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RNA加工至关重要， 降解RNA外泌体有三种主要组分-核心、核酸外切酶和外切酶。 辅因子;和辅因子赋予RNA底物特异性。我们目前的大多数生物化学和结构 对RNA外泌体的理解来自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 非常低的水平。哺乳动物中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外泌体的主要辅因子， 细胞质是含有RNA解旋酶SKIV 2L和辅因子TTC 37的“超级杀伤（SKI）”复合物。 SKIV 2L或TTC 37基因突变与遗传性常染色体隐性遗传病相关， 肝胆肠综合征（THES），表现出严重的原发性B细胞免疫缺陷和其他临床表现， 异常在初步研究中，我们产生了Skiv 21 KO和Ttc 37 KO小鼠B细胞系以及B细胞系。 特异性Skiv 21条件性敲除小鼠。我们发现，Skiv 2l缺陷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，滑雪- RNA外泌体通过加工或降解未知的RNA靶标来调节B细胞中的基因组稳定性。 我们将使用最先进的下一代测序技术确定SKI-RNA外泌体靶RNA种类 total RNA-seq、RIP-seq、DRIP-seq等技术与强大的生物信息学分析相结合 (Aim 1）。细胞质RNA外泌体的RNA监视对于其他组织和细胞也可能是重要的。 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性敲除（Aim 2）。总之，这里提出的研究将揭示 哺乳动物细胞质RNA外泌体在B细胞中的生理功能，并支持 治疗B细胞免疫缺陷疾病的策略，如THES。