MESSENGER RNA PROCESSING IN SCHIZOSACCHAROMYCES

尽管指导信使RNA剪接的信号 体内的前体一直被描绘出来，直到最近 开发可靠的体外系统，其机制是 插入序列被移除在很大程度上仍然是一个谜。 十年前，剪接技术被发现后不久， 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有帽RNA，是 负责对拼接信号进行解码的因素。这些 最初基于氢的假设方案的模型- 用拼接基板粘合，似乎大致正确，如 从无细胞系统的初步结果来判断。一大笔钱 细节的细化仍然是必要的，以带来整个 然而，过程成为焦点。途径，如剪接，在其中 一组大分子聚集在一起，是一个很好的目标 遗传方法，但易于处理的遗传系统明显 在高等真核生物中缺乏的。 这项提议的目标是利用强大的基因 酵母中可用的方法来实现对 有助于准确和高效地进行分子接触 信使核糖核酸剪接庞氏裂殖酵母被选为 我们的研究是因为，就像酿酒酵母一样，它是顺从的 通过经典和转化来进行基因操作 方法，但现有证据表明它的剪接 这种机制与哺乳动物的机制非常相似。我们 正在采用三个互补的战略。首先，我们 将分析SnRNA基因的空等位基因和条件等位基因 确定它们对RNA加工的影响。中的其他组件 然后，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。一秒钟 方法将是改变S.pombe拼接信号，这将 与SnRNA工作融合，以实现最终的测试 建议的功能模型，例如氢键 在U1SnRNA和5‘剪接接头之间。最终的目标是 我们将继续开发从S.pombe中提取的物质 它能在体外有效地剪接mRNA前体。终极的 目标将是使用这样的系统来进行生化表征 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.