Translational Control of Retroviral Unspliced mRNA

逆转录病毒未剪接 mRNA 的翻译控制

• 批准号: 7039375
• 负责人: Kathleen A. Boris-Lawrie
• 金额: $ 20.35万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-02-03 至 2011-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7039375
• 关键词: DNA binding protein; RNA interference; Retroviridae; biological models; gene expression; genetic enhancer element; genetic translation; helicase; mass spectrometry; posttranscriptional RNA processing; posttranslational modifications; protein protein interaction; protein structure; protein structure function; site directed mutagenesis; translation factor

DESCRIPTION (provided by applicant): Retroviruses vary widely in their ability to cause neoplastic transformation or immunodeficiency. In addition, retroviruses are used as a backbone for constructing nonpathogenic vectors for gene transfer applications. However, in all cases, retroviruses recruit host cell proteins to achieve cytoplasmic expression of their unspliced genome-length RNA. We have identified sequences adjacent to the 5' cap of spleen necrosis virus (SNV) that facilitate Rev/Rev responsive element (RRE)-independent expression of HIV-1 unspliced reporter RNA. The RU5 region of the SNV long terminal repeat functions as a distinct position- and orientation-dependent cap-dependent translational enhancer of intron-containing HIV-1 gag RNA as well as nonviral luciferase (luc) RNA. Designated the SNV post-transcriptional control element (PCE). polysome analyses indicate that its functional mechanism is to stimulate translation initiation, although the PCE is not an internal ribosome entry sequence. Recently, we identified PCE activity in the 5' RNA terminus of two divergent retroviruses and in a cellular protooncogene mRNA. Our novel hypothesis is that 5' PCEs are a feature shared among divergent retroviruses and selected cellular mRNAs to achieve efficient translation in the face of multiple barriers to efficient cytoplasmic expression. Combined results of site-directed mutagenesis, RNA affinity chromatography and MALDI-TOF mass spectroscopy have determined redundant stem-loop motifs are necessary for PCE activity and that the structural features of the PCE present unpaired nucleotides for interaction with RNA helicase A (RHA). Knockdown of endogenous RHA by RNA silencing eliminates PCE translation stimulation and demonstrates that RHA is necessary for PCE activity. Our findings have generated the following essential questions: i) What conserved motifs necessary for translation stimulation are shared among retroviral PCEs? ii) What residues in RHA are necessary for interaction with the PCE and with translation factors or auxiliary proteins that mediate PCE activity? iii) What step of translation is stimulated? The overall goal of this proposal is to characterize the biochemical mechanism of PCE-RHA translational enhancement. Three integrated Specific Aims for this proposal are: 1) to define conserved features in PCEs among divergent retroviruses; 2) to define the domains of RNA helicase A necessary for PCE translational enhancement; and 3) to evaluate the function of the PCE-RHA interaction in translation initiation. Our results will illuminate a unique control mechanism of eukaryotic post-transcriptional gene expression and define virus-host interactions that are important for viral replication and progression to disease. Our new fundamental knowledge of translational control will define the process by which cellular mRNAs become committed to cytoplasmic expression and produce new strategies to optimize vector systems for diverse gene transfer applications.

描述（由申请人提供）：逆转录病毒在引起肿瘤转化或免疫缺陷的能力方面差异很大。此外，逆转录病毒被用作构造基因转移应用的非人病载体的骨干。然而，在所有情况下，逆转录病毒募集宿主细胞蛋白以实现其未剪接的基因组长度RNA的细胞质表达。我们已经确定了与脾脏坏死病毒（SNV）相邻的序列，该序列促进了Rev/Rev响应元件（RRE）无plicecectectiondectiondement rna的反应元件（RRE）非依赖性表达。 SNV长末端重复的RU5区域是含有内含子内含子的HIV-1 GAG RNA以及非病毒荧光素酶（LUC）RNA的独特位置和方向依赖性帽的翻译增强子。指定了SNV转录后控制元件（PCE）。多层分析表明，其功能机制是刺激翻译起始，尽管PCE不是内部核糖体的进入序列。最近，我们鉴定了两个发散逆转录病毒的5'RNA末端和细胞原子元mRNA中的PCE活性。我们的新假设是，5'PCE是发散逆转录病毒和选定的细胞mRNA中共享的特征，面对有效的细胞质表达的多个障碍，可以实现有效的翻译。位置定向诱变，RNA亲和力色谱和MALDI-TOF质谱的组合结果确定了PCE活性的冗余茎环基序，并且PCE的结构特征呈现不成对的核苷酸与RNA Helicase A（RHA）相互作用。通过RNA沉默来敲除内源性RHA可以消除PCE的平移刺激，并证明RHA对于PCE活性是必不可少的。我们的发现产生了以下基本问题：i）在逆转录病毒PCE中共享翻译刺激所需的保守基序？ ii）RHA中的残基与PCE相互作用以及与介导PCE活性的翻译因子或辅助蛋白相互作用是必需的？ iii）刺激了哪个翻译步骤？该提案的总体目标是表征PCE-RHA转化增强的生化机制。该提案的三个集成特定目标是：1）在发散逆转录病毒中定义PCE的保守特征； 2）定义RNA解旋酶的结构域是PCE转化增强所必需的； 3）评估PCE-RHA相互作用在翻译启动中的功能。我们的结果将阐明真核后转录基因表达的独特控制机制，并定义对病毒复制和发展为疾病至关重要的病毒宿主相互作用。我们对翻译控制的新基本知识将定义细胞mRNA致力于细胞质表达的过程，并产生新的策略，以优化矢量系统以用于多种基因转移应用。