ASSEMBLY AND FUNCTION OF THE U4/U6 SPLICEOSOMAL SNRNP

U4/U6 剪接体 SNRNP 的组装和功能

• 批准号: 2857235
• 负责人: DAVID A BROW
• 金额: $ 13.69万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-01-01 至 2000-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2857235
• 关键词: RNA biosynthesis; RNA splicing; SDS polyacrylamide gel electrophoresis; cell cycle; conformation; gene expression; gene rearrangement; intermolecular interaction; messenger RNA; molecular cloning; nucleic acid sequence; nucleic acid structure; polymerase chain reaction; protein structure function; radionuclides; small nuclear ribonucleoproteins; spliceosomes; suppressor mutations; temperature sensitive mutant; ultraviolet spectrometry; yeasts

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.

U4/U6小核核糖核蛋白颗粒（SNRNP）的组装 并且其在剪接体中的功能将通过遗传检查 抑制分析。两个不同的主要突变将用于 探测前MRNA剪接周期的不同部分。一个突变 （在U6 RNA中的单基替代）抑制构象开关 U4和U6 RNA的碱基配对所必需的。超过100独立 冷敏感生长表型的自发抑制剂 与该突变相关的已经分离出来。抑制器 预计菌株将在其产物的基因中含有突变 参加U4/U6 SNRNP的组装或与U6 RNA相互作用 剪接周期的后期步骤。抑制器的表征 突变将揭示U4/U6 SNRNP组件的机制，并将 定义U6 RNA与其他剪接因子之间的相互作用。 第二个主要突变（U4 RNA中的三基碱基取代） 结果掩盖了U6 RNA中基本序列的掩盖，包括 在组装和激活期间，绝对保守的“ Acaga Box” 剪接体。我们已经隔离了25个独立的自发抑制器 该突变引起的冷敏感生长。抑制器 预计菌株将在其产物的基因中含有突变 组装过程中与U4/U6 SNRNP相互作用和激活 剪接体。 他们的进一步表征将揭示机制 剪接体激活期间U4/U6 SNRNP拆卸。 在拼接的因素中的作用，被确定为抑制器 通过遗传，生化，将进一步研究两个主要突变 以及对抑制剂等位基因产物的物理分析。这 抑制器基因座确定为两个剪接体RNA的日期代码（U4） 和U6）和两个剪接因子（PRP8和PRP24）。其他几个基因座 尚未确定。 从拟议的研究中获得的信息将显着 促进我们对真核基因表达的关键步骤的理解。 作为细胞生长的潜在限速步骤，剪接几乎是 当然参与细胞周期进程的调节。的确， 剪接因子PRP8中的某些突变，这是一个主题之一 这项研究导致在G1/s相变的细胞周期停滞。 由于失去细胞周期调节在肿瘤中起主要作用 进展，详细了解剪接途径很重要 理解致癌作用。此外，U4/U6 RNA 相互作用是RNA动力学研究的范式，一种新的 和生化研究的重要领域。