Molecular Genetic Analysis of Extracellular RNAs in C. elegans

线虫细胞外 RNA 的分子遗传学分析

基本信息

  • 批准号:
    8788416
  • 负责人:
  • 金额:
    $ 37.59万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 财政年份:
    2009
  • 资助国家:
    美国
  • 起止时间:
    2009-09-30 至 2017-11-30
  • 项目状态:
    已结题

项目摘要

DESCRIPTION (provided by applicant): Extracellular RNAs found in body fluids (serum, saliva, breast milk, etc.) are being investigated as sensitive sequence-specific markers for disease and as possible intercellular messengers. Understanding how specific RNAs are selected and exported and whether and how they are imported into target cells is integral to understanding their significance as biomarkers, as mediators of cellular functions, and as potential therapeutics. The discovery and characterization of RNA transporters that function to export and import double-stranded RNA (dsRNA) silencing signals in the model system C. elegans is providing molecular and genetic insights into this emerging field. In C. elegans, introduced dsRNA induces RNA interference (RNAi) that can spread between cells and tissues. Genetic analysis of systemic RNAi defective (Sid) mutants has identified not only RNA transporters, but regulatory and biogenesis factors that function in universal cell biological processes to support intercellular RNA transport. SID-1, a dsRNA-gated dsRNA-specific channel, confers the key dsRNA import and export activity to cells. Analysis of cultured cells expressing wild-type and mutant SID-1 proteins shows that nucleic acid transport by SID-1 is specific for dsRNA and sensitive to even minor chemical modifications to the dsRNA. Similar assays indicate that fish, mouse, and human SID-1 homologs also transport dsRNA. If the human homologs are equally specific for dsRNA and intolerant to modification, then the myriad of RNA modifications designed to stabilize therapeutic silencing RNAs in sera and reduce their immunogenicity may contribute to the difficulty of delivering these RNAs into cells. In AIM 1 we will determine the effect of common natural and unnatural RNA modifications on dsRNA transport in both C. elegans and cultured cells. These results will be directly applicable to the design of effective human therapeutic siRNAs. Mammalian extracellular RNAs are often found within exosomes, secreted vesicles derived from late endosome/multivesicular bodies (LE/MVB). SID-5 is a novel LE/MVB associated protein required for efficient export of RNA silencing signals, indicating that exosomes may transport RNA silencing signals in C. elegans. In AIM 2 we propose to characterize the activity, localization, and function of new SID proteins, including SID-1 and SID-5 interacting proteins to gain insight into the biological processes important for RNA export. These results will provide a genetic foundation and mechanistic understanding for the emerging fields of exosome biology and extracellular RNAs. In AIM 3 we propose to exploit the experimental expertise and resources developed in the Hunter lab to directly isolate endogenous extracellular RNAs and then to use the well established molecular genetic tools available in C. elegans to characterize their expression, transport, and function. Understanding how extracellular RNA signals contribute to animal physiology is key to understanding their significance as biomarkers and therapeutic targets for cancer therapy and other biological processes.
描述(由申请人提供):正在研究体液(血清、唾液、母乳等)中发现的细胞外 RNA 作为疾病的敏感序列特异性标记物和可能的细胞间信使。了解特定 RNA 是如何选择和输出的,以及它们是否以及如何输入到靶细胞中,对于理解它们作为生物标志物、细胞功能介质和潜在疗法的重要性至关重要。线虫模型系统中具有输出和输入双链 RNA (dsRNA) 沉默信号功能的 RNA 转运蛋白的发现和表征,为这一新兴领域提供了分子和遗传学见解。 在秀丽隐杆线虫中,引入的 dsRNA 会诱导 RNA 干扰 (RNAi),从而在细胞和组织之间传播。对系统性 RNAi 缺陷 (Sid) 突变体的遗传分析不仅鉴定了 RNA 转运蛋白,还鉴定了在通用细胞生物过程中发挥作用以支持细胞间 RNA 转运的调节和生物发生因子。 SID-1 是一种 dsRNA 门控 dsRNA 特异性通道,赋予细胞关键的 dsRNA 导入和导出活性。对表达野生型和突变型 SID-1 蛋白的培养细胞的分析表明,SID-1 的核酸转运对 dsRNA 具有特异性,并且对 dsRNA 的微小化学修饰也敏感。类似的测定表明鱼、小鼠和人类 SID-1 同源物也转运 dsRNA。如果人类同源物对 dsRNA 同样具有特异性并且不能耐受修饰,那么旨在稳定血清中治疗性沉默 RNA 并降低其免疫原性的无数 RNA 修饰可能会导致将这些 RNA 递送到细胞中的困难。在 AIM 1 中,我们将确定常见的天然和非天然 RNA 修饰对线虫和培养细胞中 dsRNA 转运的影响。这些结果将直接应用于有效的人类治疗性 siRNA 的设计。哺乳动物细胞外 RNA 经常存在于外泌体中,外泌体是源自晚期内体/多囊泡体 (LE/MVB) 的分泌囊泡。 SID-5 是有效输出 RNA 沉默信号所需的新型 LE/MVB 相关蛋白,表明外泌体可能在秀丽隐杆线虫中转运 RNA 沉默信号。在 AIM 2 中,我们建议表征新 SID 蛋白(包括 SID-1 和 SID-5 相互作用蛋白)的活性、定位和功能,以深入了解对 RNA 输出重要的生物过程。这些结果将为外泌体生物学和细胞外 RNA 的新兴领域提供遗传基础和机制理解。在 AIM 3 中,我们建议利用 Hunter 实验室开发的实验专业知识和资源来直接分离内源性细胞外 RNA,然后使用线虫中现有的成熟分子遗传工具来表征其表达、运输和功能。了解细胞外 RNA 信号如何影响动物生理学是了解它们作为癌症治疗和其他生物过程的生物标志物和治疗靶点的重要性的关键。

