MESSENGER RNA PROCESSING IN SCHIZOSACCHAROMYCES

尽管指导信使RNA剪接的信号 前体在体内已经划定，直到最近 开发可靠的体外系统， 插入序列被移除仍然是一个谜。 在十年前发现剪接后不久， 假设U类小核RNA，一组 在真核生物中普遍存在的代谢稳定的加帽RNA， 负责解码拼接信号的因素。 这些 模型，最初基于氢的假设方案， 与拼接基板结合，似乎是广泛正确的， 根据无细胞系统的初步结果判断。 大量 细节的细化仍然是必要的，使整个 过程成为焦点。 途径如剪接，其中 一系列的大分子聚集在一起， 遗传方法，但易处理的遗传系统是明显的 在高等真核生物中缺乏。 这项提议的目的是利用强大的基因 酵母中可用的方法，以实现精确的理解 分子接触，有助于准确和有效地 mRNA剪接。 粟酒裂殖酵母已被选择用于 我们的研究，因为，像酿酒酵母， 通过传统的和转化的基因操作 方法，但现有的证据表明，它的剪接 这一机制与哺乳动物的机制更为相似。 我们 采用了三种互补的策略。 第一，我们 将分析snRNA基因的无效和条件等位基因， 确定它们对RNA加工的影响。 中的其他组件 与snRNA相互作用的细胞可以通过 选择这些突变的基因外回复突变体。 第二 方法是改变S。pombe拼接信号，这将 与snRNA的工作融合， 提出的功能模型，例如，氢键 在U1 snRNA和5'剪接点之间。 最终目标， 我们将追求的是从S中提取的发展。pombe 其在体外有效地拼接mRNA前体。 最终 目标将是使用这样一个系统来生化表征 在上述实验中产生的突变。

Although the signals which direct splicing of messenger RNA precursors in vivo had been delineated, until the recent development of reliable in vitro systems, the mechanism by which intervening sequences are removed remained largely a mystery. Soon after the discovery of splicing a decade ago, it was postulated that U-class small nuclear RNAs, a group of metabolically stable capped RNAs ubiquitous in eukaryotes, were the factors responsible for decoding splicing signals. These models, based initially on hypothetical schemes for hydrogen- bonding with splicing substrates, appear to be broadly correct as judged by initial results with the cell-free systems. A great deal of refinement of the details is still necessary to bring the entire process into focus, however. Pathways such as splicing, in which an array of macromolecules assemble, are excellent targets for a genetic approach, but tractable genetic systems are distinctly lacking among the higher eukaryotes. The goal of this proposal is to exploit the powerful genetic methods available in yeast to achieve a precise understanding of the molecular contacts which contribute to accurate and efficient mRNA splicing. Schizosaccharomyces pombe has been chosen for our studies because, like Saccharomyces cerevisiae, it is amenable to genetic manipulation by both classical and transformation methods, but available evidence suggests that its splicing mechanism bears a closer resemblance to that of mammals. We are employing three complementary strategies. In the first, we will analyze null and conditional alleles of snRNA genes and determine their effects on RNA processing. Other components in the cell which interact with the snRNAs can then be identified by selecting for extragenic revertants of these mutations. A second approach will be to alter S. pombe splicing signals, which will converge with the snRNA work to allow definitive tests of proposed functional models, for example, hydrogen bonding between U1 snRNA and 5' splice junctions. The final goal which we will pursue is the development of an extract from S. pombe which splices mRNA precursors efficiently in vitro. The ultimate aim will be to use such a system to biochemically characterize mutations generated in the experiments described above.