ASSEMBLY AND FUNCTION OF THE U4/U6 SPLICEOSOMAL SNRNP

U4/U6 剪接体 SNRNP 的组装和功能

• 批准号: 2023277
• 负责人: DAVID A BROW
• 金额: $ 13.35万
• 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/2023277
• 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 它在剪接体中的功能将通过遗传学方法来检测。 抑制分析将使用两种不同的主要突变来 探测前体mRNA剪接周期的不同部分。一个突变 （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个独立 冷敏感生长表型的自发抑制因子 与这种突变相关的基因抑制器 预计菌株中含有基因突变， 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。抑制子的表征 突变将揭示U4/U6 snRNP组装的机制， 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中的三碱基取代） 导致U6 RNA中必需序列的掩蔽，包括 绝对保守的“ACAGA盒”，在组装和激活过程中， 剪接体我们已经分离出25个独立的自发抑制因子 这种突变引起的对冷敏感的生长。抑制器 预计菌株中含有基因突变， 与U4/U6 snRNP在组装和激活过程中相互作用， 剪接体 他们的进一步表征将揭示机制 U4/U6 snRNP在剪接体激活过程中的解体。 在剪接的作用，确定为抑制因子的基因， 两个主要突变将通过遗传，生物化学， 和抑制等位基因产物的物理分析。的 迄今为止鉴定的抑制基因座编码两种剪接体RNA（U4 和U6）和两个剪接因子（Prp 8和Prp 24）。其他几个位点 还有待确认 从拟议的研究中获得的信息将大大 推进我们对真核基因表达关键步骤的理解。 作为细胞生长中潜在的限速步骤，剪接几乎是 肯定参与了细胞周期进程的调节。的确， 剪接因子Prp 8中的某些突变， 本研究结果表明，细胞周期阻滞在G1/S期。 由于细胞周期调控的丧失在肿瘤中起着重要作用， 随着研究的进展，对剪接途径的详细了解非常重要 来理解致癌作用。此外，U4/U6 RNA 相互作用作为RNA动力学研究的一个范例，一个新的 也是生物化学研究的重要领域。