Cap binding and gene expression in trypanosomes

锥虫中的帽子结合和基因表达

• 批准号: 8538532
• 负责人: DAVID A CAMPBELL
• 金额: $ 5.26万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8538532
• 关键词: 5' Untranslated Regions; Address; Affinity; African Trypanosomiasis; Antibodies; Award; Binding; Binding Proteins; Biological; Biological Assay; Biological Process; Biology; Brazil; Budgets; Cell Cycle; Cell Extracts; Cell Line; Cells; Chagas Disease; Clinical; Co-Immunoprecipitations; Code; Collaborations; Complex; Coupled; Cytosol; Development; Distal; Elements; Eukaryota; Eukaryotic Initiation Factor-4E; Event; Exons; Family; Family member; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Genetic Translation; Goals; Homologous Gene; Human; In Vitro; Individual; Internships; Kinetoplastida; Knock-out; Laboratories; Leishmania; Leishmaniasis; Life Cycle Stages; Link; Mass Spectrum Analysis; Mediating; Medical; Messenger RNA; Methods; Methylation; Mini-Exons; Nature; Nuclear; Nucleotides; Organism; Orthologous Gene; Parasites; Participant; Peptide Initiation Factors; Play; Polyadenylation; Primer Extension; Process; Protein Binding; Proteins; RNA; RNA Binding; RNA Cap-Binding Proteins; RNA Interference; RNA Processing; RNA Splicing; Recombinant Proteins; Regulation; Relative (related person); Research; Role; Small RNA; Spliced Leader RNA; Spliced Leader Sequences; Staging; Structure; Students; System; Testing; Therapeutic Agents; Trans-Splicing; Transcript; Transcriptional Regulation; Translation Process; Translations; Travel; Trypanosoma; Trypanosoma brucei brucei; United States National Institutes of Health; Validation; Variant; Visit; Work; Yeasts; base; combinatorial; finesse; human disease; hybrid protein; in vivo; interest; mRNA Expression; mRNA capping; parent grant; pathogen; preference; protein complex; protein protein interaction; yeast two hybrid system

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.

描述（由申请人提供）：锥虫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是最小的，这是令人感兴趣的，因为几个物种是负责人类疾病的寄生虫。单个mRNA通过3'多聚腺苷酸化和5'反式剪接分解成典型的真核单顺反子mRNA，其结果是存在含有高甲基化帽4结构的通用外显子序列。帽4对于寄生虫是独特的，并且由剪接的前导RNA转录物提供，剪接前的前导RNA转录物是一种小RNA，其可以在其成熟过程中穿过胞质溶胶。锥虫具有六个蛋白质相关的真核细胞帽结合防御起始因子eIF 4 E，相比之下，在人类中的三个。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，我们解决的作用和功能TbeIF 4 E5，它结合三种变体的锥虫翻译起始因子eIF 4G，否则uncharacterized。本申请的目的是探索锥虫特异性的基因控制机制介导的独特的mRNA帽结构。TbeIF 4 E家族是我们的发射台，具有eIF 4 E结合蛋白eIF 4G的六个同源物和eIF 4A的两个同源物，这增加了组合的可能性，可以为基因表达的调节提供精细度。我们开始验证和扩展我们的酵母双杂交蛋白质-蛋白质相互作用的结果，在体外系统将确认这些结论，然后通过竞争试验，以确定协会的喜好。将在布氏锥虫的基因敲除和敲除中测试eIF 4 E5和eIF 4 E6直系同源物的功能冗余，目的是确定eIF 4 E5的功能。将通过纯化eIF 4 E5复合物，然后进行灵敏的质谱分析，鉴定体内与eIF 4 E5相关的蛋白质;将通过引物延伸检查结合的RNA及其帽结构。该应用是加州大学洛杉矶分校的坎贝尔/斯特姆实验室与Aggeu Magalhaes研究中心（巴西）的Osvaldo P de梅洛内托博士实验室合作的基础。我们的研究小组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，而巴西的工作则来自翻译的角度。预算包括一名巴西学生到加州大学洛杉矶分校实习，并在巴西进行实验室参观和研讨会。加州大学洛杉矶分校的研究小组获得了NIH奖（AI 056034; 7/01/09-6/30/14），以研究动质体剪接前导RNA的成熟。这个FIRCA应用程序的目标是没有直接关系到父母补助金。