AUTOANTIBODY PROBES FOR MAMMALIAN GENE EXPRESSION

我们的长期目标是阐明 细胞核中存在各种小的RNA-蛋白质(RNP)复合体 哺乳动物细胞的核仁(SnoRNPs)、核仁(SnoRNPs)或细胞质(ScRNPs)。 患者自身抗体(针对这些病毒的蛋白质成分 颗粒)和在RNA处理或其他步骤中活跃的体外系统 基因表达是要使用的主要工具之一。现在， 含有U1、U2、U4/U6和U5 RNA的Sm SnRNPs最丰富 被确认为剪接体组件，它们在前mRNA剪接中的作用将 更准确的定义是：检查热休克对 U4/U6-U5 SnRNP复合体；对剪接体组装的早期步骤进行排序 U2 SnRNP亲和层析柱；筛选“抗生素”的能力 产生剪接中间体，并定义金精羧酸如何 抑制体外剪接；试图获得正确的替代方案 为揭示体外剪接位点配对调控的基础 剪接；确定与SnRNP相关的70kd 3‘剪接点的作用 在剪接中结合蛋白质；以及评估构建体的能力 含有SnRNA和底物序列进行部分剪接 在没有蛋白质的情况下的反应。最近人们意识到，反式- 拼接前导RNA是含有5‘外显子和SnRNA的分子嵌合体。 就像与Sm蛋白(一种新的Sm SnRNP亚类)组装的部分一样 被用来研究哺乳动物细胞 同样，参与反式剪接。SL单链RNPs与其相互作用的研究 其他剪接成分将在HeLa核提取物中进行探测，以确定 顺式和反式剪接机制之间的重叠。的结构 &gt；10个新发现的低丰度Sm SNRNPs将被阐明，并将其 利用寡核苷酸确定信使核糖核酸生物发生中的可能功能 检查它们的组织分布和测试它们的活性的探针 以及在体外与其他SNRNPs的相互作用。推定的多聚腺苷 将通过寻找疱疹病毒的细胞类似物来鉴定SNRNPs Saimiri U RNA，它与AAUAAA具有互补性。碱基配对 U7SnRNP与U7下游保守信号的相互作用 组蛋白前mRNAs将被操纵以询问特定的碱基对 是3‘端成熟所必需的；辅助蛋白质因子调节 组蛋白信使核糖核酸的加工也将被表征。核糖体蛋白L5， 在核糖体组装之前结合5S rRNA的哪一个将被检测其 将类病毒RNA运送到其核仁复制位置的能力(通过 对致病性的影响)。大豆的蛋白质组成 7SK SnRNP将使用自身抗体进行定义，并暗示它可能 将努力实现与Sm SNRNPs的协调运作。了解 自身免疫的小型RNP靶标将不仅提供对 风湿病还可以转化为基因的进化和调控 在正常细胞和恶性细胞中均有表达。

Our long-term objectives are to elucidate the structures and functions of a variety of small RNA-protein (RNP) complexes present in the nucleus (snRNPs), nucleolus (snoRNPs), or cytoplasm (scRNPs) of mammalian cells. Patient autoantibodies (directed against the protein components of these particles) and in vitro systems active in RNA processing or other steps in gene expression are among the primary tools to be employed. Now that the most abundant Sm snRNPs containing U1, U2, U4/U6 and U5 RNAs have been identified as spliceosome components, their roles in pre-mRNA splicing will be more precisely defined by: examining the effect of heat shock on the U4/U6-U5 snRNP complex; ordering early steps in spliceosome assembly using U2 snRNP affinity columns; screening "antibiotics" for their ability to generate splicing intermediates and defining how aurintricarboxylic acid acts to inhibit in vitro splicing; attempting to obtain correct alternative splice site pairing in vitro in order to uncover the basis of regulated splicing; ascertaining the role of the snRNP-associated 70kd 3' splice site binding protein in splicing; and assessing the ability of constructs containing snRNA and substrate sequences to carry out partial splicing reactions in the absence of proteins. The recent realization that trans- spliced leader RNAs are molecular chimeras containing a 5' exon and snRNA- like moiety that assemble with Sm proteins (a novel Sm snRNP subclass) will be exploited in experiments designed to ask whether mammalian cells likewise engage in trans-splicing. The interactions of SL snRNPs with other splicing components will be probed in HeLa nuclear extracts to define the overlaps between the cis- and trans-splicing machinery. Structures of >10 newly identified low abundance Sm snRNPs will be elucidated and their presumed functions in mRNA biogenesis established using oligonucleotide probes to examine their tissue distribution and to test their activities and interactions with other snRNPs in vitro. Putative polyadenylation snRNPs will be identified by seeking cellular analogues of Herpesvirus saimiri U RNAs, which exhibit complementarity to AAUAAA. Base-pairing interactions between the U7 snRNP and the downstream conserved signal of histone pre-mRNAs will be manipulated to ask whether specific base pairs are required for 3' end maturation; auxiliary protein factors regulating histone mRNA processing will also be characterized. Ribosomal protein L5, which binds 5S rRNA prior to ribosome assembly will be tested for its ability to deliver viroid RNAs to their nucleolar site of replication (with consequent implications for pathogenicity). The protein composition of the 7SK snRNP will be defined using autoantibodies, and hints that it may function coordinately with Sm snRNPs will be pursued. Understanding the small RNP targets of autoimmunity will provide insights not only into rheumatic disease but also into the evolution and regulation of gene expression in normal and malignant cells.