MECHANISMS OF RNA TRANSPORT

该提案旨在探索控制的有效机制 核质RNA转运。 其主要目标包括：1） RNA转录物选择中内含子区室化的表征 用于运输。 将使用cDNA检测内含子的代谢 用特定的多核苷酸引物合成， 通过聚合酶链反应。 拼接接头的改变将 检查以下A）用化学致癌物处理， 核质区室化和核结构;和B）在 用仓鼠细胞系进行转染实验， 人类免疫缺陷病毒Rev蛋白，其调节宿主细胞 核质转运机制通过直接相互作用与 核支架（NS）核苷三磷酸酶（NTR）。 2)定义 46 kD NTR在核质RNA转运中的作用。 这 酶由核纤层蛋白A/C产生，核纤层蛋白A/C是核纤层蛋白的主要结构蛋白。 NS，通过在接近末端的保守酪氨酸残基（aa 376）处切割， 线圈-2。 克隆核纤层蛋白C制剂的大量ATP结合已经被证实， 记录了合作（K D = 3x 10 -6 M）和非合作（K D = 3x 10 -6 M）和非合作（K D = 2x 10 -5 M）站点， 组氨酸残基。 然而，克隆的核纤层蛋白C制剂没有显示出 NTR活性，表明翻译后修饰或其他 因素是重要的。 因此，各种表达系统和体外 将检查修改是否与活性物质的生产相关。 新台币。 由于NS NTR活性和46 KD核纤层蛋白片段是 CCl 4或硫代乙酰胺处理后增加300%，而不增加 核纤层蛋白mRNA，由于46 kD的N-末端缺乏核 决定核纤层蛋白核内输入的定位信号，我们 假设新发现的NS蛋白酶参与后 NTR的翻译生产。 这些蛋白酶显示出明显的 对核纤层蛋白A/C的选择性，受到Ca 2+的严格调节， 为了控制有丝分裂再生过程中NTR的产生， 致癌物治疗 许多单克隆抗体针对 46 kD蛋白已经被开发出来。 这些抗体调节NS NTR 活性，并显示出在体外对RNA转运的平行调节。 抗NTR抗体将在体外使用， 研究确定46 kD NTR在RNA转运中的作用， 将直接探索NTT的特性。 NS的属性 蛋白酶将被定义，并用于探索其在生产中的作用， 使用克隆的核纤层蛋白和NS制备物，从核纤层蛋白A/C获得NTT。

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.