TRANS-SPLICING IN C ELEGANS

描述（改编自申请人的摘要）：C. elegans从事 三种类型的核前mRNA加工：正常顺式剪接和 两个不同前导序列的反式剪接：靠近前前导序列5'末端的SL 1， 多顺反子前体mRNA内部反式剪接位点的SL 2 然后分裂成基因长度的mRNA。 这三个过程是 机械上紧密相关，但功能上不同。 C. 秀丽线虫剪接体对5'剪接位于顺式与内含子3' 剪接位点，但只利用适当的SL的两种类型的 反式剪接位点 有人认为，高度保守的扩展 在3'剪接位点的共有序列UUUCAG是 开始剪接。 这个序列中的单碱基变化将是 在体内测试剪接位点选择的改变。 比照 哺乳动物剪接，似乎U2 F是负责 来识别这个序列。 C.秀丽隐杆线虫U2 AF 结合RNA并为剪接所需的大亚基， 最近的结果表明它是选择性剪接的。 将完成U2 F基因的表征， 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， 将选择改变或破坏U2 AF功能的突变。 u2AF 在E.将检测大肠杆菌与UUUCAG的特异性结合 序列使用凝胶迁移率，移位竞争和SELEX测定。 基因在多顺反子系统中下游位置的位置 转录单位（操纵子）足以引起其转录的反式剪接。 mRNA与SL 2。 本项目的主要重点是了解 操纵子内部位点的SL 2特异性反式剪接机制。 近年来发展了一种新的体外反式剪接系统。elegans 胚胎提取物将用于研究SL 2是如何被指定的。 将测试多顺反子转录物的反式- 体外剪接。 反式剪接的特异性也将进行研究 在转基因动物中，通过改变基因间隔，5'端帽，附近的 poly（a）信号和顺反子间序列。 SL 2的位置 还将研究特异性决定簇所在的RNA。 3'端形成所需的序列，包括GU框和 将确定转录终止信号。 差异 在操纵子内的3'末端形成和在操纵子的3'末端形成之间， 将研究转录单位。 新的操纵子将继续 以确定它们是否涉及基因的共表达 他们的产品功能在一起，并了解有多少变化， 操纵子结构是容许的。 操纵子的进化和功能 将研究的多顺反子单位的存在搜索， 其他属。 将鉴定参与反式剪接的分子 通过遗传和生化技术。 突变体失去了 在限制性温度下SL 1和SL 2反式剪接的能力将 被选中。 与U2 AF或与SL 1或SL 2 RNA相互作用的蛋白质 将通过体外结合实验鉴定。 此外该 将研究U2 AF亚基过表达的影响，并且显性的 将通过U2 AF缺失RNA的过表达来寻找阴性突变体， 结合或亚基相互作用结构域。

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.