RNA polymerase II transcription in trypanosomes

描述（申请人提供）：布鲁氏锥虫、克氏锥虫和利什曼原虫分别是人类主要疾病非洲昏睡病、恰加斯病和利什曼病的病因。由于治疗这些疾病的药物很少、有毒且难以施用，而且寄生虫对这些药物的耐药性正在上升，因此开发新的治疗策略变得越来越重要。虽然这些寄生虫已经发展出独特的宿主-寄生虫相互作用，但它们都具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。这个过程中的一个关键分子是剪接前导RNA (SL)，其中5‘末端部分被切割并融合到每个mRNA的5’端。由于SL RNA在反式剪接中被消耗，因此寄生虫的生存能力至关重要地依赖于持续强的SL RNA合成。因此，抑制SLRNA基因（SLRNA）转录似乎是一种很有前途的针对锥虫寄生虫的宽带策略。到目前为止，我们已经鉴定并表征了布鲁氏体中包含25种蛋白质的四种转录因子，这些转录因子对该过程至关重要：启动子结合复合物TRF4/SNAPc/TFIIA， TFIIB， TFIIH和TFIIE的复合物，以及锥虫介质。我们的数据表明，这些蛋白质在SLRNA启动子处形成转录起始前复合物，并招募RNA聚合酶II进行准确的转录起始。相反，我们没有发现证据表明TFIIB与已知启动蛋白质编码基因阵列的RNA聚合酶II转录的发散链开关区（dSSRs）结合。因此，我们建议在Aim 1中继续我们的RNA聚合酶II转录因子的生化表征，以确定最有希望的目标进行进一步分析；除了特定的因子，这些包括与RNA pol II共纯化的新蛋白，以及新的TFIIA相关复合物和cdk相关激酶，两者似乎在pre-mRNA合成中具有特异性作用。对于这些因子表征，我们计划采用大量的遗传和生化实验，包括蛋白质复合物的串联亲和纯化，条件基因沉默实验和体外转录分析。在目标2中，我们将使用系统的方法来研究dSSRs的转录起始机制，这将基于RNA聚合酶II占用的全基因组分析和dssr驱动报告基因表达的突变分析。总的来说，这些实验可能揭示了一个基本重要过程中独特而必要的因素或因素结构域，即RNA聚合酶II向DNA的募集。此外，它们将提供对蛋白质表达第一步的机制理解，这将有助于长期控制锥虫病。

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.