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

基本信息

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
项目类别：
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

项目摘要

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外泌体是一种进化保守的核糖核酸酶，对RNA加工和降解。 RNA外泌体的三个主要组成部分 - 核心，外切核酸酶和辅助因子；和辅因子会议RNA底物特异性。我们目前的大多数生化和结构对RNA外泌体的理解来自在酵母中进行的研究。但是，哺乳动物表达了许多更多的RNA种，尤其是各种非编码RNA，要么不存在于酵母中，要么存在于非常低的水平。对哺乳动物中RNA外泌体的功能障碍研究仍然很少。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“ Superkiller（Ski）”复合物，其中包含RNA解旋酶SKIV2L和Cofactor TTC37。 SKIV2L或TTC37基因中的突变与遗传常染色体隐性疾病有关， Trichohepatoenteric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-细胞特定的SKIV2L条件敲除小鼠。我们发现SKIV2L缺陷导致细胞周期停滞和dsDNA B细胞的损害以及早期的B细胞发育停滞在小鼠中。因此，我们假设滑雪 RNA外泌体通过处理或降解未知RNA靶标调节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来确定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学分析结合（目标1）。细胞质RNA外泌体的RNA监测对于其他组织和细胞也可能很重要类型。这是我们还将研究成熟B细胞功能是否需要滑雪rna外泌体和其他免疫细胞通过有条件的敲除（AIM 2）。一起，这里提出的研究将揭示 B细胞中哺乳动物细胞质RNA外泌体的生理功能，并支持治疗B细胞免疫缺陷疾病（例如THES）的策略。