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

在这个新申请中概述的实验延续了过去11年来这个项目的中心主题。从一开始，我们的项目就涉及到家蚕（Bombyx mori）中U1和U2基因的结构和功能特征、转录物和剪接体蛋白。该项目的主要研究目标是阐明昆虫物种的前mrna剪接。多年来，这个科学项目一直发挥着特别好的作用，并将继续成为教育和激励有前途的少数民族学生从事生物医学科学事业的绝佳工具。U1和U2 rna是六种丰富的u系列小核rna （snrna）中的两种，U1- u6已知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-snRNPs），其功能是将hnRNA（前mRNA）剪接成mRNA /蛋白质复合物形成小核糖核蛋白颗粒（U-snRNPs），其功能是将hnRNA（前mRNA）剪接成mRNA。我们的实验室已经在蚕蛾中对U1和U2的变体进行了结构表征，并开始研究与它们不同结合的蛋白质。U1和U2变体是发育和组织特异性转录的，表明在基因表达控制中起作用。本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。它们在mRNA前剪接中的功能将在发育阶段的蚕丝腺、卵泡和几种家蚕细胞系中进行研究。紫外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。在紫外交联实验之后，将进行免疫沉淀来鉴定和分离所讨论的蛋白质。此外，我们实验室鉴定的U1和U2同工异构体将被用于重组实验，其中它们与剪接体蛋白组装成snRNP/hnRNP颗粒的差异相互作用，与体外转录含内含子的丝素蛋白和绒毛膜rna的关联，以及剪接能力将被确定。这些研究应该有助于确定U1和U2变体在差异剪接和发育中的发育和组织特异性表达的可疑作用。家蚕以多种组织特异性、受发育调控的U1和U2变异形式表现出的极端和无与伦比的多态性，以及高度专门化生产特定蛋白质（丝素或蚕丝）的组织的存在，使家蚕成为一个理想的模型系统。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.