STRUCTURE AND FUNCTION OF YEAST SMALL NUCLEAR RNPS

酵母小核RNPS的结构和功能

• 批准号: 3072924
• 负责人: Manuel Ares
• 金额: $ 6.99万
• 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/3072924
• 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; transcription factor; 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.

虽然需要含有丰富的snRNA的snRNP才能删除 来自Pre-MRNA的内含子，对作用机理知之甚少 snrnps。 虽然已经确定了SNRNA的主要序列，并且 已经开发了二级结构模型，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的作用机理 最初专注于U2 SNRNP。 基于U2序列的比较 来自不同生物体和初始化学修饰实验， 我们怀疑U2 snRNA被折叠成有趣的RNA结构 被称为伪诺。 伪诺在反应中心OS中发现 I组自剪接的内含子和16S rRNA。 我们的假设是 结构在反应中心起重要作用 剪接体。 我们将使用寡核苷酸定向诱变来改变 酵母U2基因的克隆副本，并将突变体U2基因引入 缺乏其他美国基因的酵母细胞。 允许正常的美国突变 剪接和增长将被认为不影响U2功能，并且 那些不允许增长或仅允许异常或 有条件的生长与剪接中的缺陷被认为会影响 U2功能在体内。 我们将使用化学结构探针确定 U2突变对U2结构的影响。 我们还将追求 旨在识别与U2相互作用的蛋白质的方法。 搜索 对于冷敏感的U2突变的基本抑制剂，可能会识别 编码这种蛋白质的基因。