Pre-mRNA Processing and Function of Alternatively Spliced Isoforms of TFPI

TFPI 选择性剪接亚型的前 mRNA 加工和功能

• 批准号: 10664506
• 负责人: Amy Siebert-McKenzie
• 金额: $ 11.63万
• 依托单位: VERSITI WISCONSIN, INC.
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-15 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10664506
• 关键词: 3' Splice Site; Adult; Affect; Alternative Splicing; American; Anticoagulants; Area; Basic Science; Binding; Biochemical; Biochemical Pathway; Biological; Biology; Blood Coagulation Disorders; Blood Platelets; Blood Proteins; Blood Vessels; Blood coagulation; Cell Line; Cells; Cessation of life; Coagulation Process; Conserved Sequence; Coupled; Cues; Defect; Development; Disease; Elements; Embryo; Embryonic Development; Endothelium; Equilibrium; Event; Exons; Frequencies; Generations; Genes; Genotype; Hemorrhage; Hemostatic Agents; Hemostatic function; Human; Individual; Life; Longevity; Messenger RNA; Microscopic; Molecular; Mus; Myelogenous; Pathologic; Patients; Perinatal; Perinatal mortality demographics; Phase; Phenotype; Physiological; Placenta; Plasma; Poly A; Polyadenylation; Positioning Attribute; Pregnancy; Production; Protein Isoforms; Proteins; RNA; RNA Splicing; Regulation; Regulatory Element; Research; Research Personnel; Role; Signal Transduction; Site; Source; Spontaneous abortion; Surface; TFPI; Technical Expertise; Testing; Thrombin; Thromboplastin; Thrombosis; Training; Transcript; Variant; Woman; career; cell type; defined contribution; factor V Leiden; human disease; improved; in vivo; inhibitor; innovation; mRNA Expression; mRNA Precursor; mutant mouse model; prevent; progenitor; recruit; skills

Hemostasis is a constant balancing act between pro- and anticoagulant factors, platelets, and the vasculature that is required to prevent excessive bleeding or pathological clotting. The anticoagulant, Tissue Factor Pathway Inhibitor (TFPI), is a vital factor in this balance and modulates a broad range of bleeding and clotting disorders through inhibition of TF-FVIIa, FXa, and prothrombinase (FXa-FVa). The TFPI gene is evolutionarily conserved and due to alternative splicing, different TFPI isoforms are predominant within distinct pools. While the specific inhibitory function of each TFPI isoform has been characterized, little is known regarding differences in isoform- specific contributions under prothrombotic disease conditions such as Factor V Leiden (FVL) and during embryonic development. Further, the pre-mRNA splicing and processing mechanisms dictating expression of each isoform are unknown. As a causal relationship exists between aberrant splicing of FV and TFPI isoform- specific function in human bleeding disorders, these mechanisms, coordinated by precise cues directed at maintaining the hemostatic balance, are highly relevant. Thus, the long-term objective of this proposal is to differentiate the physiological, site-specific production of each TFPI isoform at a molecular level and define their anticoagulant function in embryonic development and disease. TFPIα is the only isoform present in platelets and the only isoform that inhibits prothrombinase during the initiation of blood coagulation. Additionally, global TFPI deficiency results in prothrombotic perinatal lethality in FVL mice, and TFPIα prothrombinase inhibitory activity is reduced in the presence of FVL. To this end, K99 phase studies probe the physiological role of TFPIα as a regulator of FV/FVL, particularly in prothrombinase assembly on platelet surfaces during development (AIM 1) and characterizes biological activity of new platelet-specific TFPIα splice variants identified in mice and humans (AIM 2). The candidate will acquire technical expertise to define TFPIα anticoagulant function both in vivo using two unique isoform- and site-specific TFPIα mutant mouse models and ex vivo using human and mouse platelets. In AIM 3 (R00 phase), the candidate will take advantage of the evolutionary conservation of alternative TFPI splice forms and splicing signals embedded in highly conserved sequences to determine cis-RNA element and trans-acting splicing factor interactions regulating TFPI isoform diversity in mice and humans. Deciphering the pre-mRNA processing mechanisms that regulate site-specific TFPI isoform expression will delineate how alternative splicing contributes to the physiological and pathophysiological hemostatic balance during embryonic development and in adulthood. As there are many patients with bleeding and clotting disorders of unknown cause, the relation of aberrant splicing to these diseases represents a relatively new and unexplored area with great potential for launching a successful independent career. This proposal also outlines an intensive training plan of courses, seminars, and hands-on training for transitioning the candidate into a well-equipped independent investigator with a unique combination of research skills and a highly promising basic research pipeline.

止血是促凝因子和抗凝因子、血小板和脉管系统之间的持续平衡行为 这是防止过度出血或病理性凝血所必需的。 抑制剂 (TFPI) 是这种平衡的重要因素，可调节多种出血和凝血障碍 通过抑制 TF-FVIIa、FXa 和凝血酶原酶 (FXa-FVa) TFPI 基因在进化上是保守的。 由于选择性剪接，不同的 TFPI 同种型在不同的池中占主导地位。 每种 TFPI 同工型的抑制功能已得到表征，但对于同工型之间的差异知之甚少。 在血栓性疾病条件下的特定贡献，例如因子 V Leiden (FVL) 和期间 此外，前体 mRNA 剪接和加工机制决定了 mRNA 的表达。 每个亚型都是未知的，因为 FV 和 TFPI 亚型的异常剪接之间存在因果关系。 这些机制在人类出血性疾病中具有特定功能，通过针对特定线索的精确线索进行协调 维持止血平衡具有高度相关性，因此，该提案的长期目标是 在分子水平上区分每种 TFPI 同工型的生理学、位点特异性生产，并定义它们的 TFPIα 在胚胎发育和疾病中具有抗凝功能，是血小板和疾病中存在的唯一亚型。 在血液凝固起始过程中抑制凝血酶原酶的唯一亚型。 缺乏导致 FVL 小鼠血栓前围产期死亡，以及 TFPIα 凝血酶原酶抑制活性 FVL 存在时会降低。为此，K99 阶段研究探讨了 TFPIα 作为一种药物的生理作用。 FV/FVL 调节剂，特别是发育过程中血小板表面凝血酶原酶组装 (AIM 1) 并表征了在小鼠和人类中发现的新血小板特异性 TFPIα 剪接变体的生物活性 (AIM 2) 候选人将获得技术专业知识来定义 TFPIα 体内抗凝功能。 两种独特的亚型和位点特异性 TFPIα 突变小鼠模型，以及使用人和小鼠血小板的离体模型。 在AIM 3（R00阶段）中，候选者将利用替代TFPI的进化守恒 嵌入高度保守序列中的剪接形式和剪接信号以确定顺式RNA元件和 反式作用剪接因子相互作用调节小鼠和人类的 TFPI 同工型多样性。 调节位点特异性 TFPI 同工型表达的前 mRNA 加工机制将描述如何 选择性剪接有助于胚胎时期的生理和病理生理止血平衡 由于许多患者患有未知的出血和凝血障碍。 原因，异常剪接与这些疾病的关系代表了一个相对较新且未经探索的领域 该提案还概述了开展成功的独立职业的巨大潜力。 将候选人转变为装备精良的独立人员的课程、研讨会和实践培训计划 具有独特的研究技能和非常有前途的基础研究管道的研究员。