MOBILE GROUP I INTRONS IN THE NUCLEUS

第一组内含子是典型的催化RNA或核酶之一。的 在100多个已知的第一组内含子中，可能有10%是移动的： 它们可以作为DNA元件在同一基因的未被占据的等位基因中“回家” 它们通常居住的地方。归位是由高度特定的 内含子本身编码的内切酶。大多数第一类内含子位于 细胞器DNA，但在细胞核中发现了一小部分。全核族I 内含子插入核糖体DNA(RDNA)。只有三个 原子核中可移动的I族内含子的例子，其中只有两个 已经被刻画出来。它们是PpLSU3和DiSSU1，来自脱细胞 粘菌分别为多头绒泡菌和虹膜假单胞菌。自.以来 内含子驻留在rRNA中，嵌入其中的内切酶基因是 由RNA聚合酶I转录。因此这些基因是独一无二的 它们推测的信使RNA显然来自PolI 文字记录。 这项拟议研究的第一个主要目标是阐明基因是如何 对于I-PPO内切酶，从PpLSU3中表达。这项工作将使 使用我们以前分离的对他有抵抗力的酵母菌株 核酸内切酶的致死作用。我们将决定是什么顺序 这种蛋白的成功表达需要一些元件。为 例如，I-PPO的mRNA是从Pol I转录本加工而来的，还是 它来自Pol II的一份次要的文字记录？是否需要有功能的核糖体 用来表达？核仁RNA结合蛋白是否在 表情？人工放入该内含子的其他基因是否也可以 表达？ DiSSU1是一个自剪接内含子，具有一个独特的性质：两个独立的 可分离的催化RNA元件在剪接过程中以某种方式协同工作 反应。按照顺序，上游元素属于组的类 我插话道。下游元件(新型剪接核酶，NSR)，通过 本身可以切割3‘端的剪接位点，似乎与其他已知的无关 催化核糖核酸。 拟议研究的第二个主要目标是描述NSR的特征 并理解它如何与I组元素合作来协调 外显子结扎法。我们将使用突变策略，包括体外诱变 进化和反转变种的选择，询问NSR中的哪些序列 对它的功能至关重要。NSR能在TRANS中切割其他RNA吗？可以吗？ 酵母细胞的体内功能？我们将研究NSR如何与 上游组I核酶。当表达为单独的分子时，可以 这两种核酶形成了一种复合体，它能够连接外显子 变性人？如果反式剪接在体外有效，我们将尝试建立一个 活体系统将允许在这些合作中选择突变体 核酶。

Group I introns are one of the prototypic catalytic RNAs, or ribozymes. Of the more than 100 known group I introns, perhaps ten percent are mobile: they can"home" as DNA elements to unoccupied alleles of the same gene in which they normally reside. Homing is mediated by a highly specific endonuclease encoded in the intron itself. Most group I introns reside in organellar DNA, but a few have been found in nuclei. All nuclear group I introns are inserted in ribosomal DNA (rDNA). There are only three examples of mobile group I introns in the nucleus, and only two of these have been characterized. They are PpLSU3 and DiSSU1, from the acellular slime molds Physarum polycephalum and Didymium iridis, respectively. Since the introns reside in rRNA, the endonuclease genes embedded in them are transcribed by RNA Polymerase I. Hence these genes are unique in that their presumed messenger RNA apparently is derived from the Pol I transcript. The first major goal of the proposed research is to elucidate how the gene for the I-Ppo endonuclease is expressed from PpLSU3. The work will make use of our previously isolated yeast strains that are resistant tot he lethal effects of the endonuclease. We will determine what sequence elements are needed for successful expression of this protein. For example, is the mRNA for I-Ppo processed from the Pol I transcript, or is it derived from a minor Pol II transcript? Is a functional ribosome needed for expression? Do nucleolar RNA binding proteins play a role in expression? Can other genes artificially placed into this intron also be expressed? DiSSU1 is a self splicing intron with a unique property: Two independent and separable catalytic RNA elements somehow cooperate in the splicing reaction. By sequence the upstream element belongs to the class of group I introns. The downstream element (novel splicing ribozyme, NSR), which by itself can cleave the 3' splice site, appears unrelated to other known catalytic RNAs. The second major goal of the proposed research is to characterize the NSR and to understand how it cooperates with the group I element to orchestrate exon ligation. We will use mutagenesis strategies, including in vitro evolution and selection of revertants, to ask what sequences in the NSR are critical for its function. Can the NSR cleave other RNAs in trans? Can it function in vivo in yeast cells? We will study how the NSR interacts with the upstream group I ribozyme. When expressed as separate molecules, can the two ribozymes form a complex that is competent to ligate exons in trans? If trans splicing works in vitro, we will attempt to set up an in vivo system that will allow selection of mutants in these cooperating ribozymes.