STRUCTURE AND FUNCTION OF YEAST SMALL NUCLEAR RNPS

酵母小核RNPS的结构和功能

• 批准号: 3072923
• 负责人: Manuel Ares
• 金额: $ 6.96万
• 依托单位: UNIVERSITY OF CALIFORNIA SANTA CRUZ
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-07-01 至 1994-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3072923
• 关键词: DNA binding protein; DNA footprinting; fungal genetics; genetic manipulation; genetic regulation; nuclear magnetic resonance spectroscopy; nucleic acid sequence; optical polarization; protein structure function; small nuclear RNA; spliceosomes; temperature sensitive mutant; yeasts

Although snRNPs containing the abundant snRNAs are required for removing introns from pre-mRNA, little is known about the mechanism of action of snRNPs. While the primary sequences of snRNAs have been determined, and secondary structure models have been developed, the higher order structures of snRNAs and their relationship to snRNP function remain mysterious. We propose to analyze the mechanism of action of snRNPs focusing initially on the U2 snRNP. Based on comparison of U2 sequences from different organisms, and initial chemical modification experiments, we suspect that U2 snRNA is folded into an intriguing RNA structure called a pseudoknot. Pseudoknots are found in the reaction center os group I self-splicing introns and 16S rRNA. Our hypothesis is that this structure plays an important role at the reaction center of the spliceosome. We will use oligonucleotide directed mutagenesis to alter a clones copy of the yeast U2 gene, and introduce the mutant U2 gene into yeast cells lacking any other US gene. US mutations that allow normal splicing and growth will be considered not to affect U2 function, and those mutations that do not allow growth or allow only abnormal or conditional growth with defects in splicing will be considered to affect U2 function in vivo. We will use chemical structure probes to determine the effect of U2 mutations on U2 structure. We will also pursue approaches designed to identify proteins that interact with U2. A search for extragenic suppressors of a cold sensitive U2 mutation may identify genes encoding such proteins.

尽管去除需要含有丰富 snRNA 的 snRNP 来自前mRNA的内含子，但对其作用机制知之甚少 snRNP。 虽然 snRNA 的一级序列已确定，并且 二级结构模型已经开发出来，高阶 snRNA 的结构及其与 snRNP 功能的关系仍然存在 神秘。 我们建议分析 snRNP 的作用机制 最初关注 U2 snRNP。 基于U2序列的比较 来自不同的生物体，以及最初的化学修饰实验， 我们怀疑 U2 snRNA 被折叠成一个有趣的 RNA 结构 称为假结。 反应中心操作系统中发现假结 I 组自剪接内含子和 16S rRNA。 我们的假设是，这 结构在反应中心起着重要作用 剪接体。 我们将使用寡核苷酸定向诱变来改变 克隆酵母U2基因的拷贝，并将突变的U2基因引入 酵母细胞缺乏任何其他美国基因。 美国突变允许正常 剪接和生长将被认为不会影响U2功能，并且 那些不允许生长或只允许异常或 剪接缺陷的条件生长将被认为影响 U2在体内发挥作用。 我们将使用化学结构探针来确定 U2突变对U2结构的影响。 我们也将追求 旨在识别与 U2 相互作用的蛋白质的方法。 一次搜索 对于冷敏感 U2 突变的外源抑制因子可能会识别 编码此类蛋白质的基因。