genética , Perfilação da Expressão Gênica/métodos , RNA , Microambiente Tumoral/genéticaRESUMO
Leber congenital amaurosis (LCA) and early-onset retinal degeneration (EORD) are inherited retinal diseases (IRD) characterized by early-onset vision impairment. Herein, we studied 15 Saudi families by whole exome sequencing (WES) and run-of-homozygosity (ROH) detection via AutoMap in 12/15 consanguineous families. This revealed (likely) pathogenic variants in 11/15 families (73%). A potential founder variant was found in RPGRIP1. Homozygous pathogenic variants were identified in known IRD genes (ATF6, CRB1, CABP4, RDH12, RIMS2, RPGRIP1, SPATA7). We established genotype-driven clinical reclassifications for ATF6, CABP4, and RIMS2. Specifically, we observed isolated IRD in the individual with the novel RIMS2 variant, and we found a retina-enriched RIMS2 isoform conserved but not annotated in mouse. The latter illustrates potential different phenotypic consequences of pathogenic variants depending on the particular tissue/cell-type specific isoforms they affect. Lastly, a compound heterozygous genotype in GUCY2D in one non-consanguineous family was demonstrated, and homozygous variants in novel candidate genes ATG2B and RUFY3 were found in the two remaining consanguineous families. Reporting these genes will allow to validate them in other IRD cohorts. Finally, the missing heritability of the two unsolved IRD cases may be attributed to variants in non-coding regions or structural variants that remained undetected, warranting future WGS studies.