项目成果

期刊论文数量(0)
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科研奖励数量(0)
会议论文数量(0)
专利数量(0)

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CRAIG Patrick HUNTER其他文献

CRAIG Patrick HUNTER的其他文献

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{{ truncateString('CRAIG Patrick HUNTER', 18)}}的其他基金

Molecular Genetic Analysis of Extracellular RNAs in C. Elegans
线虫细胞外 RNA 的分子遗传学分析
  • 批准号:
    10160923
  • 财政年份:
    2009
  • 资助金额:
    $ 37.59万
  • 项目类别:
Molecular Genetic Analysis of Extracellular RNAs in C. Elegans
线虫细胞外 RNA 的分子遗传学分析
  • 批准号:
    9924608
  • 财政年份:
    2009
  • 资助金额:
    $ 37.59万
  • 项目类别:
Molecular Genetic Analysis of Extracellular RNAs in C. elegans
线虫细胞外 RNA 的分子遗传学分析
  • 批准号:
    8130855
  • 财政年份:
    2009
  • 资助金额:
    $ 37.59万
  • 项目类别:
Molecular Genetic Analysis of Extracellular RNAs in C. elegans
线虫细胞外 RNA 的分子遗传学分析
  • 批准号:
    8325718
  • 财政年份:
    2009
  • 资助金额:
    $ 37.59万
  • 项目类别:
Molecular Genetic Analysis of Extracellular RNAs in C. elegans
线虫细胞外 RNA 的分子遗传学分析
  • 批准号:
    8628210
  • 财政年份:
    2009
  • 资助金额:
    $ 37.59万
  • 项目类别:
Molecular Genetic Analysis of Extracellular RNAs in C. elegans
线虫细胞外 RNA 的分子遗传学分析
  • 批准号:
    7936354
  • 财政年份:
    2009
  • 资助金额:
    $ 37.59万
  • 项目类别:
Genetic and Biochemical Analysis of SID-1 and SID-2
SID-1 和 SID-2 的遗传和生化分析
  • 批准号:
    6838163
  • 财政年份:
    2004
  • 资助金额:
    $ 37.59万
  • 项目类别:
Genetic and Biochemical Analysis of SID-1 and SID-2
SID-1 和 SID-2 的遗传和生化分析
  • 批准号:
    7173262
  • 财政年份:
    2004
  • 资助金额:
    $ 37.59万
  • 项目类别:
Genetic and Biochemical Analysis of SID-1 and SID-2
SID-1 和 SID-2 的遗传和生化分析
  • 批准号:
    6990570
  • 财政年份:
    2004
  • 资助金额:
    $ 37.59万
  • 项目类别:
Genetic and Biochemical Analysis of SID-1and SID-2
SID-1和SID-2的遗传和生化分析
  • 批准号:
    7659988
  • 财政年份:
    2004
  • 资助金额:
    $ 37.59万
  • 项目类别:

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