STRUCTURE AND FUNCTION OF YEAST SMALL NUCLEAR RNPS

尽管含有大量snRNA的snRNP是去除RNA所必需的， 内含子的作用机制知之甚少， snRNP。 虽然snRNA的一级序列已经确定， 二级结构模型已经开发，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 首先关注U2 snRNP。 基于U2序列的比较 从不同的生物体，和最初的化学改造实验， 我们怀疑U2 snRNA被折叠成一种有趣的RNA结构， 叫做假结 在反应中心os处发现假结 I组自我剪接内含子和16S rRNA。 我们的假设是 结构在反应中心起着重要的作用， 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基因导入 缺乏其他US基因的酵母细胞。 美国突变，允许正常 剪接和生长将被认为不影响U2功能， 那些不允许生长或只允许异常或 有条件的增长与缺陷的拼接将被认为是影响 U2在体内的功能。 我们将使用化学结构探针来确定 U2突变对U2结构的影响。 我们还将寻求 旨在鉴定与U2相互作用的蛋白质的方法。 搜索 对于冷敏感性U2突变的基因外抑制因子， 编码这些蛋白质的基因。

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.