TRNA SPLICING

这项工作的长期目标是了解tRNA在酵母、酿酒酵母和脊椎动物中的剪接。TRNA剪接在酵母、人类和可能所有真核生物中都是必不可少的，并且与其他类别的剪接有独特的不同。从表面上看，tRNA在酵母中的剪接很简单：内切酶切除内含子，RNA连接酶连接半分子离开剪接连接2‘-磷酸，2’-磷酸转移酶将磷酸转移到NAD形成ADP-核糖1“-2”环磷酸(appr&gt；p)，这是一种以前未知的代谢物。这些酶在脊椎动物中高度保守，最后一步在非洲爪哇卵母细胞中起作用。然而，还有惊人的复杂性：首先，在剪接过程中等摩尔量的appr；gt；p的形成意味着一条使其返回已知新陈代谢的途径，并表明酵母作为传感器具有调节功能。其次，脊椎动物显然还有第二种连接酶，它与tRNA剪接有关，它使用不同的化学物质，不需要2‘-磷酸转移酶。这是同一生物体中罕见的两条看似多余的途径，这意味着某种形式的调控，或者其中一条途径的另一种用途。第三，在酵母中，催化tRNA剪接的连接酶出人意料地也需要HAC1 mRNA剪接来介导未折叠的蛋白质反应，这表明连接酶的这一功能在其他真核生物中也是可能的。第四，在大肠杆菌中发现了一种功能齐全的2‘-磷酸转移酶，进化分析表明，它在细菌中已经存在了10亿多年。这是一个惊喜，因为目前还不知道大肠杆菌有内含子，细菌也不会通过这种机制剪接RNA。它在那里的保留表明了一种与剪接无关的重要功能，延伸到真核生物中也可能发生。由于该机制的第一步与许多毒素(如白喉和霍乱)催化的ADP-核糖化非常相似，而细菌蛋白的过量生产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。这项建议旨在研究：通过调节其在酵母中的水平和分析细胞功能，APPR&GT；P在细胞中的作用；通过同时检测这两个途径的分析，以及通过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；以及通过构建和分析突变的磷酸转移酶蛋白，以及分析在大肠杆菌中的生长缺陷及其可能的原因，来研究磷酸转移酶在酵母和大肠杆菌中的作用。

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.