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

描述（由申请人提供）：本提案是我们对家蚕（Bombyx mori）小核rna （snrna）及其转录控制序列的结构和功能特征研究的延续。本提案中概述的实验旨在阐明snRNA变异体在pre-mRNA剪接水平上调控基因表达的作用。他们参与前mrna剪接将评估分阶段蚕丝腺和卵泡。多年来，我们观察到：(1)蚕蛾被赋予了U1、U2、U4和U6 snRNA亚型群体；(2)这些变异在组织和发育阶段的表达差异；(3)特异性单链RNA结合蛋白与某些snRNA异构体的差异结合；(4)某些变异优先与高分子量剪接体复合物相关。综上所述，这些数据提出了以下假设：snrna在功能上很重要，并有助于差异的pre-mRNA剪接、组织分化和发育。或者，这些同工异构体可能在功能上是等同的。

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.