BIOSYNTHESIS OF RNAS

尽管在许多实验室进行了深入的研究，但我们对 分子机制，指定准确和有效的剪接， 含内含子的mRNA核转录物仅略有进展 超越现象学。 这同样适用于 信息的成熟，包括3'末端的产生， 聚腺苷酸化 最近，一个由六个小的 核RNA（snRNA），其非凡的结构保守性， 加上各种间接证据，表明他们的 参与真核基因的功能保守步骤 表情 例如，假设U1 RNA介导内含子去除 通过与剪接点处的保守序列互补。 由于高等教育缺乏易操作的实验系统， 真核生物，这些假设还有待严格检验。 我们提出 利用酵母独有的强大遗传技术， 实现这些目标。 我们采用了两种互补的战略。 第一次利用我们的 最近证实了在酿酒酵母中存在snRNAs 由单拷贝基因编码;这使我们能够进行明确的测试， 这些模型通过分析无效和条件等位基因的后果， 在每一个基因中。 此外，基因外抑制因子的分离 这些突变将确定细胞成分， snRNA;这些应该包括snRNP蛋白，以及潜在的 “靶”RNA：前mRNA底物。 我们的第二种方法 我们最近鉴定的突变5'剪接点的优势， 酵母mRNA内含子。 在这种情况下，分离外源基因的能力 抑制子应该允许直接测试RNA的“UI指导模型”， 剪接

Despite intensive study in numerous laboratories, our understanding of the molecular mechanisms which specify the accurate and efficient splicing of intron-containing mRNA nuclear transcripts has progressed only slightly beyond the phenomenological. The same is true for other steps in the maturation of messages, including the generation of 3' termini and polyadenylation. Attention has recently been drawn to a group of six small nuclear RNAs (snRNAs), whose extraordinary structural conservation, together with a variety of circumstantial evidence, suggest their participation in functionally conserved steps in eukaryotic gene expression. For example, U1 RNA is hypothesized to mediate intron removal via complementarity to conserved sequences at the splice junction(s). Because of the lack of tractable experimental systems among the higher eukaryotes, these hypotheses have yet to be critically tested. We propose to exploit the powerful genetic techniques uniquely available in yeast to achieve these ends. We are employing two complementary strategies. The first exploits our recent demonstration of the existence in Saccharomyces cerevisiae of snRNAs encoded by single copy genes; this allows us to perform definitive tests of these models by analyzing the consequences of null and conditional alleles in each of these genes. Moreover, the isolation of extragenic suppressors of these mutations will identify cellular components which interact with the snRNAs; these should include the snRNP proteins, as well as potential "target" RNAs: the pre-mRNA substrates. Our second approach takes advantage of our recent identification of a mutant 5' splice junction in a yeast mRNA intron. In this case, the ability to isolate extragenic suppressors should allow a direct test of the "Ul guide model" for RNA splicing