RNA Processing Regulation of Immunoglobulin Gene Expression

摘要Peterson MCB-9808637是小鼠免疫球蛋白Mu基因，是研究B淋巴细胞发育过程中调节核糖核酸加工的已建立的模型系统。该基因包含一个切割-聚腺苷酸化或剪接的位点；这两个相互竞争的反应的效率必须是平衡的。当B细胞成熟为浆细胞时，这两种加工方式的相对使用受到调节；浆细胞产生相对更多的在Mu-S Polya位点被裂解和多腺化的RNAs。到目前为止的证据表明，调控所需要的是基因结构，而不是Ig基因特有的序列。因此，调控机制必须涉及改变一般RNA加工机制的组件。当B细胞成熟为浆细胞时，裂解-多聚腺苷的活性增加。此外，初步证据表明，B细胞和浆细胞之间的剪接活性不同。因此，调节MU加工的机制可能涉及切割-多腺化和剪接活性的变化。这个项目将扩展目前的研究，以进一步了解调节u替代RNA加工的机制，剪接位点的选择，以及一般的切割-聚腺苷酸化位点的使用。为了达到这些目的，将通过两种不同的方法来探索剪接活动的变化。有助于差异剪接活性的序列将使用需要剪接增强子的基因进行鉴定。这些序列将在Mu基因和嵌合基因中鉴定，嵌合基因的剪接在B细胞和浆细胞中受到调控。任何激活剪接的序列都将用于通过RNA凝胶移动、UV交联和抗体鉴定程序的组合来鉴定RNA结合蛋白。第二种方法将使用抑制消减杂交技术来寻找编码与RNA加工相关的产物的基因，这些基因的表达在B细胞和浆细胞之间存在差异。切割-聚腺苷酸化反应将通过以新的方式检查可能影响切割-聚腺苷酸化活性的u-S多聚腺苷酸位点的序列来进一步研究。远离Polya位点下游和上游的序列都对其活性有贡献。为了更好地了解这些序列的功能，还将进行额外的定点突变分析和体外切割-聚腺苷酸化和结合分析。为了确定上游序列是否参与剪接和切割-聚腺苷酸化反应之间的相互作用，将使用耦合这些反应的条件进行体外分析。由于调控被预测涉及到一般剪接和切割-多聚腺苷酸化因子的变化，这些建议的方法将提供关于剪接和切割-多腺化促进序列和一般因素的信息，并有助于理解淋巴细胞成熟过程中的调控机制。另一种RNA加工是真核细胞扩大其基因组编码能力的一种机制：单个基因可以产生不止一个产物。替代的RNA加工经常受到调控，不同的mRNA在不同的细胞类型中或在细胞发育的不同阶段产生。人们才刚刚开始了解这种替代的RNA加工过程是如何受到监管的。该项目研究了免疫球蛋白基因的替代加工--替代产物要么是分泌蛋白，要么是膜相关蛋白，其相对丰度在B淋巴细胞发育过程中发生变化。从这项研究中获得的信息将有助于我们了解RNA加工反应以及这些反应的调节。

Abstract Peterson MCB-9808637 The mouse immunoglobulin mu gene is an established model system for studying regulated RNA processing during B lymphocyte development. This gene contains a site subject to either cleavage-polyadenylation or splicing; the efficiencies of these two competing reactions must be balanced. The relative use of these two processing options is modulated as B cells mature to plasma cells; plasma cells produce relatively more RNA cleaved and polyadenylated at the mu-s polyA site. Evidence to date has suggested that the gene structure, rather than Ig gene-specific sequences, is required for regulation. The regulatory mechanism must, therefore, involve changes in components of the general RNA processing machinery. Cleavage-polyadenylation activity increases as B cells mature to plasma cells. In addition, preliminary evidence suggests that splicing activity differs between B cells and plasma cells. Thus, the mechanism regulating mu processing likely involves changes in both the cleavage-polyadenylation and splicing activities. This project will extend current studies to further the understanding of the mechanism regulating mu alternative RNA processing, of splice site choice, and of cleavage-polyadenylation site usage in general. Toward these ends, the changes in splicing activity will be explored by two different approaches. Sequences that contribute to differential splicing activity will be identified using a splice enhancer-requiring gene. These sequences will be identified in the mu gene and in a chimeric gene whose splicing is regulated in B cells and plasma cells. Any sequences that activate splicing will be used to identify RNA binding proteins by a combination of RNA gel shift, UV cross-linking, and antibody identification procedures. A second approach will use the suppression subtraction hybridization technique to find genes encoding products relevant to RNA processing whose expression differs between B cells and plasma cells. The cleavage-polyadenylation reaction will be further studied by examining sequences from the mu-s polyA site that potentially affect cleavage-polyadenylation activity in novel ways. Sequences far downstream and upstream from the polyA site both contribute to its activity. To understand better the function of these sequences, additional site-directed mutational analyses and in vitro cleavage-polyadenylation and binding assays will be performed. To determine if the upstream sequences are involved in interactions between splice and cleavage-polyadenylation reactions, in vitro assays using conditions that couple these reactions will be performed. As regulation is predicted to involve changes in general splicing and cleavage-polyadenylation factors, these proposed approaches will provide information on splice and cleavage-polyadenylation enhancing sequences and factors in general, as well as contribute to the understanding of the regulatory mechanisms operating during lymphocyte maturation. Alternative RNA processing is a mechanism by which eukaryotic cells expand the coding capacity of their genomes: more than one product can be made from a single gene. Alternative RNA processing is often regulated such that different mRNA's are made in different cell types or during different stages of cellular development. How this alternative RNA processing is regulated is only beginning to be understood. This project examines the alternative processing of the immunoglobulin gene - the alternate products are either a secreted protein or a membrane-associated protein whose relative abundance changes during B lymphocyte development. Information gained from this research will contribute to our knowledge of RNA processing reactions in general and of the regulation of these reactions.