Expression/Function of U1/2/6 Isoforms during Developmen

这项建议代表了我们继续努力从结构和功能上表征蚕蛾的小核RNA(SnRNAs)和剪接体蛋白。该研究计划的总体目标是提供有关昆虫物种中前mRNA剪接调控的信息。我们实验室已经对蚕蛾U1、U2和U6的变异体进行了结构鉴定，并开始研究与它们有差异结合的蛋白质。家蚕的U1、U2和U6变种被发现是发育和组织特异性转录的。此外，我们还通过紫外光交联证明了 特定核蛋白与U1变异序列和某些U1存在差异 异构体优先组装成剪接体复合体。基于上述结果，可以考虑两个假设。一是这些SnRNA变异体在功能上具有重要意义，并有助于差异前mRNA剪接。另一种假设认为，SnRNA变异体在功能上是等价的，它们的组织和发育特异性表达是其转录控制元件和细胞类型特异性反式作用因子之间独特相互作用的结果。家蚕表现出极端和无与伦比的多态，表现为多种组织特异性的、发育调节的U1、U2和U6变体，以及高度专门生产特定蛋白质(丝素或丝素)的组织的存在，使丝蛾成为区分这两种假说的理想模式系统。这项建议描述了旨在研究U1、U2和U6变体以及SnRNP蛋白在剪接过程中控制基因表达的潜在作用的实验。将使用发育阶段的丝腺、卵泡和几个家蚕细胞系来研究它们在前mRNA剪接中的功能。与SnRNA变体差异相互作用的蛋白质将被识别和测序。本实验室鉴定的异构体将用于重组实验，以确定它们与剪接体蛋白的差异相互作用，组装成SnRNP/hnRNP颗粒，与体外转录的含内含子的丝素和绒毛膜RNA的结合，以及剪接能力。由于所问问题的普遍性，在这些实验过程中得到的答案可能具有深远的生物医学意义。

This proposal represents a continuation of our efforts to structurally and functionally characterize small nuclear RNAs (snRNAs) and spliceosomal proteins in the silk moth Bombyx mori. The research program's overall objective has been to provide information on the regulation of pre-mRNA splicing in an insect species. Our laboratory has structurally characterized variants of U1, U2 and U6 in the silk moth, and has started to study the proteins that differentially bind to them. The B. mori U1, U2 and U6 variants were found to be developmentally and tissue-specifically transcribed. In addition, we have demonstrated by UV cross-linking that specific nuclear proteins associate differentially with U1 variant sequences and certain U1 isoforms assemble into spliceosomal complexes preferentially. Based on the above results, two hypotheses can be entertained. One is that these snRNA variants are functionally significant and contribute to differential pre-mRNA splicing. The other postulates that the snRNA variants are functionally equivalent and that their tissue and developmentally specific expression results from unique interactions between their transcriptional controlling elements and cell-type specific trans-acting factors. The extreme and unparalleled polymorphism exhibited by B. mori in the form of multiple, tissue-specific, developmentally regulated U1, U2 and U6 variants, and the existence of tissues highly specialized for the production of a specific protein (fibroin or silk) make the silk moth an ideal model system to discriminate between the two hypothesis. This proposal describes experiments designed to investigate potential roles of U1, U2 and U6 variants as well as snRNP proteins in the control of gene expression during splicing. Their function in pre-mRNA splicing will be investigated using developmentally staged silk glands, follicles and several B. mori cell lines. Proteins differentially interacting with snRNA variants will be identified and sequenced. The 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. Due to the universal nature of the questions being asked, the answers obtained during the course of these experiments may have profound biomedical implications.