AUTOANTIBODY PROBES FOR MAMMALIAN GENE EXPRESSION

我们的长期目标是阐明 存在于细胞核中的各种小RNA-蛋白质（RNP）复合物 在一些实施方案中，所述细胞可以是哺乳动物细胞的核内核糖核酸（snRNP）、核仁（snoRNP）或细胞质（scRNP）。 患者自身抗体（针对这些抗体的蛋白质组分） 颗粒）和在RNA加工或其他步骤中有活性的体外系统， 基因表达是所采用的主要工具。 现在 含有U1、U2、U4/U6和U 5 RNA的最丰富的Sm snRNP已经被发现， 作为剪接体成分，它们在前体mRNA剪接中的作用将 更准确地定义为：检查热休克对 U4/U6-U 5 snRNP复合物;使用 U2 snRNP亲和柱;筛选“抗生素”的能力， 产生剪接中间体，并定义金精三羧酸 抑制体外剪接;试图获得正确的替代 体外剪接位点配对，以揭示调控的基础 剪接;确定snRNP相关的70 kd 3'剪接位点的作用 结合蛋白的剪接;和评估构建体的能力 含有snRNA和底物序列以进行部分剪接 在没有蛋白质的情况下发生反应。 最近认识到，跨- 剪接的前导RNA是含有5'外显子和snRNA的分子嵌合体， 与Sm蛋白组装的类似部分（一种新的Sm snRNP亚类）将 被用于研究哺乳动物细胞 同样参与反式剪接。 SL snRNP与 将在HeLa细胞核提取物中探测其他剪接组分，以确定 顺式和反式剪接机制之间的重叠 结构 >10个新鉴定的低丰度Sm snRNP将被阐明， 使用寡核苷酸建立的mRNA生物发生中的假定功能 探针来检查它们的组织分布并测试它们的活性 以及与其他snRNP的体外相互作用。 推定的多聚腺苷酸化 snRNP将通过寻找疱疹病毒的细胞类似物来鉴定 saimiri U RNA与AAUAAA具有互补性。 碱基配对 U 7 snRNP和下游保守信号之间的相互作用 组蛋白前mRNA将被操纵，以询问特定的碱基对是否 是3'端成熟所必需的;辅助蛋白因子调节 还将表征组蛋白mRNA加工。 核糖体蛋白L5， 在核糖体组装之前结合5S rRNA的蛋白质将被测试其 将类病毒RNA递送至其核仁复制位点的能力（ 对致病性的影响）。 的蛋白质组成 7SK snRNP将使用自身抗体定义，并暗示它可能 将追求与Sm snRNP协调功能。 了解 自身免疫的小RNP靶点将不仅提供对 风湿性疾病还涉及到基因的进化和调控 在正常和恶性细胞中表达。

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.