Cap binding and gene expression in trypanosomes

描述(申请人提供)：锥体使用类似于细菌范例的多顺反子转录，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微乎其微，这是很有意义的，因为有几个物种是导致人类疾病的寄生虫。单个mRNAs通过3‘聚腺苷酸化和5’反式剪接被分解成典型的真核单顺反子mRNAs，其结果是存在一个普遍的外显子序列，该外显子序列包含一个超甲基化的4帽结构。帽4是寄生虫所特有的，由剪接的前导RNA转录本提供，这是一种小的RNA，在剪接之前的成熟过程中可能会穿过细胞质。锥虫拥有六种与真核生物帽子结合翻译起始因子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，这些元件是寄生虫所特有的，因此为临床靶向攻击提供了潜在的途径。在这里，我们讨论了TbeIF4E5的角色和功能，它与锥虫翻译起始因子eIF4G的三个变体结合，在其他方面没有特征。这一应用的目的是探索锥虫特有的基因控制机制，这种机制是通过其独特的mRNA帽子结构介导的。TbeIF4E家族是我们的发射台，有六个eIF4E结合蛋白eIF4G的同源基因和两个eIF4A的同源基因，增加了可能为基因表达调控提供巧妙手段的组合可能性。我们首先验证和扩展我们的酵母双杂交蛋白质-蛋白质相互作用结果；体外系统将确认这些结论，随后进行竞争分析以确定结合偏好。为了确定eIF4E5和eIF4E6的功能，将在布鲁氏锥虫的基因敲除和敲除中测试eIF4E5和eIF4E6同源基因的功能冗余。体内与eIF4E5结合的蛋白质将通过eIF4E5复合体的纯化和灵敏的质谱分析来鉴定；结合的RNA及其帽子结构将通过引物延伸来检测。这项申请是加州大学洛杉矶分校Campbell/Sturm实验室与巴西Aggeu Magalhaes研究中心Osvaldo P de Melo Neto博士的实验室合作的基础。我们的团队从相反的方向处理这些问题，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，而巴西的工作则是从翻译的角度进行的。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)，以研究动态体剪接前导RNA的成熟。这项FIRCA申请的目标与母公司的拨款没有直接关系。 与公共卫生相关：动塑形目包含几种具有重要医学意义的病原体，包括人类利什曼病、非洲锥虫病和恰加斯病的病原体。这些病原体不是使用基因表达的转录调控，而是使用不同寻常的转录后调控机制。动质体具多种mRNA帽结合蛋白，其中许多在哺乳动物宿主中没有对应的蛋白。这项研究的目的是确定与寄生虫特异性TbeIF4E5蛋白相关的独特成分，以及它在锥虫生物学中的作用。

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.