RNA polymerase II transcription in trypanosomes

描述(申请人提供)：锥虫寄生虫布氏锥虫、克氏锥虫和利什曼原虫。导致人类主要疾病非洲睡眠病、恰加斯病和利什曼病。由于治疗这些疾病的药物很少，有毒且难以管理，而且寄生虫对这些药物的抗药性正在上升，因此开发新的治疗策略变得越来越重要。虽然这些寄生虫发展了独特的宿主-寄生虫相互作用，但它们都有相同的不寻常的基因表达模式，包括蛋白质编码基因的多顺反子转录和核前mRNA的反式剪接。在这一过程中的一个关键分子是剪接前导(SL)RNA，它的5‘端部分被切割并与每个mRNA的5’端融合。由于SL RNA在反式剪接中被消耗，寄生虫的生存关键取决于持续强劲的SL RNA合成。因此，抑制SL RNA基因(SLRNA)转录似乎是一种很有前途的防治锥虫寄生虫的宽带策略。到目前为止，我们已经能够在布氏锥虫中鉴定出四种转录因子，包括25种蛋白质，它们是启动子结合复合体TRF4/SNAPc/TFIIA，TFIIB，TFIIH和TFIIE的复合体，以及锥虫介体。我们的数据表明，这些蛋白质在SLRNA启动子上形成转录前起始复合体，并招募RNA聚合酶II进行准确的转录起始。相反，我们没有发现任何证据表明TFIIB与已知的启动蛋白质编码基因阵列的RNA聚合酶II转录的发散链切换区(DSSR)结合。因此，在目标1中，我们建议继续我们对RNA聚合酶II转录因子的生化表征，以确定最有希望进行进一步分析的靶点；除了指定的因子外，这些因子还包括与RNA PolII共纯化的新蛋白，以及新的TFIIA相关复合体和CDK相关激酶，这两个蛋白似乎都在前mRNA合成中发挥特殊功能。对于这些因子的表征，我们计划使用大量的遗传和生化实验，包括蛋白质复合体的串联亲和纯化、条件性基因沉默实验和体外转录分析。在目标2中，我们将使用一种系统的方法来研究dSSR中转录启动的机制，这将基于对RNA聚合酶II占据的全基因组分析和对dSSR驱动的报告基因表达的突变分析。总体而言，这些实验可能会发现一个根本重要的过程中的独特和必要的因素或因素域，即RNA聚合酶II到DNA的招募。此外，它们将提供对蛋白质表达的第一步的机械性理解，这将有助于长期控制锥虫。

DESCRIPTION (provided by applicant): The trypanosomatid parasites Trypanosoma brucei, T. cruzi and Leishmania spp. cause the major human diseases African Sleeping Sickness, Chagas' disease, and Leishmaniasis, respectively. Since drugs for these diseases are few, toxic and difficult to administer, and parasite resistance to these drugs is on the rise, it becomes increasingly important to develop new therapeutic strategies. While these parasites have developed unique host-parasite interactions, they all share the same unusual mode of gene expression involving polycistronic transcription of protein coding genes and trans splicing of nuclear pre-mRNA. A key molecule in this process is the spliced leader (SL) RNA from which the 5' terminal part is cleaved and fused to the 5' end of each mRNA. Since SL RNA is consumed in trans splicing, parasite viability crucially depends on continuously strong SL RNA synthesis. Therefore, inhibition of SL RNA gene (SLRNA) transcription appears to be a promising broadband strategy against trypanosomatid parasites. So far, we have been able to identify and characterize four transcription factors comprising 25 proteins in T. brucei that are essential for the process: the promoter-binding complex TRF4/SNAPc/TFIIA, TFIIB, a complex of TFIIH and TFIIE, and the trypanosome mediator. Our data show that these proteins form a transcription pre-initiation complex at the SLRNA promoter and recruit RNA polymerase II for accurate transcription initiation. Conversely, we found no evidence that TFIIB binds to divergent strand switch regions (dSSRs) known to initiate RNA polymerase II transcription of the protein coding gene arrays. We therefore propose in Aim 1 to continue our biochemical characterization of RNA polymerase II transcription factors to identify the most promising targets for further analysis; besides the specified factors, these include new proteins that co-purified with RNA pol II as well as a novel TFIIA- associated complex and a CDK-related kinase both of which appear to function specifically in pre-mRNA synthesis. For these factor characterizations we plan to employ a plethora of genetic and biochemical experiments which include tandem affinity purification of protein complexes, conditional gene silencing experiments and in vitro transcription assays. In Aim 2, we will use a systematic approach to investigate the mechanism of transcription initiation in dSSRs which will be based on a genome-wide analysis of RNA polymerase II occupancy and on a mutational analysis of dSSR-driven reporter gene expression. Overall, these experiments may uncover unique and essential factors or factor domains in a fundamentally important process, namely the recruitment of RNA polymerase II to DNA. Moreover, they will provide a mechanistic understanding of the first step in protein expression which will help to control trypanosomatids in the long term.