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

这一建议代表了我们的努力，在结构和功能上的特点小核RNA（snRNA）和剪接体蛋白在蚕蛾家蚕的延续。该研究计划的总体目标是提供有关昆虫物种中前体mRNA剪接调节的信息。我们的实验室已经在结构上表征了蚕蛾中U1，U2和U6的变体，并已开始研究与它们差异结合的蛋白质。B。发现桑U1、U2和U6变异体是发育和组织特异性转录的。此外，我们还通过UV交联证实了 特异性核蛋白与U1变异序列和某些U1 同种型优先组装成剪接体复合物。基于上述结果，可以接受两个假设。一个是这些snRNA变体在功能上是重要的，并有助于差异前mRNA剪接。另一种假设snRNA变体在功能上是等同的，并且它们的组织和发育特异性表达是由它们的转录控制元件和细胞类型特异性反式作用因子之间的独特相互作用引起的。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的、发育调节的U1、U2和U6变体，并且存在高度专门化的用于产生特定蛋白质（丝心蛋白或丝）的组织，这使得蚕蛾成为区分这两种假说的理想模型系统。该提案描述了旨在研究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B研究它们在前mRNA剪接中的功能。Mori细胞系。将鉴定和测序与snRNA变体差异相互作用的蛋白质。在我们的实验室中表征的同种型将用于重建实验，其中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心蛋白和绒毛膜RNA的结合，以及剪接能力将被确定。由于所问问题的普遍性，在这些实验过程中获得的答案可能具有深刻的生物医学意义。

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.