SPLICING OF A RIBOSOMAL RNA PRECURSOR

在单细胞真核生物的大核核糖体RNA基因中， 嗜热四膜虫，26S rRNA编码区被413区打断 碱基对插入序列(IVS)。IVS包含在 最初的转录本，但随后通过剪接从RNA中切下。 我们最近开发了一种完全定义的体外剪接系统。 我们发现，剪接活动是固有的IVS部分 RNA，而且不需要酶或其他蛋白质。该计划的目标是 提出的研究是为了进一步表征前rRNA的机制。 剪接和介导反应所需的RNA的结构。这个 外显子(RRNA)连接过程中形成的连接结构 与IVS相邻的序列)将通过RNA指纹图谱确定，以及 该连接反应的动力学和辅因子要求如下 调查过了。鸟苷辅助因子在RNA中的结合位置将 以鸟苷类似物的动力学研究为特征，通过 光亲和标记法。IVS的二级结构将是 使用化学和酶探针，计算机计算和 序列的系统发育比较。切除的IVS RNA的结构 当它是前体的一部分时，将与静脉输液系统进行比较。 重组质粒DNA的泛化和定点突变 随后的体外转录将用于产生改变的Pre-rRNA 将在体外剪接系统中进行测试的分子；这将 允许将剪接反应特定步骤中的缺陷关联起来 在RNA序列中有特定的变化。最后，本文介绍了 四膜虫Pre-rRNA剪接机制将被研究。这个 四膜虫Pre-rRNA剪接系统异常地服从于详细的 学习。希望其中的一些发现将适用于 更不用说人类信使核糖核酸剪接的问题，其中的缺陷是 已知会导致贝塔+地中海贫血等疾病。

In the macronuclear ribosomal RNA genes fo the unicellular eucaryote, Tetrahymena thermophila, the 26S rRNA coding region is interrupted by a 413 base pair intervening sequence (IVS). The IVS is contained within the primary transcript but is subsequently excised from the RNA by splicing. We have recently developed a completely defined, in vitro splicing system. We found that the splicing activity is intrinsic to the IVS portion of the RNA, and that no enzyme or other protein is required. The objective of the proposed research is to further characterize the mechanism of pre-rRNA splicing and the structure of the RNA needed to mediate the reaction. The structure of the junction formed during ligation of the exons (rRNA sequences bordering the IVS) will be determined by RNA fingerprinting, and the kinetics and cofactor requirements of this ligation reaction will be investigated. The binding site of the guanosine cofactor in the RNA will be characterized by kinetic studies with guanosine analogs and by photoaffinity labeling. The secondary structure of the IVS will be determined using chemical and enzymatic probes, computer calculations and phylogenetic comparison of sequences. The structure of the excised IVS RNA will be compared to that of the IVS when it is part of the precursor. Generalized and sitespecific mutagenesis of recombinant plasmid DNA followed by in vitro transcription will be used to produce altered pre-rRNA molecules which will be tested in the in vitro splicing system; this will allow defects in specific steps of the splicing reaction to be correlated with specific changes in the RNA sequence. Finally, the generality of the Tetrahymena pre-rRNA splicing mechanism will be investigated. The Tetrahymena pre-rRNA splicing system is unusually amenable to detailed study. It is hoped that some of the findings will be applicable to the much less tractable problem of human mRNA splicing, defects in which are known to cause diseases such as Beta+-thalassemia.