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）：秀丽隐杆线虫参与 三种类型的核前MRNA处理：正常的顺式切片和 对两个不同的领导者的移序：SL1附近的前5'末端 mRNA和SL2在多物种前MRNA中的内部变形位点处 然后分成基因长度mRNA。 这三个过程是 机械上密切相关，但功能上不同。 C. 秀丽隐杆线虫的剪接组合配对位于顺式的5'剪接与内含子3' 剪接网站，但仅利用适当的SL进行两种类型的SL 移板网站？ 有人认为高度保守的扩展 共识，uuucag，在3'拼接位点是关键序列 启动剪接。 此序列中的单个基础变化将是 测试了体内剪接站点选择的改变。 类似于 哺乳动物的剪接，U2F似乎是分子负责的 为了识别此序列。 秀丽隐杆线虫U2AF的基因 大型亚基结合RNA并且是剪接所必需的，已经是 克隆并最近的结果表明它是剪接的。 U2F基因的表征将完成，蛋白质或 将表达蛋白质，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函数的突变。 U2AF 在大肠杆菌中制造的将对与UUUCAG的结合进行测试 使用凝胶移动性，转移竞赛和SELEX分析的序列。 基因位于多物种中下游位置的位置 转录单元（操纵子）足以引起其移动 sl2 mRNA。 该项目的主要重点是了解 在操纵子中内部位置的SL2特异性移序机制。 秀丽隐杆线虫的最近开发的体外移植系统 胚胎提取物将用于研究如何指定SL2。 多处理转录本将进行转言的特异性测试 体外剪接。 也将研究跨式特异性 在转基因动物中，通过改变基因间距，5'帽，附近 poly（a）信号和跨发言序列。 SL2的位置 还将研究特异性决定因素的RNA。 3'端形成所需的顺序，包括GU框和 将确定转录终止信号。 差异 在操纵子内的3'末端形成之间和在3'的末端 转录单元将进行研究。 新操纵子将继续 确定以确定它们是否涉及基因共表达 谁的产品功能在一起，并了解多少变化 操纵子结构被耐受。 操纵子的演变和功能 将通过寻找在 其他属。 将确定参与跨拼布的分子 通过遗传和生化技术。 失去的突变体 SL1和SL2在限制性温度下进行移动的能力将 被选中。 与U2AF相互作用的蛋白质，或与SL1或SL2 RNA相互作用的蛋白质 将通过体外结合实验来识别。 另外， 将研究U2AF亚基过度表达的效果，并主要 通过过表达U2AF缺失RNA-将寻求阴性突变体 结合或亚基相互作用域。