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

snRNA 变体在蚕蛾中的组织特异性和发育表达

• 批准号: 7425708
• 负责人: RENE J HERRERA
• 金额: $ 28万
• 依托单位: FLORIDA INTERNATIONAL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-05-14 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7425708
• 关键词: Alternative Splicing; Amino Acid Sequence; Binding; Biological Models; Bombyx mori; Cells; Collaborations; Complex; Data; Development; Development Plans; Exhibits; Fibroins; Funding; Gene Expression Regulation; Genetic Polymorphism; Gland; Graph; Heterogeneous Nuclear RNA; Heterogeneous-Nuclear Ribonucleoproteins; Human; In Vitro; Introns; Laboratories; Learning; Letters; Mainstreaming; Manuscripts; Minority; Molecular Biology; Molecular Weight; Morus; Moths; Nature; Nuclear Extract; Nuclear Protein; Nuclear Proteins; Numbers; Organism; Peptide Sequence Determination; Peptides; Play; Population; Procedures; Production; Productivity; Professional Practice; Protein Binding; Protein Isoforms; Proteins; Publications; RNA Splicing; RNA-Binding Proteins; Relative (related person); Research; Resources; Role; Science; Silk; Small Nuclear RNA; Small Nuclear Ribonucleoproteins; Staging; Structure; Students; Testing; Time; Tissue Differentiation; Tissues; Training and Education; Transcript; Transcriptional Regulation; Travel; U1 small nuclear RNA; U6 small nuclear RNA; Variant; Work; Writing; cDNA Library; career; design; hnRNP Complexes; improved; in vivo; mRNA Precursor; research study; stem

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.

描述（由申请人提供）：本提案是我们在结构和功能上表征丝蛾 Bombyx mori 中的小核 RNA (snRNA) 及其转录控制序列的工作的延续。本提案中概述的实验旨在阐明 snRNA 变体在前 mRNA 剪接水平的基因表达调节中所起的作用。将使用分阶段的丝腺和毛囊来评估它们在前 mRNA 剪接中的参与。多年来，我们观察到：（1）蚕蛾具有U1、U2、U4和U6 snRNA亚型种群； (2)这些变异在组织和发育阶段有差异表达； (3) 特异性单链RNA结合蛋白与一些snRNA亚型有差异性结合； (4)某些变体优先与高分子量剪接体复合物结合。总而言之，这些数据提出了以下假设：snRNA 具有重要的功能，有助于差异化的前 mRNA 剪接、组织分化和发育。或者，这些同工型可以在功能上等效。 为了检验这两个假设，我们提出以下具体目标：（1）评估不同组织、发育阶段和剪接体复合物中snRNA亚型的相对丰度； (2) 鉴定和表征与 snRNA 变体结合的蛋白质； (3) 利用不同组织和发育阶段的核提取物确定snRNA变体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。家蚕以多种、组织特异性、发育调节的 U1、U2、U4 和 U6 变体的形式表现出极端且无与伦比的多态性，以及高度专门用于生产特定蛋白质（丝心蛋白或丝）的组织的存在，使丝蛾成为区分这两种假设的理想模型系统。由于所提出的问题具有普遍性，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，选择性前 mRNA 剪接是非常重要的。因此，这些变体的预期差异功能可能会调节包括人类在内的多种生物体的发育和组织分化。此外，PI还制定了详细的专业发展计划，旨在提高其在获得主流资金方面的竞争力。