STRUCTURE AND FUNCTION OF YEAST SMALL NUCLEAR RNPS

虽然去除含有大量SnRNAs的SnRNPs是必需的 来自前-mRNA的内含子，对其作用机制知之甚少 SNRNPs。虽然已经确定了SnRNA的初级序列，并且 已经开发了二级结构模型，更高阶的 SnRNAs的结构及其与SnRNP功能的关系仍然存在 很神秘。我们建议分析SNRNPs的作用机制 最初重点放在U2 snRNP上。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U2SnRNA被折叠成一种有趣的RNA结构 被称为伪结。在反应中心os中发现了伪结点。 I组自剪接内含子和16S rRNA。我们的假设是，这是 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突变来改变 克隆酵母U2基因的拷贝，并将突变的U2基因导入 缺乏任何其他US基因的酵母细胞。允许正常的美国突变 剪接和生长将被认为不影响U2功能，并且 那些不允许生长或只允许异常或 剪接缺陷的条件性生长将被认为影响 U2在体内发挥作用。我们将使用化学结构探头来确定 U2突变对U2结构的影响。我们还将继续追查 旨在识别与U2相互作用的蛋白质的方法。一次搜查 对于寒冷敏感的U2突变的基因外抑制物可能识别 编码这类蛋白质的基因。

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.