TRANS-SPLICING IN C ELEGANS

线虫中的反式剪接

• 批准号: 6180223
• 负责人: Thomas Blumenthal
• 金额: $ 38.84万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-05-01 至 2003-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6180223
• 关键词: Caenorhabditis elegans; RNA binding protein; RNA splicing; RNase protection assay; chemical binding; expression cloning; gel mobility shift assay; genetic library; genome; immunoprecipitation; nucleic acid sequence; operon; polymerase chain reaction; posttranscriptional RNA processing; precursor mRNA; protein sequence; protein structure function; small nuclear RNA; small nuclear ribonucleoproteins; western blottings; yeast two hybrid system

DESCRIPTION (Adapted from Applicant's Abstract): C. elegans engages in three types of nuclear pre-mRNA processing: normal cis-splicing and trans-splicing of two different leaders: SL1 near the 5' end of pre- mRNAs, and SL2 at internal trans-splice sites in polycistronic pre-mRNAs to divide then into gene-length mRNAs. These three processes are mechanistically closely related,yet functionally distinct. How do C. elegans splicesomes pair 5' splice located in cis with an intron 3' splice site, but utilize only the appropriate SL for the two types of trans-splice site? It is argued that the highly conserved extended consensus, UUUCAG, at the 3' splice site is the key sequence for initiating splicing. Single base changes in this sequence will be tested for alterations in splice site choice in vivo. By analogy with mammalian splicing, it seems likely that U2F is the molecular responsible for recognition of this sequence. The gene for the C. elegans U2AF large subunit, which binds RNA and is required for splicing, has been cloned and recent results indicate it is alternatively spliced. Characterization of the U2F gene will be completed, the protein or proteins will be expressed, and antibodies obtained. The small subunit gene will also be cloned. Their map locations will be determined, and mutations that alter or destroy U2AF function will be selected. U2AF made in E. coli will be tested for binding specifically to the UUUCAG sequence using gel mobility, shift competitions and SELEX assays. Locations of a gene in a downstream position in a polycistronic transcription unit (operon) is sufficient to cause trans-splicing of its mRNA with SL2. The major focus of this project is to understand the mechanism of SL2 specific trans-splicing at internal sites in operons. A recently-developed in vitro trans-splicing system from C. elegans embryo extracts will be used to study how SL2 is specified. Polycistronic transcripts will be tested for specificity of trans- splicing in vitro. Trans-splicing specificity will also be investigated in transgenic animals, by altering gene spacing, the 5' cap, the nearby poly(a) signals, and the intercistronic sequence. The location of SL2 RNA where the specificity determinants reside will also be studied. Sequence required for 3' end formation, including the GU box and transcription termination signals will be determined. Differences between 3' end formation within an operon and at the 3' ends of transcription units will be investigated. New operons will continue to be identified to determine whether they involve co-expression of genes whose product function together, and to learn how much variation in operon structure is tolerated. The evolution and function of operons will be studied by a search for existence of polycistronic units in other genera. Molecules involved in trans-splicing will be identified by both genetic and biochemical techniques. Mutants that have lost the capacity for SL1 and SL2 trans-splicing at restrictive temperature will be selected. Proteins that interact with U2AF, or with SL1 or SL2 RNA's will be identified by in vitro binding experiments. In addition, the effect of overexpression of U2AF subunits will be studied, and dominant negative mutants will be sought by overexpression of U2AF missing RNA- binding or subunit interaction domain.

描述（改编自申请人的摘要）：线虫从事 核前 mRNA 加工的三种类型：正常顺式剪接和 两个不同前导序列的反式拼接：SL1 靠近前导序列 5' 端 mRNA 和 SL2 位于多顺反子前体 mRNA 的内部转剪位点 然后分成基因长度的 mRNA。 这三个过程是 机制上密切相关，但功能上却截然不同。 C 怎样做？ 线虫剪接体对 5' 剪接位于顺式，具有 3' 内含子 剪接位点，但仅利用适合两种类型的 SL 反式剪接位点？ 有人认为，高度保守的扩展 3' 剪接位点的共有序列 UUUCAG 是关键序列 开始拼接。 该序列中的单碱基变化将是 测试体内剪接位点选择的改变。 类推 哺乳动物剪接，U2F 似乎是负责的分子 用于识别该序列。 线虫 U2AF 基因 结合RNA并且是剪接所必需的大亚基已被 克隆和最近的结果表明它是选择性剪接的。 U2F基因的表征将完成，蛋白质或 蛋白质将被表达，并获得抗体。 小亚基 基因也将被克隆。 他们的地图位置将被确定，并且 将选择改变或破坏 U2AF 功能的突变。 U2AF 将测试大肠杆菌中制造的与 UUUCAG 的特异性结合 使用凝胶迁移率、移位竞争和 SELEX 测定进行序列。 多顺反子下游位置的基因位置 转录单位（操纵子）足以引起其转拼 带有 SL2 的 mRNA。 该项目的主要重点是了解 操纵子内部位点 SL2 特异性反式剪接机制。 最近开发的线虫体外转拼系统 胚胎提取物将用于研究 SL2 是如何被指定的。 将测试多顺反子转录物的反式特异性 体外剪接。 还将研究转剪接特异性 在转基因动物中，通过改变基因间距、5'帽、附近的 Poly(a) 信号和顺反子间序列。 SL2的位置 特异性决定子所在的 RNA 也将被研究。 3'端形成所需的序列，包括GU盒和 将确定转录终止信号。 差异 操纵子内的 3' 末端形成和操纵子的 3' 末端之间 将研究转录单位。 新的操纵子将继续 被鉴定以确定它们是否涉及基因的共表达 哪些产品可以一起发挥作用，并了解其中的差异有多大 操纵子结构是可以耐受的。 操纵子的进化和功能 将通过寻找多顺反子单元的存在来研究 其他属。 将鉴定参与转拼的分子 通过遗传和生化技术。 失去了基因的突变体 限制温度下 SL1 和 SL2 反式拼接的能力 被选中。 与 U2AF、或与 SL1 或 SL2 RNA 相互作用的蛋白质 将通过体外结合实验来鉴定。 此外， 将研究 U2AF 亚基过度表达的影响，并确定显性 阴性突变体将通过U2AF缺失RNA的过度表达来寻找 结合或亚基相互作用域。