RNA Surveillance in B cell by the Mammalian Cytoplasmic RNA Exosome

哺乳动物细胞质 RNA 外泌体对 B 细胞的 RNA 监视

• 批准号: 10041640
• 负责人: Nan Yan
• 金额: $ 24.51万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-16 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10041640
• 关键词: Antibody Affinity; Antibody Response; Antigens; B-Cell Development; B-Lymphocytes; Biochemical; Bioinformatics; Biological Process; CRISPR/Cas technology; Cell Count; Cell Cycle; Cell Cycle Arrest; Cell Degranulation; Cell Line; Cell physiology; Cells; Childhood; Clinical; Complex; Cytoplasm; DNA; DNA Repair; Data; Development; Disease; Enhancers; Exhibits; Exonuclease; Exploratory/Developmental Grant; Genes; Genetic Predisposition to Disease; Genetic Transcription; Genome Stability; Hereditary Disease; Hybrids; Immune; Immune response; Immunity; Immunoglobulin Class Switching; Immunologic Deficiency Syndromes; Impairment; Inherited; Interferon Type II; Investigation; Knockout Mice; Mammalian Cell; Mammals; Mature B-Lymphocyte; Measures; Memory B-Lymphocyte; Messenger RNA; MicroRNAs; Molecular; Mus; Mutation; Natural Killer Cells; Nature; Nuclear RNA; Patients; Phase; Phenotype; Physiological; Production; RNA; RNA Degradation; RNA Helicase; RNA Processing; RNA immunoprecipitation sequencing; Ribonucleases; Role; SKIV2L gene; Specificity; Stimulus; Structure; Substrate Specificity; T-Cell Proliferation; T-Lymphocyte; Technology; Tissues; Trichohepatoenteric syndrome; Untranslated RNA; Vaccination; WD Repeat; Yeasts; cell type; cofactor; conditional knockout; congenital immunodeficiency; cytokine; ds-DNA; exosome; experimental study; hypogammaglobulinemia; next generation sequencing; recurrent infection; response; therapeutic development; transcriptome sequencing

The RNA exosome is an evolutionarily conserved ribonuclease complex critical for RNA processing and degradation. There are three major components of the RNA exosome – the core, the exonuclease and the cofactors; and cofactors confer RNA substrate specificity. The majority of our current biochemical and structural understanding of the RNA exosome are from studies performed in yeast. However, mammals express many more RNA species, especially various kinds of non-coding RNAs, that either does not exist in yeast or exist at very low levels. Lost-of-function studies of RNA exosome in mammals are still scarce. We propose to study the physiological function of the mammalian cytoplasmic RNA exosome. The major cofactor of the RNA exosome in the cytoplasm is the ‘superkiller (SKI)’ complex, which contains RNA helicase SKIV2L and cofactor TTC37. Mutations in either SKIV2L or TTC37 gene are associated with an inherited autosomal recessive disorder, trichohepatoenteric syndrome (THES), which exhibits severe primary B-cell immunodeficiency and other clinical abnormalities. In preliminary studies, we generated Skiv2lKO and Ttc37KO mouse B cell lines as well as B-cell- specific Skiv2l conditional knockout mice. We found that Skiv2l-deficiency results in cell cycle arrest and dsDNA damage in B cells as well as early B-cell developmental arrest in mice. Therefore, we hypothesize that the SKI- RNA exosome regulates genomic stability in B cells through processing or degradation of unknown RNA targets. We will determine SKI-RNA exosome target RNA species using state-of-the-art next-gen sequencing technologies such as total RNA-seq, RIP-seq, DRIP-seq, in combination with powerful bioinformatic analysis (Aim 1). RNA surveillance by the cytoplasmic RNA exosome may be also important for other tissues and cell types. Thus, we will also investigate whether the SKI-RNA exosome is required for functions of mature B cell and other immune cells by conditional knockout (Aim 2). Together, studies proposed here will reveal the physiological function of mammalian cytoplasmic RNA exosome in B cells and support the development of strategies to treat B-cell immunodeficiency diseases such as THES.

RNA 外泌体是一种进化上保守的核糖核酸酶复合物，对于 RNA 加工和 降解。 RNA外泌体由三个主要成分组成——核心、核酸外切酶和 辅助因子；和辅因子赋予RNA底物特异性。我们目前的大多数生化和结构 对 RNA 外泌体的了解来自于在酵母中进行的研究。然而，哺乳动物表达许多 更多的RNA种类，特别是各种非编码RNA，它们要么不存在于酵母中，要么存在于酵母中 非常低的水平。哺乳动物 RNA 外泌体功能丧失的研究仍然很少。我们建议研究 哺乳动物细胞质RNA外泌体的生理功能。 RNA外泌体的主要辅助因子 细胞质是“超级杀手（SKI）”复合物，其中含有RNA解旋酶SKIV2L和辅因子TTC37。 SKIV2L 或 TTC37 基因的突变与遗传性常染色体隐性遗传病有关， 毛肝肠综合征 (THES)，表现出严重的原发性 B 细胞免疫缺陷和其他临床症状 异常。在初步研究中，我们生成了 Skiv2lKO 和 Ttc37KO 小鼠 B 细胞系以及 B 细胞系 特异性Skiv2l条件敲除小鼠。我们发现 Skiv2l 缺陷会导致细胞周期停滞和 dsDNA B 细胞损伤以及小鼠早期 B 细胞发育停滞。因此，我们假设 SKI- RNA 外泌体通过加工或降解未知的 RNA 靶标来调节 B 细胞的基因组稳定性。 我们将使用最先进的下一代测序确定 SKI-RNA 外泌体目标 RNA 种类 总RNA-seq、RIP-seq、DRIP-seq等技术与强大的生物信息分析相结合 （目标 1）。细胞质 RNA 外泌体的 RNA 监测可能对其他组织和细胞也很重要 类型。因此，我们还将研究 SKI-RNA 外泌体是否是成熟 B 细胞功能所必需的 和其他免疫细胞通过条件敲除（目标 2）。这里提出的研究将共同​​揭示 B 细胞中哺乳动物细胞质 RNA 外泌体的生理功能并支持 B 细胞的发育 治疗 B 细胞免疫缺陷疾病（例如 THES）的策略。