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(改编自申请者摘要)：线虫从事 核前mRNA加工的三种类型：正常顺式剪接和 两个不同的引导子的反式剪接：SL1靠近Pre-5‘端 多顺反子前mRNAs的内部反式剪接位点的mRNAs和SL2 然后分裂成基因长度的mRNAs。这三个过程是 机械上紧密相关，但在功能上截然不同。C.如何做到？ Elgans剪接位于顺式基因内含子3‘的5’剪接对 拼接位置，但仅使用两种类型的适当SL 反式剪接位点？有人认为，高度保守的延伸体 3‘剪接位点上的共识UUUCAG是 启动拼接。该序列中的单碱基变化将是 在体内测试了剪接位点选择的变化。以…为类比 哺乳动物剪接，似乎U2F是负责的分子 用于识别该序列。线虫U2AF基因 结合RNA并进行剪接所需的大亚基已经被 克隆和最近的结果表明它是交替剪接的。 U2F基因的鉴定将完成，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 改变或破坏U2AF功能的突变将被选中。U2AF 在大肠杆菌中制造的将被测试与UUUCAG特异性结合 使用凝胶迁移率、移位竞争和SELEX分析进行测序。 基因在多顺反子下游位置的位置 转录单位(操纵子)足以引起其反式剪接 带有SL2的mRNA。本项目的主要关注点是了解 操纵子内部位点SL2特异性反式剪接的机制。 一种新开发的线虫体外反式剪接系统 胚胎提取物将被用来研究SL2是如何被指定的。 多顺反子转录本将被测试反式- 体外拼接。反式剪接的特异性也将被研究 在转基因动物中，通过改变基因间隔，5‘帽，附近的 Poly(A)信号和顺反子间序列。SL2的位置 还将研究特异性决定因素所在的RNA。 3‘末端形成所需的序列，包括gu盒和 转录终止信号将被确定。差异 在操纵子内的3‘端形成和在操纵子的3’端形成之间 将对转录单位进行调查。新的歌剧将继续 以确定它们是否涉及基因的共表达 他们的产品一起发挥作用，并了解在 操纵子结构是可以容忍的。操纵子的演化和功能 将通过寻找是否存在多顺反子单元来研究 其他属。参与反式剪接的分子将被确定 通过基因和生化技术。已经失去了 限制温度下SL1和SL2反式剪接的能力将 被选中。与U2AF或SL1或SL2 RNA相互作用的蛋白质 将通过体外结合实验进行鉴定。此外， U2AF亚基过表达的影响将被研究，并占优势 通过过度表达U2AF缺失的RNA来寻找阴性突变体 结合域或亚单位相互作用域。