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: 8576682
• 负责人: Peter John Myler
• 金额: $ 55.73万
• 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 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8576682
• 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; Deoxyribonucleases; Development; Elements; Enzymes; Eukaryota; Fluorescent in Situ Hybridization; Gene Expression; Genes; Genetic; Genetic Transcription; 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; 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; 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; Deoxyribonucleases; Development; Elements; Enzymes; Eukaryota; Fluorescent in Situ Hybridization; Gene Expression; Genes; Genetic; Genetic Transcription; 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; 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）：Leishmania寄生虫在全球感染了人类，导致了大量的发病率和死亡率。利什曼原虫和其他锥虫中蛋白质编码基因的转录在少数大型多余的转录单元（PTU）和成熟的mRNA的5'末端启动，是通过基本转录物的转换来产生的。但是，对于利什曼尼亚终止转录的方式知之甚少。碱基J是一种过度修饰的DNA碱基，在利什曼尼亚和相关生物中取代了约1％的t中T。当tarentolae promastigotes中涉及J生物合成的基因之一（JBP2）时，它们损失了约70％的J，从而导致PTUS之间的收敛性链开关区域的大量转录读取（以及在转录启动的虚假启动中增加）。此外，JPB2 DKO寄生虫对溴脱氧尿苷（BRDU）的生长过敏，从而导致J进一步降低J，甚至更多的读取和细胞死亡。我们假设IJ是转录终止的关键信号，并且缺乏J的细胞中正确终止的损失会杀死瘫痪。在这个项目中，我们将通过使用最先进的方法结合进一步阐明利什曼尼亚的J生物合成和功能，从而批判性地评估这一假设。在AIM 1中，我们将使用RNA-Seq分析JBP2和JBP1基因中断后在不同时间分离的样品，以及BRDU和DMOG治疗，以识别IJ损失后的水平增加或下降的mRNA（潜在的致命后果），以及在基因表达中的补偿性变化，以及与L. l. partentolae cope cope cope cope cope copepers的基因表达变化。 AIM 2将利用单分子实时（SMRT）测序来识别转换为J的确切t残基，并将使用这些序列的系统修改来确定从头j综合所需的最小信息，以及j“扩散”。 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将使用DNA亲和力和PTP标记的蛋白亲和力方法，再加上基于质谱的蛋白质组学，以识别与IJ插入相关的蛋白质以及IJ在转录终止位点募集的蛋白质。这些实验的结果将结合起来，以建立与J介导的转录终止相关的分子过程的更完整的模型。拟议的研究将增加我们对J控制这些生物转录作用的理解，并可以深入了解其他真核生物中类似基础修饰的可能作用。此外，由于在利什曼尼亚的哺乳动物宿主（包括人类）中找不到碱基J，因此该信息将为开发新型化学治疗剂针对这些重要的寄生虫提供新的机会。