Tissue Specific and Developmental Expression of snRNA Variants in the Silk Moth

描述(由申请人提供)：这项建议是我们工作的继续，目的是从结构和功能上表征蚕蛾小核RNA(SnRNAs)及其转录控制序列。这项提案中概述的实验旨在阐明SnRNA变体在mRNA剪接前水平对基因表达的调节中所起的作用。我们将利用分期的丝腺和卵泡来评估它们参与前mRNA剪接的情况。多年来，我们观察到：(1)蚕蛾具有U1、U2、U4和U6SnRNA亚型的种群；(2)这些变体在组织和发育阶段存在差异表达；(3)特定的单链RNA结合蛋白与某些SnRNA亚型存在差异结合；(4)某些变体优先与高分子量剪接体复合体结合。综上所述，这些数据提出了以下假设：SnRNAs在功能上具有重要意义，并有助于差异前mRNA剪接、组织分化和发育。或者，这些异构体在功能上可能是相同的。 为了验证这两个假设，我们提出了以下具体目标：(1)评估不同组织、发育阶段和剪接体复合体中SnRNA异构体的相对丰度；(2)鉴定和表征与SnRNA变体结合的蛋白质；(3)确定SnRNA变体与不同组织和发育阶段核提取物的体外组装和剪接效率；(4)分析SnRNA变体在体内的组装和剪接效率。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、U4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型系统。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远的生物医学意义，因为在许多人类疾病中，替代的前mRNA剪接是非常重要的。因此，这些变体的预期差异功能可能调节包括人类在内的不同生物体的发育和组织分化。此外，私隐专员公署已制订详细的专业发展计划，以提高其在取得主流资助方面的竞争力。

DESCRIPTION (provided by applicant): This proposal is a continuation of our work to structurally and functionally characterize the small nuclear RNAs (snRNAs) and their transcriptional controlling sequences in the silk moth Bombyx mori. The experiments outlined in this proposal are designed to elucidate the role that snRNA variants play in the regulation of gene expression at the pre-mRNA splicing level. Their involvement in pre-mRNA splicing will be assessed employing staged silk glands and follicles. Over the years, we have observed that: (1) the silk moth is endowed with populations of U1, U2, U4 and U6 snRNA isoforms; (2) these variants are expressed differentially in tissues and developmental stages; (3) specific single-stranded RNA binding proteins differentially bind to some snRNA isoforms; (4) certain variants preferentially associate with high molecular weight spliceosomal complexes. Taken together, these data suggest the following hypothesis: snRNAs are functionally significant and contribute to differential pre-mRNA splicing, tissue differentiation and development. Alternatively, these isoforms may be functionally equivalent. To test these two hypotheses, we propose the following specific aims: (1) to assess the relative abundance of snRNA isoforms in different tissues, stages of development and spliceosomal complexes; (2) to identify and characterize proteins that bind to snRNA variants; (3) to ascertain the in vitro assembly and splicing efficiency of snRNA variants with nuclear extracts from different tissues and developmental stages; (4) to analyze the assembly and splicing efficiency of snRNA variants in vivo. The extreme and unparalleled polymorphism exhibited by B. mori in the form of multiple, tissue-specific, developmentally regulated U1, U2, U4 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 hypotheses. Due to the universal nature of the questions being asked, the answers obtained during the course of these experiments may have profound biomedical implications given the number of human maladies in which alternative pre-mRNA splicing is highly significant. Thus, the expected differential functions of these variants may modulate development and tissue differentiation in diverse organisms including humans. In addition, the PI has formulated a detailed professional development plan designed to improve his competitiveness in procuring mainstream funding.