Base J and transcription termination in Leishmania

描述（由申请方提供）：利什曼原虫寄生虫感染全球人类，导致大量发病率和死亡率。利什曼原虫和其他锥虫中蛋白质编码基因的转录起始于少量大的多顺反子转录单位（PTU）的5'末端，并且成熟mRNA通过初级转录物的反式剪接产生。然而，很少有人知道转录是如何终止在利什曼原虫。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体的核DNA中取代约1%的T。当参与J生物合成的基因（JBP 2）在L.在tarentolae前鞭毛体中，它们失去约70%的J，导致在PTU之间的会聚链转换区域处的大量转录通读（以及转录起始处的错误起始的增加）。此外，JPB 2 dKO寄生虫对溴脱氧尿苷（BrdU）的生长变得超敏感，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作为一个关键的信号转录终止，并在J-缺陷细胞的损失适当终止杀死寄生虫。在这个项目中，我们将批判性地评估这一假设，通过使用一个国家的组合的方法，以进一步阐明J的生物合成和功能，利什曼原虫。在目标1中，我们将使用RNA-seq分析JBP 2和JBP 1基因破坏后不同时间分离的样本，以及BrdU和DMOG处理，以鉴定iJ丧失后水平增加或减少的mRNA（具有潜在的致命后果），以及使L. tarentolae来科普这种侮辱。目标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J合成以及J“扩散”所需的最小信息。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、通过诱导染色质变化或通过将染色质募集到特定的亚核“沉默结构域”来终止转录。目的4将使用DNA亲和性和PTP标记的蛋白质亲和性方法，再加上定量质谱为基础的蛋白质组学，以确定与iJ插入和iJ在转录终止位点招募的蛋白质。这些实验的结果将结合起来，建立一个更完整的模型与J介导的转录终止相关的分子过程。拟议的研究将增加我们的理解J在控制这些生物体中的转录的作用，并可能提供洞察到其他真核生物中类似的碱基修饰的可能作用。此外，由于碱基J在利什曼原虫的哺乳动物宿主（包括人类）中未发现，因此这一信息将为开发针对这些重要寄生虫的新型化疗剂提供新的机会。

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.