MOBILE GROUP I INTRONS IN THE NUCLEUS

细胞核中的移动基团 I 内含子

• 批准号: 2022930
• 负责人: Volker M. Vogt
• 金额: $ 16.25万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-01-01 至 1998-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2022930
• 关键词: RNA binding protein; RNA splicing; cell nucleus; endonuclease; fungal genetics; gene expression; introns; messenger RNA; mutant; nucleic acid sequence; nucleolus; oligonucleotides; open reading frames; polymerase chain reaction; protein structure function; ribozymes; spectrometry; transposon /insertion element; yeasts

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.

I 组内含子是原型催化 RNA 或核酶之一。 的 超过 100 个已知的 I 组内含子，大约有 10% 是可移动的： 它们可以作为DNA元件“归巢”到同一基因的未占据等位基因上 他们通常居住的地方。 归巢是由高度特异性介导的 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，但也有少数在细胞核中发现。 全核组I 内含子插入核糖体 DNA (rDNA) 中。 只有三个 细胞核中移动组 I 内含子的例子，其中只有两个 已被表征。 它们是 PpLSU3 和 DiSSU1，来自非细胞 粘菌分别是多头绒泡菌和虹膜镨菌。 自从 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，这些基因的独特之处在于 他们推测的信使 RNA 显然源自 Pol I 成绩单。 拟议研究的第一个主要目标是阐明基因如何 I-Ppo 核酸内切酶由 PpLSU3 表达。 这项工作将使 使用我们之前分离的具有抗性的酵母菌株 核酸内切酶的致死作用。 我们将确定什么顺序 该蛋白质的成功表达需要一些元件。 为了 例如，I-Ppo 的 mRNA 是从 Pol I 转录本加工而来，还是 它源自较小的 Pol II 转录本？ 是否需要功能性核糖体 为了表达？ 核仁RNA结合蛋白是否发挥作用 表达？ 是否也可以人工将其他基因放入该内含子中？ 表达？ DiSSU1 是一个自剪接内含子，具有独特的性质：两个独立的内含子 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组的类 我内含子。下游元件（新型剪接核酶，NSR）， 本身可以切割 3' 剪接位点，似乎与其他已知的无关 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来协调 外显子连接。 我们将使用诱变策略，包括体外 回复体的进化和选择，询问 NSR 中的序列是什么 对其功能至关重要。 NSR 可以反式切割其他 RNA 吗？ 可以吗 在酵母细胞体内发挥作用？ 我们将研究 NSR 如何与 上游 I 组核酶。 当表达为单独的分子时，可以 这两种核酶形成一​​个复合物，能够连接外显子 反式？ 如果反式剪接在体外起作用，我们将尝试建立一个体内 体内系统将允许在这些合作中选择突变体 核酶。