MECHANISMS OF RNA TRANSPORT

这项提议旨在探索控制 核质RNA转运。它的主要推力包括1) RNA转录本选择中内含子区隔的特征 用于运输。内含子的新陈代谢将使用cDNA进行检测 用特定的多核苷酸启动子合成并随后扩增 通过聚合酶链式反应。拼接连接中的更改将是 在A)用化学致癌物治疗后进行检查，这些致癌物会改变 核质分隔和核结构；和B)在 利用可产生功能性细胞的仓鼠细胞系进行的转基因实验 调节宿主细胞的人类免疫缺陷病毒Rev蛋白 核质转运机制通过与细胞直接相互作用 核支架(NS)核苷三磷酸酶(NTPase)。2)定义 46kD NTPase在核质RNA转运中的作用这 酶是由层蛋白A/C产生的，层蛋白A/C是 NS，通过在接近末端的保守酪氨酸残基(Aa376)上裂解 线圈-2。克隆的层蛋白C制剂具有很强的ATP结合能力 已记录，表示合作(K D=3x10-6M)和非合作(K D=3x10-6M)和非合作(KD=2x10-5M)站点 组氨酸残留物。然而，克隆的层粘连蛋白C制剂没有显示 NTPase活性，提示翻译后修饰或其他 因素很重要。因此，不同的表达系统和体外 将检查修改是否与Active的生产相关 NTPase。由于NS NTPase活性和46kD的Lamin片段 在CCl4或硫代乙酰胺处理后增加300%，没有增加 在lamin mRNA中，由于46kD的N末端没有核 决定板层蛋白核内导入的定位信号，我们 假设新发现的NS蛋白水解酶参与后 NTPase的翻译产物。这些蛋白水解酶显示出明显的 对层蛋白A/C的选择性，受到钙离子的严格调节，并出现 在有丝分裂再生和后续过程中控制NTPase的产生 致癌物治疗。一些针对人的单抗 46kD蛋白已被开发出来。这些抗体调节NS NTPase 活性，并在体外表现出平行的RNA转运调节。 将在体外使用抗NTPase抗体，并进行卵母细胞显微注射 研究确定46kD的NTPase在RNA运输中的作用，以及 我们将直接探索NTPase的性质。NS的性质 将对蛋白酶进行定义，并将其用于探索它们在生产 使用克隆的Lamins和NS制备的Lamins A/C中的NTPase。

This proposal is designed to explore mechanisms operative in control of nucleocytoplasmic RNA transport. Its major thrusts encompass 1) characterization of intron compartmentation in selection of RNA transcripts for transport. Metabolism of introns will be examined using cDNA synthesized with specific polynucleotide promers and subsequently amplified by the polymerase chain reaction. Alterations in splice junctions will be examined following A) treatment with chemical carcinogens, which alter nucleocytoplasmic compartmentation and nuclear structure; and B) in transfection experiments with a hamster cell line which produces functional human immunodeficiency virus Rev protein, which modulates host cell nucleocytoplasmic transport mechanisms via direct interaction with the nuclear scaffold (NS) nucleoside triphosphatase (NTPase). 2) Definition of the role of the 46kD NTPase in nucleocytoplasmic RNA transport. This enzyme is produced from lamins A/C, the major structural proteins of the NS, by cleavage at a conserved tyrosine residue (aa376) near the end of coil-2. Substantial ATP-binding by cloned lamin C preparations has been documented, which shows cooperative (K D =3x10-6 M) and non-cooperative (K D =3x10-6 M) and non-cooperative (K D =2x10-5 M) sites which involve histidine residues. However, cloned lamin C preparations do not show NTPase activity, suggesting that post-translational modifications or other factors are important. Therefore various expression systems and in vitro modifications will be examined for relevance in production of active NTPase. Since NS NTPase activity and the 46 KD lamin fragment are increased 300% following CCl 4 or thioacetamide treatment, without increase in lamin mRNA, and since the 46 kD N-terminus lacks the nuclear localization signal which dictates intranuclear import of lamins, we hypothesize that newly discovered NS proteases are involved in post- translational production of NTPase. These proteases show a marked selectivity for lamins A/C, are stringently regulated by Ca2+, and appear to control NTPase production during mitotic regeneration and following carcinogen treatment. A number of monoclonal antibodies directed against the 46 kDprotein have been developed. These antibodies modulate NS NTPase activity and have shown parallel modulation of RNA transport in vitro. Anti-NTPase antibodies will be used in vitro, and inoocyte microinjection studies to determine the role of the 46 kD NTPase in RNA transport, and the properties of the NTPase will be directly explored. Properties of the NS proteases will be defined, and used to explore their role in production of the NTPase from lamins A/C, using both cloned lamins and NS preparations.