TRNA SPLICING

TRNA剪接

基本信息

批准号：
6180615
负责人：
Eric M. Phizicky
金额：
$ 25.43万
依托单位：
UNIVERSITY OF ROCHESTER
依托单位国家：
美国
项目类别：
财政年份：
1995
资助国家：
美国
起止时间：
1995-05-01 至 2003-04-30
项目状态：
已结题

项目摘要

The long-term goal of this work is to understand tRNA splicing in the yeast S. cerevisiae and vertebrates. tRNA splicing is essential in yeast, man and likely all eukaryotes, and is uniquely different from other classes of splicing. Superficially, tRNA splicing is simple in yeast: an endonuclease excises the intron, an RNA ligase joins the half-molecules to leave a splice junction 2'-phosphate, and a 2'-phosphotransferase transfers the phosphate to NAD to form ADP-ribose 1"-2" cyclic phosphate (Appr>p), a previously unknown metabolite. The enzymes are highly conserved in vertebrates, and the last step works in vivo in Xenopus oocytes. Yet there are striking layers of complexity: First, the formation of Appr>p in equimolar amounts during splicing implies a pathway to return it to known metabolism and suggests a regulatory function in yeast as a sensor. Second, vertebrates apparently also have a second ligase that has been implicated in tRNA splicing, which uses different chemistry and does not require a 2'-phosphotransferase. This is a rare occurrence of two seemingly redundant pathways in the same organism, and suggests some form of regulation, or another use of one of the pathways. Third, the ligase that catalyzes tRNA splicing is unexpectedly also required in yeast for HAC1 mRNA splicing to mediate the unfolded protein response, suggesting that this function of ligase is also possible in other eukaryotes. Fourth, a fully functional form of the 2'-phosphotransferase is found in E. coli, and evolutionary analysis indicates that it has been in bacteria for more than a billion years. This is a surprise, since E. coli is not known to have introns, and bacteria do not splice RNA by this mechanism. Its retention there suggests an important function unrelated to splicing which, by extension, may also occur in eukaryotes. Since the first step of the mechanism is strikingly similar to the ADP-ribosylation catalyzed by a number of toxins (such as Diphtheria and cholera), and overproduction of the bacterial protein causes a distinct growth defect, the enzyme may have important regulation function. This proposal aims to study: the role of Appr>p in the cell through modulation of its levels in yeast and analysis of cellular function; how tRNA splicing is catalyzed in vertebrates, by use of an assay that detects both pathways simultaneously, and by studying expression of mouse phosphotransferase in different tissues; and the role of the phosphotransferase in yeast and E. coli, by the construction and analysis of mutated phosphotransferase proteins, coupled with analysis of the growth defect and its likely cause in E. coli.

这项工作的长期目标是了解酿酒酵母和脊椎动物中的 tRNA 剪接。 tRNA 剪接在酵母、人类以及可能所有真核生物中都是必需的，并且与其他类别的剪接有独特的不同。从表面上看，酵母中的 tRNA 剪接很简单：核酸内切酶切除内含子，RNA 连接酶连接半分子以留下剪接点 2'-磷酸，2'-磷酸转移酶将磷酸转移到 NAD 形成 ADP-核糖 1 “-2”环状磷酸盐（Appr>p），一种以前未知的代谢物。这些酶在脊椎动物中高度保守，最后一步在非洲爪蟾卵母细胞体内发挥作用。然而，存在着惊人的复杂性：首先，剪接过程中等摩尔量 Appr>p 的形成意味着一条使其返回已知代谢的途径，并表明酵母作为传感器的调节功能。其次，脊椎动物显然还具有与 tRNA 剪接有关的第二种连接酶，它使用不同的化学物质并且不需要 2'-磷酸转移酶。在同一生物体中出现两条看似冗余的途径的情况很少见，这表明存在某种形式的调节，或其中一条途径的另一种用途。第三，出乎意料的是，酵母中的 HAC1 mRNA 剪接也需要催化 tRNA 剪接的连接酶来介导未折叠的蛋白质反应，这表明连接酶的这种功能在其他真核生物中也可能存在。第四，在大肠杆菌中发现了 2'-磷酸转移酶的全功能形式，进化分析表明它已经在细菌中存在了十亿多年。这是一个惊喜，因为我们不知道大肠杆菌有内含子，而且细菌也不会通过这种机制剪接 RNA。它的保留表明了一种与剪接无关的重要功能，推而广之，这种功能也可能发生在真核生物中。由于该机制的第一步与许多毒素（如白喉和霍乱）催化的 ADP-核糖基化惊人相似，并且细菌蛋白的过量产生会导致明显的生长缺陷，因此该酶可能具有重要的调节功能。本提案旨在研究：通过调节酵母中 Appr>p 的水平并分析细胞功能，研究 Appr>p 在细胞中的作用；通过使用同时检测两条途径的检测方法以及研究小鼠磷酸转移酶在不同组织中的表达，如何在脊椎动物中催化 tRNA 剪接；以及磷酸转移酶在酵母和大肠杆菌中的作用，通过构建和分析突变的磷酸转移酶蛋白，并分析大肠杆菌中的生长缺陷及其可能的原因。