EXPRESSION /FUNCTION--U1/U2 RNA ISOFORMS DURING DEVE

这一更新申请中概述的实验延续了过去11年来该项目的中心主题。从一开始，我们的项目就涉及了家蚕U1和U2基因、转录本和剪接体蛋白的结构和功能特征。该计划的主要研究目标是阐明昆虫物种中的前mRNA剪接。多年来，这一科学计划一直服务得特别好，并将继续成为一个很好的工具，教育和激励有前途的少数民族学生在生物医学科学追求事业。U1和U2 RNA是六种丰富的U系列小核RNA（snRNA）U1-U6中的两种，已知它们与六到十种蛋白质相关。这些RNA/蛋白质复合物形成小核糖核蛋白颗粒（U-snRNP），其在将hnRNA（前mRNA）剪接成mRNA /蛋白质复合物中起作用。我们的实验室已经在结构上表征了蚕蛾中U1和U2的变体，并开始研究与它们差异结合的蛋白质。U1和U2变体在发育和组织特异性转录，表明在基因表达控制中的作用。该提案描述了旨在阐明U1，U2和snRNP蛋白变体在基因表达控制中的作用的实验。将使用发育阶段的丝腺、滤泡和几种B研究它们在前mRNA剪接中的功能。Mori细胞系。UV交联实验将用于检测与不同U1和U2以及snRNA变体具有差异相互作用的蛋白质。在UV交联实验之后，将进行免疫沉淀以鉴定和分离所讨论的蛋白质。此外，我们实验室表征的U1和U2亚型将用于重建实验，其中将确定它们与剪接体蛋白组装成snRNP/hnRNP颗粒的差异相互作用、与体外转录的含内含子丝心蛋白和绒毛膜RNA的缔合以及剪接能力。这些研究应有助于确定U1和U2变异体在差异剪接和发育中发育和组织特异性表达的可疑作用。B所表现出的极端的、无与伦比的多态性。家蚕具有多种组织特异性、发育调节的U1和U2变体，并且存在高度特化的组织以产生特定的蛋白质（丝心蛋白或丝），这使得蚕蛾成为理想的模型系统。由于所问问题的普遍性和中心性，在这些实验过程中获得的答案可能具有深远的生物医学意义。

The experiments outlined in this renewed application continue the central theme of this project over the past 11 years. Since the start, our project has involved the structural and functional characterization of U1 and U2 genes, their transcripts and spliceosomal proteins in the silkmoth, Bombyx mori. The program's primary research objective has been to elucidate pre-mRNA splicing in an insect species. Over the years, this scientific program has served particularly well and will continue to be an excellent vehicle to educate and motivate promising minority students to pursue careers in the biomedical sciences. U1 and U2 RNAs are two of six abundant U-series small nuclear RNAs (snRNAs), U1-U6, which are known to associate with six to ten proteins. These RNA/protein complexes form small ribonucleoprotein particles (U-snRNPs) which function in splicing hnRNA (pre-mRNA) into mRNA /protein complexes form small ribonucleoprotein particles (U-snRNPs) which function in splicing hnRNA (pre-mRNA) into mRNA. Our laboratory has structurally characterized variants of U1 and U2 in the silkmoth, and started to examine the proteins that differentially bind to them. The U1 and U2 variants are developmentally and tissue-specifically transcribed, suggesting a role in control of gene expression. This proposal describes experiments designed to elucidate the role U1, U2 and snRNP protein variants play in the control of gene expression. Their function in pre- mRNA splicing will be investigated using developmentally staged silk glands, follicles and several B. mori cell lines. UV cross-linking experiments will be used to detect proteins with differential interactions to different U1 and U2 and snRNA variants. Following the UV cross- linking experiments, immunoprecipitation will be performed to identify and isolate the proteins in question. In addition, U1 and U2 isoforms characterized in our laboratory will be used in reconstitution experiments in which their differential interaction with spliceosomal proteins assembly into snRNP/hnRNP particles, association with in vitro transcribed intron- containing fibroin and chorion RNAs, and splicing capabilities will be ascertained. These studies should help define the suspected role of developmental and tissue-specific expression of U1 and U2 variants in differential splicing and development. The extreme and unparalleled polymorphism exhibited by B. mori in the form of multiple, tissue- specific, developmentally regulated U1 and U2 variants, and the existence of tissues highly specialized for the production of a specific protein (fibroin or silk) make the silkmoth an ideal model system. Due to the universal and central nature of the questions being asked, the answers obtained during the course of these experiments may have profound biomedical implications.