ASSEMBLY AND FUNCTION OF THE U4/U6 SPLICEOSOMAL SNRNP

U4/U6核糖核蛋白小颗粒的组装 而它在剪接体中的功能将通过基因 抑制分析。两种不同的初级突变将被用于 探索前信使核糖核酸剪接周期的不同部分。一种突变 (U6 RNA中的单碱基取代)抑制构象转换 U4和U6 RNA的碱基配对所需。超过100家独立公司 冷敏感生长表型的自发抑制因子 与这种突变相关的基因已经被分离出来。抑制者 预计菌株在其产物的基因中存在突变 参与U4/U6 snRNP的组装或与U6 RNA相互作用 拼接循环的后续步骤。抑制者的特征 突变将揭示U4/U6 SnRNP组装的机制，并将 定义U6 RNA和其他剪接因子之间的相互作用。 第二个主要突变(U4 RNA中的三个碱基替换) 导致U6 RNA中的基本序列被屏蔽，包括 绝对保守的“ACAGA盒”，在组装和激活 剪接体。我们已经分离出了25个独立的自发抑制者 这种突变引起的对冷敏感的生长。抑制者 预计菌株在其产物的基因中存在突变 在组装和激活过程中与U4/U6 SnRNP相互作用 剪接体。它们的进一步表征将揭示这一机制 在剪接体激活过程中U4/U6 SnRNP的拆解。 在被确认为抑制因子的剪接中的作用 两个主要的突变将进一步通过遗传，生化， 以及对抑制基因产物的物理分析。这个 迄今发现的两个剪接体RNA(U4)的抑制子基因座 和U6)和两个剪接因子(Prp8和Prp24)。其他几个基因座 都还没有确定。 从拟议的研究中获得的信息将显著地 促进我们对真核基因表达的一个关键步骤的理解。 作为细胞生长的潜在限速步骤，剪接几乎是 当然参与了细胞周期进程的调节。的确， 剪接因子Prp8的某些突变，是 本研究使细胞周期停滞于G1/S期。 由于细胞周期调控的丧失在肿瘤中起主要作用 进展，剪接途径的详细知识是重要的 以了解癌症的发生。此外，U4/U6 RNA 相互作用作为一种研究RNA动力学的范式，是一种新的 也是生化研究的重要领域。

Assembly of the U4/U6 small nuclear ribonucleoprotein particle (snRNP) and its function in the spliceosome will be examined by genetic suppression analysis. Two different primary mutations will be used to probe different portions of the pre-mRNA splicing cycle. One mutation (a single-base substitution in U6 RNA) inhibits a conformational switch required for base-pairing of U4 and U6 RNAs. Over 100 independent spontaneous suppressors of the cold-sensitive growth phenotype associated with this mutation have been isolated. The suppressor strains are expected to harbor mutations in genes whose products participate in assembly of the U4/U6 snRNP or interact with U6 RNA in later steps of the splicing cycle. Characterization of the suppressor mutations will reveal the mechanism of U4/U6 snRNP assembly and will define interactions between U6 RNA and other splicing factors. The second primary mutation (a triple-base substitution in U4 RNA) results in the masking of essential sequences in U6 RNA, including the absolutely conserved "ACAGA box", during assembly and activation of the spliceosome. We have isolated 25 independent spontaneous suppressors of the cold-sensitive growth caused by this mutation. The suppressor strains are expected to harbor mutations in genes whose products interact with the U4/U6 snRNP during assembly and activation of the spliceosome. Their further characterization will reveal the mechanism of U4/U6 snRNP disassembly during activation of the spliceosome. The roles in splicing of the factors identified as suppressors of the two primary mutations will be further examined by genetic, biochemical, and physical analyses of the products of the suppressor alleles. The suppressor loci identified to date code for two spliceosomal RNAs (U4 and U6) and two splicing factors (Prp8 and Prp24). Several other loci have yet to be identified. The information obtained from the proposed study will significantly advance our understanding of a key step in eukaryotic gene expression. As a potential rate-limiting step in cell growth, splicing is almost certainly involved in the regulation of cell cycle progression. Indeed, certain mutations in the splicing factor Prp8, one of the subjects of this study, result in cell cycle arrest at the G1/S phase transition. Since loss of cell cycle regulation plays a major role in tumor progression, detailed knowledge of the splicing pathway is important for understanding carcinogenesis. Furthermore, the U4/U6 RNA interaction serves as a paradigm for the study of RNA dynamics, a new and important field of biochemical investigation.