GENETIC AND MOLECULAR STUDIES OF RNA SPLICING

这项研究的长期目标是：1）开发新的见解 酵母线粒体DNA第二组内含子的结构及其作用 在体内和体外剪接反应中的那些结构; 2） 研究由IIA组编码的成熟酶蛋白的功能 内含子剪接; 3）开发和测试两种遗传模型 依赖于IIA组内含子的现象;和4）纯化和 表征由组II编码的突变体和野生型蛋白质 内含子 预测的三级结构的有效性将 通过将相关突变等位基因转化为mtDNA进行体内测试; 将确定体内表型， 分离并表征回复突变体。 我们已经发现了一种 组的结构域5和结构域1之间的关键三级相互作用 II内含子，这是必要的反应，在两个剪接 交叉点 进一步了解的功能重要性 将获得结构域5内的特定序列和结构 通过筛选随机化的含有结构域5的RNA， 使用反式剪接测定法测定其功能。 结构域5的突变体将 转化为线粒体和第二位点抑制子 其特征在于，为了定义内含子的区域， 它相互作用。 IIA组内含子的阅读框架类似于 逆转录酶，并且还含有锌指样结构域; 几乎所有具有阅读框架的II组内含子都具有这些特征 以及其他高度保守的片段， 存在已知蛋白质。 我们会让这些区域发生变异， 将突变转化为线粒体， 关于拼接 将测试阅读框突变体的效应 两种遗传现象似乎依赖于该内含子， 即，通过内含子切除使剪接缺陷突变体回复 (RIE)和II组内含子迁移率。 生化实验将 来测试这些过程的模型预测。 最后，野生型和突变形式的蛋白质编码的 将从线粒体提取物中纯化II组内含子， 表征;剪接、核酸剪接的体外试验 和/或逆转录酶进行检测。

The long-term goals of this research are to 1) develop new insights to the structure of group II introns of yeast mtDNA and roles of those structures in splicing reactions in vivo and in vitro; 2) study the function of a maturase protein encoded by a group IIA intron in splicing; 3) develop and test models for two genetic phenomena dependent on the group IIA introns; and 4) purify and characterize mutant and wild-type proteins encoded by group II introns. The validity of predicted tertiary structures will be tested in vivo by transforming relevant mutant alleles into mtDNA; the in vivo phenotypes will be determined and second-site revertants isolated and characterized. We have discovered a crucial tertiary interaction between domain 5 and domain 1 of group II introns that is necessary for reactions at both splice junctions. Further insight to the functional importance of specific sequences and structures within domain 5 will be obtained by screening randomized domain 5-containing RNAs for retained function using a trans-splicing assay. Mutants of domain 5 will be transformed into mitochondria and second-site suppressors characterized in order to define regions of the intron with which it interacts. The reading frame of the group IIA intron resembles reverse transcriptase and also contains a zinc-finger like domain; nearly all group II introns with reading frames have those features and additionally other highly conserved segments not resembling a known protein are present. We will mutate each of those regions, transform the mutations into mitochondria and determine the effect on splicing. Reading frame mutants will be tested for their effect on two genetic phenomena that appear dependent on that intron, namely, reversion of splicing defective mutants by intron excision (RIE) and group II intron mobility. Biochemical experiments will be carried out to test predictions of models for those processes. And finally, wild-type and mutant forms of proteins encoded by group II introns will be purified from mitochondrial extracts and characterized; in vitro assays for splicing, nucleic acid splicing and/or reverse transcriptase will be tested.