Cap binding and gene expression in trypanosomes

描述（由申请人提供）：锥虫使用类似于细菌范例的多顺反电子转录，但缺乏由转录单位连接的基因之间的功能关联。因此，基因表达的转录控制是最小的，这是有趣的，因为一些物种是负责人类疾病的寄生虫。单个mrna通过3‘聚腺苷化和5’反式剪接被分解成典型的真核单顺反子mrna，其结果是存在一个普遍的外显子序列，其中包含一个超甲基化的cap 4结构。帽4是寄生虫特有的，由剪接的先导RNA转录物提供，这是一种小RNA，可以在剪接前的成熟过程中穿过细胞质。锥虫拥有六种与真核生物帽结合翻译起始因子eIF4E相关的蛋白质，而人类只有三种。该家族具有在寄生虫特有的剪接前导RNA加工和mRNA翻译的基因表达中发挥主要作用所需的元件，因此为靶向临床攻击提供了潜在途径。在这里，我们讨论了beif4e5的作用和功能，它与锥虫翻译起始因子eIF4G的三个变体结合，在其他方面是未知的。本应用程序的目的是探索锥虫独特的mRNA帽结构介导的基因控制机制。theif4e家族是我们的发射台，eIF4E结合蛋白eIF4G有6个同源物，eIF4A有2个同源物，增加了组合的可能性，可能为基因表达的调节提供了技巧。我们从酵母双杂交蛋白-蛋白相互作用结果的验证和扩展开始；体外系统将证实这些结论，其次是竞争分析，以确定协会的偏好。eIF4E5和eIF4E6同源基因的功能冗余将在布鲁氏锥虫基因敲除和敲除中进行测试，目的是确定eIF4E5的功能。体内与eIF4E5相关的蛋白将通过纯化eIF4E5复合物，然后采用灵敏质谱法进行鉴定；结合的RNA及其帽结构将通过引物延伸检查。该应用程序是加州大学洛杉矶分校Campbell/Sturm实验室和巴西Aggeu Magalhaes研究中心Osvaldo P de Melo Neto博士实验室合作的基础。我们的团队从相反的方向研究这些问题，加州大学洛杉矶分校历史上专注于导致反式剪接的RNA加工事件，而巴西的研究则从翻译的角度出发。预算包括一名来自巴西的学生到加州大学洛杉矶分校实习，并在巴西参观实验室和举办研讨会。加州大学洛杉矶分校的研究小组获得了美国国立卫生研究院奖（AI056034; 7/01/09-6/30/14），以研究着丝质体剪接前导RNA的成熟。本FIRCA申请的目标与父基金没有直接关系。

DESCRIPTION (provided by applicant): Trypanosomes use polycistronic transcription akin to the bacterial paradigm, but lacking the functional association between genes linked by transcription units. As such, transcriptional control of gene expression is minimal, which is of interest as several species are parasites responsible for human disease. Individual mRNAs are resolved into typical eukaryotic monocistronic mRNAs via 3' polyadenylation and 5' trans-splicing, the result of which is the presence of a universal exon sequence containing a hypermethylated cap 4 structure. The cap 4 is unique to the parasite, and is provided by the spliced leader RNA transcript, a small RNA that may travel through the cytosol in the course of its maturation prior to splicing. Trypanosomes possess six proteins related to the eukaryotic cap-binding translation-initiation factor eIF4E, as compared to three in humans. This family has the elements required to play major roles in gene expression for both spliced leader RNA processing and mRNA translation that are unique to the parasite, and hence provide a potential avenue for targeted clinical attack. Here we address the role and function of TbeIF4E5, which binds to three variants of the trypanosome translation initiation factor eIF4G and is otherwise uncharacterized. The goal of this application is to explore trypanosome-specific mechanisms of gene control mediated by their unique mRNA cap structure. The TbeIF4E family is our launching pad, with six homologues for eIF4E binding protein eIF4G and two for eIF4A adding to the combinatorial possibilities that may provide the finesse for modulation of gene expression. We begin with validation and expansion of our yeast two-hybrid protein-protein interaction results; an in vitro system will confirm these conclusions, followed by competition assays to determine association preferences. Functional redundancy of the eIF4E5 and eIF4E6 orthologs will be tested in gene knockdown and knockouts in Trypanosoma brucei with the goal of determining the function of eIF4E5. The proteins associating with eIF4E5 in vivo will be identified by purification of eIF4E5 complex followed by sensitive mass spectrometry; bound RNA and their cap structures will be examined by primer extension. This application is the basis of a collaboration between the Campbell/Sturm laboratory at UCLA and the laboratory of Dr. Osvaldo P de Melo Neto at the Research Center Aggeu Magalhaes (Brazil). Our groups approach these questions from opposing directions, with UCLA historically focused on RNA processing events leading up to trans-splicing and the Brazil work coming from the angle of translation. The budget includes the internship of a student from Brazil to UCLA, with a laboratory visit and seminar presented in Brazil. The UCLA group has an NIH award (AI056034; 7/01/09-6/30/14) to study the maturation of the kinetoplastid spliced leader RNA. The goals of this FIRCA application are not related directly to the parent grant. PUBLIC HEALTH RELEVANCE: The Order Kinetoplastida contains several pathogens of medical importance including the causative agents of human leishmaniasis, African trypanosomiasis and Chagas disease. Rather than using transcriptional regulation of gene expression, these pathogens employ unusual post-transcriptional mechanisms of regulation. Kinetoplastids possess multiple mRNA cap binding proteins, many of which do not have counterparts in the their mammalian hosts. The goal of this study is to identify unique components associated with the parasite-specific TbeIF4E5 protein, and its role in trypanosome biology.