CHIMERIC IG MRNAS AND HUMAN ISOTYPE SWITCHING

描述：（改编自研究者摘要）总体 本提案的目的是阐明一个关键过程， 免疫球蛋白（IG）同种型转换：免疫球蛋白的作用机制 确定并最终实现IG同种型转换特异性。 我们 将检验嵌合IG生殖系mRNA转录物作为 确定IG类转换特异性的特异性模板 重组，重点是切换到IgE。 我们已经证明， 嵌合mRNA来源于C的种系IG前mRNA的反式剪接 mu和下游同种型的种系前mRNA。 这些嵌合IG 生殖系mRNA，通过Hoogsteen碱基配对， 链DNA区域，是理想的模板作为“桥 模板”。 这种桥接将使mu交换机和所涉及的下游 同种型转换区域非常接近，从而直接IG转换 重组 为了验证这一假设，我们将在人类原发性 B细胞是否转染产生特异性嵌合IG的构建体 反义方向的生殖系mRNA转录物阻断特异性IG 开关重组和同种型生产以及是否转染 有义方向嵌合IG种系mRNA特异性增强开关 重组和IG产生。 我们将定量地确定 嵌合IG生殖系mRNA转录物出现的动力学及其 与IG同种型转换的关系。 为了更好地定义嵌合体的作用， IG生殖系转录本在IG转换中的作用，我们将确定 已知的调节反式剪接的开关信号和由此产生的产物 嵌合IG生殖系MRNAS。 作为开关的选择和方式， 重组位点（即初级重组与次级重组）具有重要的 IG同种型生产和稳定的影响，我们将扩大我们的 通过检验嵌合IG种系的特异性结构 转录物（3 '-5'与5 '-3'嵌合体）具有不同的介导 一级或二级重组预测3 '-5'嵌合体（例如I ε-C μ转录物）将介导初级重组，而5 '-3' 嵌合体（例如，I μ-C β转录物）将介导继发性 重组 最后，我们将检验重复的G碱基 在RNA（“G4-RNA”）中发现IG开关区域在 促进IG种系前mRNA转录物的反式剪接。 采用 一个无细胞的体外剪接系统，我们将分析G4-RNA的影响， 形成对IG反式剪接的影响。 此外，我们还将确定 嵌合IG生殖系mRNA在体外与相应区域配对 通过Hoogsteen碱基配对的双链DNA。

DESCRIPTION: (Adapted from the investigator's abstract) The overall objective of this proposal is to elucidate a critical process in immunoglobulin (Ig) isotype switching: the mechanism by which the specificity of Ig isotype switch is determined and ultimately achieved. We will test the hypothesis that chimeric Ig germline mRNA transcripts serve as the specific templates that determine the specificity of Ig class switch recombination with emphasis on switching to IgE. We have shown that such chimeric mRNA are derived from trans-splicing of germline Ig pre-mRNAs of C mu and the germline pre-mRNAs of downstream isotype(s). These chimeric Ig germline mRNAs, by Hoogsteen base-pairing to their corresponding double stranded DNA region, are ideal templates to function as "bridging templates". This bridging will bring mu switch and the involved downstream isotype switch regions in close proximity and thereby direct Ig switch recombination. To test this hypothesis, we will determine in human primary B cells whether transfection of constructs producing specific chimeric Ig germline MRNA transcripts in anti-sense orientation blocks specific Ig switching recombination and isotype production and whether transfection of chimeric Ig germline MRNA in sense orientation specifically enhances switch recombination and Ig production. We will quantitatively determine the kinetics of appearance of chimeric Ig germline MRNA transcripts and their relationship to Ig isotype switching. To better define the role of chimeric Ig germline transcripts in Ig switching, we will determine the ability of known switch signals to regulate trans-splicing and the resulting production of chimeric Ig germline MRNAS. As the selection and pattern of switch recombination sites (i.e. primary vs. secondary recombination) has important implications for Ig isotype production and stabilization, we will extend our hypothesis by testing if the specific structure of chimeric Ig germline transcripts (3'-5' vs 5'-3' chimeras) have distinct abilities to mediate primary or secondary recombination predicting that 3'-5' chimeras (e.g. I epsilon-C mu transcripts) will mediate primary recombination whereas 5'-3' chimeras (e.g. I mu-C epsilon transcripts) will mediate secondary recombination. Finally, we will test the hypothesis that repetitive G bases in RNA ("G4-RNA") as found in Ig switch regions plays a key role in promoting the trans-splicing of Ig germline pre-mRNA transcripts. Employing a cell free in vitro splicing system, we will analyze the effects of G4-RNA formation on Ig trans-splicing. Furthermore we will determine the ability of chimeric Ig germline mRNAs to pair in vitro to the corresponding region of double stranded DNA through Hoogsteen base pairing.