Cap binding and gene expression in trypanosomes

锥虫中的帽子结合和基因表达

• 批准号: 8720092
• 负责人: DAVID A CAMPBELL
• 金额: $ 5.37万
• 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/8720092
• 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式的多重分发转录，但缺乏通过转录单元链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，这是对人类疾病负责的寄生虫的感兴趣。单个mRNA通过3'聚腺苷酸化和5'移媒体化分辨为典型的真核一核mRNA，结果是存在含有高甲基化CAP 4结构的通用外显子序列。盖4是寄生虫独有的，由剪接的Leader RNA转录本提供，它是一种小的RNA，在拼接之前可能会在成熟过程中穿过细胞质。与人类三种相比，锥虫具有与真核帽结合引入因子EIF4E相关的六种蛋白质。该家族具有在寄生虫独有的剪接领导者RNA加工和mRNA翻译中扮演主要角色所需的要素，因此为有针对性的临床攻击提供了潜在的途径。在这里，我们解决了TBEIF4E5的作用和功能，该作用和功能与锥虫翻译起始因子EIF4G的三种变体结合，否则未经表征。该应用的目的是探索由其独特的mRNA帽结构介导的基因控制的锥虫特异性机制。 TBEIF4E家族是我们的发射板，具有六种用于EIF4E结合蛋白EIF4G的同源物，而EIF4A的两个同源物增加了组合的可能性，这些可能性可能为调节基因表达的调节提供精致。我们首先要验证和扩展酵母双杂交蛋白 - 蛋白质相互作用的结果；体外系统将确认这些结论，然后进行竞争测定，以确定关联偏好。 EIF4E5和EIF4E6直系同源物的功能冗余将在基因敲低和锥虫中的基因敲低中进行测试，目的是确定EIF4E5的功能。通过纯化EIF4E5复合物，随后是敏感的质谱法，将鉴定与体内EIF4E5相关的蛋白质。绑定的RNA及其CAP结构将通过底漆扩展检查。该应用是UCLA的Campbell/Sturm实验室与研究中心Aggeu Magalhaes（巴西）的Osvaldo P de Melo Neto实验室合作的基础。我们的小组从相反的方向上解决了这些问题，UCLA历史上着重于RNA处理事件，导致跨性别的工作以及来自翻译角度的巴西工作。预算包括从巴西到加州大学洛杉矶分校的学生实习，并在巴西举行了实验室访问和研讨会。 UCLA集团获得了NIH奖（AI056034; 7/01/09-6/30/14），以研究动力质体剪接的Leader RNA的成熟。此FIRCA申请的目标与父母赠款无直接关系。