Base J and transcription termination in Leishmania

描述(申请人提供)：利什曼原虫感染世界各地的人类，导致相当大的发病率和死亡率。在利什曼原虫和其他锥虫中，蛋白质编码基因的转录始于少数大的多顺反子转录单位(PTU)的5‘端，成熟的mRNAs是通过对初级转录产物的反式剪接产生的。然而，关于利什马尼亚的转录是如何终止的，人们知之甚少。碱基J是一种高度修饰的DNA碱基，在利什曼原虫和相关生物的核DNA中取代了~1%的T。当参与J生物合成的一个基因(JBP2)在L.tarentolae前鞭毛体中被破坏时，它们失去了~70%的J，导致PTU之间汇聚的链开关区域的大量转录通读(以及转录起始处的假启动增加)。此外，JPB2 dKO寄生虫对溴脱氧尿苷(BrdU)中的生长变得高度敏感，导致J进一步降低，甚至更多地通读和细胞死亡。我们假设IJ作为转录终止的关键信号，在J缺失的细胞中失去适当的终止会杀死母体。在这个项目中，我们将通过使用AT状态方法的组合来批判性地评估这一假说，以进一步阐明利什曼原虫中J的生物合成和功能。在目标1中，我们将使用RNA-seq来分析JBP2和JBP1基因中断后不同时间分离的样本，以及BrdU和DMOG处理，以确定IJ丢失(具有潜在的致命后果)后其水平上升或下降的mRNAs，以及使达伦托利乳杆菌能够应对这种侮辱的基因表达的补偿性变化。AIM 2将利用单分子实时(SMRT)测序来鉴定转化为J的确切T残基，并将使用这些序列的系统修饰来鉴定从头合成J以及J“扩散”所需的最少信息。在目标3中，我们将使用体内和体外转录分析、全基因组染色质图谱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、通过诱导染色质变化或通过将染色质募集到特定的亚核“沉默区域”来终止转录。AIM 4将使用DNA亲和力和PTP标记的蛋白质亲和力方法，结合基于定量质谱学的蛋白质组学，识别与IJ插入相关的蛋白质和IJ在转录终止点招募的蛋白质。这些实验的结果将被结合起来，以建立与J介导的转录终止相关的更完整的分子过程模型。拟议的研究将增加我们对J在这些生物体中控制转录的作用的理解，并可能为其他真核生物中类似的碱基修饰提供可能的作用。此外，由于在利什曼原虫(包括人类)的哺乳动物宿主中没有发现J碱基，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。

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.