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.

止血是亲和抗凝因子，血小板和脉管系统之间的恒定平衡行为 这是防止多余的出血或病理服装所必需的。抗凝剂，组织因子途径 抑制剂（TFPI）是这种平衡的重要因素，并调节了广泛的出血和闭合障碍 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 由于替代剪接，不同的TFPI同工型主要在不同的池中。而特定的 每个TFPI同工型的抑制功能已经表征，关于同工型的差异知之甚少 在促血栓性疾病条件下（例如因子V Leiden（FVL））和期间的具体贡献 胚胎发展。此外，指示表达的MRNA剪接和处理机制 每个同工型都是未知的。由于FV和TFPI同工型的异常剪接之间存在因果关系。 人类出血障碍中的特定功能，这些机制，由针对的精确线索协调 保持止血平衡非常相关。这，该提议的长期目标是 在分子水平上区分每个TFPI同工型的物理，特定于位点的产生并定义它们 抗凝作用在胚胎发育和疾病中。 TFPIα是血小板中唯一存在的同工型 在血液凝血的倡议期间，唯一抑制凝血酶酶的同工型。此外，全球TFPI 缺乏导致FVL小鼠的促血栓性围产致死性和TFPIα黑凝结酶抑制活性 在FVL存在下减少。为此，K99期研究探测了TFPIα作为A的身体作用 FV/FVL的调节剂，特别是在发育过程中血小板表面的凝血酶组合酶组件中（AIM 1） 并表征了在小鼠和人类中鉴定出的新血小板特异性TFPIα剪接变体的生物学活性 （目标2）。候选人将获得技术专长来定义TFPIα抗凝功能，既可以在体内使用 使用人和小鼠血小板的两个独特的同工型和特异性TFPIα突变小鼠模型，并在体内。 在AIM 3（R00阶段）中，候选人将利用替代TFPI的进化保护 嵌入在高度保守序列中的剪接形式和剪接信号确定顺式-RNA元素和 跨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。解密 调节特定位点特异性TFPI同工型表达的MRNA前处理机制将描述如何 替代剪接有助于胚胎期间的生理和病理生理止血平衡 发展和成年。由于有许多患者出血和闭合障碍 原因，异常剪接与这些疾病的关系代表了一个相对较新且出乎意料的区域 开展成功的独立职业的巨大潜力。该建议还概述了深入的培训 课程，半手和动手培训计划，将候选人转变为设备齐全的独立 研究人员具有独特的研究技能和高度有希望的基础研究管道的研究者。