MOBILE GROUP I INTRONS IN THE NUCLEUS

I组内含子是一种原型催化RNA或核酶。 的 超过100个已知的I组内含子，可能有10%是移动的： 它们可以作为DNA元件“归巢”到同一基因的未被占据的等位基因， 他们通常居住的地方。 归巢是由一种高度特异性的 内含子本身编码的核酸内切酶。 大多数I组内含子位于 细胞器DNA，但少数已被发现在细胞核中。 全核I组 内含子插入核糖体DNA（rDNA）中。 只有三 细胞核中移动的I组内含子的例子，其中只有两个 已经被定性。 它们是PpLSU 3和DisSU 1，来自非细胞的 黏菌为多头绒泡菌（Physarum polycephalum）和虹膜皮黏菌（Didymium iridis）。 以来 内含子位于rRNA中，嵌入其中的核酸内切酶基因是 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Pol I 成绩单。 这项研究的第一个主要目标是阐明基因是如何 I-Ppo内切核酸酶由PpLSU 3表达。 这项工作将使 使用我们以前分离的酵母菌株， 核酸内切酶的致死作用。 我们将确定 成功表达这种蛋白质需要元件。 为 例如，是从Pol I转录物加工的I-Ppo的mRNA，或者是 它是从一个次要的Pol II转录本中衍生出来的吗 是否需要功能性核糖体 为了表达？ 核仁RNA结合蛋白在 表情？ 其他被人工置入这个内含子的基因是否也能被 表达？ DiSSU 1是一个自我剪接内含子，具有独特的性质：两个独立的 和可分离的催化RNA元件以某种方式在剪接中合作 反应 按顺序，上游元素属于群类 I内含子。下游元件（新型剪接核酶，NSR）， 本身可以切割3'剪接位点，似乎与其他已知的 催化RNA。 拟议研究的第二个主要目标是表征NSR 并了解它如何与第一组元素合作， 外显子连接 我们将使用诱变策略，包括体外 回复突变体的进化和选择，以询问NSR中的序列是什么 对它的功能至关重要。 NSR能反式切割其他RNA吗？ 能 在酵母细胞中的功能 我们会研究“噪音感应强的地方”如何与 上游组I核酶。 当表达为单独的分子时， 这两种核酶形成一种复合物， 跨性别？ 如果反式剪接在体外起作用，我们将尝试建立一个 体内系统，将允许选择突变体在这些合作 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.