Base J and transcription termination in Leishmania

利什曼原虫中的碱基 J 和转录终止

• 批准号: 8853793
• 负责人: Peter John Myler
• 金额: $ 56.59万
• 依托单位: SEATTLE BIOMEDICAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-06-11 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8853793
• 关键词: Affinity; Affinity Chromatography; Anabolism; Animals; Base Composition; Biochemical; Bioinformatics; Biological Assay; Bromodeoxyuridine; Cell Death; Cell Line; Cells; ChIP-seq; Characteristics; Chromatin; Chromatin Structure; Chromosomes; Cloning; Code; Collaborations; Complex; Country; Coupled; DNA; DNA Sequence; DNA-Directed RNA Polymerase; DNase I hypersensitive sites sequencing; Development; Elements; Enzymes; Eukaryota; Fluorescent in Situ Hybridization; Gene Expression; Genes; Genetic; Genetic Transcription; Genomic DNA; Genomics; Growth; Human; Hydroxylation; In Vitro; Infection; Knock-out; Lead; Leishmania; Location; Maintenance; Maps; Mass Spectrum Analysis; Mediating; Messenger RNA; Methods; Modification; Molecular; Molecular Conformation; Molecular Models; Morbidity - disease rate; Nuclear; Nucleosomes; Organism; Parasites; Pharmaceutical Preparations; Plasmids; Process; Proteins; Proteomics; Reading; Recruitment Activity; Research Design; Role; Sampling; Signal Transduction; Site; Techniques; Testing; Time; Trans-Splicing; Transcript; Transferase; Variant; Work; base; cell killing; chemotherapeutic agent; coping; density; design; genetic information; genome-wide; histone modification; in vivo; insight; killings; molecular modeling; mortality; novel; protein complex; public health relevance; research study; single molecule; transcription termination; transcriptome sequencing

DESCRIPTION (provided by applicant): Leishmania parasites infect humans worldwide, leading to substantial morbidity and mortality. Transcription of protein-coding genes in Leishmania and other trypanosomatids initiates at the 5' end of a small number of large polycistronic transcription units (PTUs), and mature mRNAs are generated by trans-splicing of the primary transcript. However, little is known about how transcription is terminated in Leishmania. Base J is a hyper-modified DNA base that replaces ~1% of T in the nuclear DNA in Leishmania and related organisms. When one of the genes (JBP2) involved in J biosynthesis is disrupted in L. tarentolae promastigotes, they lose ~70% of their J, leading to massive transcriptional read-through at the convergent strand-switch regions between PTUs (and an increase in false-starts where transcription initiates). Furthermore, JPB2 dKO parasites become hypersensitive to growth in bromodeoxyuridine (BrdU), leading to a further reduction in J, even more read-through, and cell death. We postulate that iJ serves as a critical signal for transcription termination, and that loss of proper termination in J-deficient cells kills the paraste. In this project, we will critically assess this hypothesis by using a combination of state-of-the-at approaches to further elucidate J biosynthesis and function in Leishmania. In Aim 1, we will use RNA-seq to analyze samples isolated at various times after disruption of the JBP2 and JBP1 genes, as well as BrdU and DMOG treatment, to identify mRNAs whose levels increase or decrease after iJ loss (with potentially lethal consequences), as well compensatory changes in gene expression that enable L. tarentolae to cope with this insult. Aim 2 will utilize single-molecule real-time (SMRT) sequencing to identify the exact T residues that are converted to J, and will use systematic modification of these sequences to identify the minimal information required for de novo J synthesis, as well as for J "spreading". In Aim 3, we will use in vivo and i vitro transcription assays, genome-wide chromatin mapping, as well as fluorescence in situ hybridization and chromosome conformation capture experiments to determine whether J terminates transcription either by directly blocking the RNA polymerase, by inducing chromatin changes, or by recruitment of chromatin to specific sub-nuclear "silencing domains". Aim 4 will use DNA affinity and PTP-tagged protein affinity approaches, coupled with quantitative mass spectrometry-based proteomics, to identify proteins associated with iJ insertion and those recruited by iJ at transcription termination sites. The results from these experiments will be combined to build a more complete model of the molecular processes associated with J-mediated transcription termination. The proposed studies will increase our understanding of the role of J in controlling transcription in these organisms, and may provide insight into a possible role for similar base modifications in other eukaryotes. Furthermore, since base J is not found in the mammalian hosts of Leishmania (including humans), this information will provide a new opportunity for the development of novel chemotherapeutic agents against these important parasites.

描述（由申请人提供）：利什曼原虫寄生虫感染全世界人类，导致大量发病率和死亡率。利什曼原虫和其他锥虫动物中蛋白质编码基因的转录起始于少量大的多顺反子转录单位 (PTU) 的 5' 端，并且通过初级转录物的反式剪接生成成熟的 mRNA。然而，人们对利什曼原虫转录如何终止知之甚少。 J 碱基是一种高度修饰的 DNA 碱基，可替代利什曼原虫及相关生物体核 DNA 中约 1% 的 T。当 L. tarentolae 前鞭毛体中参与 J 生物合成的基因 (JBP2) 之一被破坏时，它们会丢失约 70% 的 J，导致 PTU 之间的聚合链开关区域发生大量转录读取（并且转录开始时的错误启动）。此外，JPB2 dKO 寄生虫对溴脱氧尿苷 (BrdU) 的生长变得高度敏感，导致 J 进一步减少，甚至更多的通读和细胞死亡。我们假设 iJ 是转录终止的关键信号，J 缺陷细胞中适当终止的丧失会杀死寄生虫。在这个项目中，我们将通过结合最新的方法来批判性地评估这一假设，以进一步阐明利什曼原虫中的 J 生物合成和功能。在目标 1 中，我们将使用 RNA-seq 分析 JBP2 和 JBP1 基因破坏以及 BrdU 和 DMOG 处理后不同时间分离的样本，以识别在 iJ 丢失后水平增加或减少的 mRNA（具有潜在的致命后果） ），以及基因表达的补偿性变化，使 L. tarentolae 能够应对这种侮辱。目标 2 将利用单分子实时 (SMRT) 测序来识别转化为 J 的确切 T 残基，并将使用这些序列的系统修改来识别从头合成 J 以及合成 J 所需的最少信息。 J“传播”。在目标 3 中，我们将使用体内和体外转录测定、全基因组染色质作图以及荧光原位杂交和染色体构象捕获实验来确定 J 是否通过直接阻断 RNA 聚合酶、通过诱导染色质来终止转录变化，或通过将染色质募集到特定的亚核“沉默域”。 Aim 4 将使用 DNA 亲和力和 PTP 标记的蛋白质亲和力方法，结合基于定量质谱的蛋白质组学，来识别与 iJ 插入相关的蛋白质以及 iJ 在转录终止位点招募的蛋白质。这些实验的结果将被结合起来，建立一个更完整的与 J 介导的转录终止相关的分子过程模型。拟议的研究将增加我们对 J 在控制这些生物体转录中的作用的理解，并可能深入了解类似碱基修饰在其他真核生物中的可能作用。此外，由于在利什曼原虫的哺乳动物宿主（包括人类）中未发现碱基J，这一信息将为开发针对这些重要寄生虫的新型化疗药物提供新的机会。