RACK1-mediated control of mRNA selectivity by poxviruses

RACK1介导的痘病毒对mRNA选择性的控制

• 批准号: 10216958
• 负责人: Madeline Grace Rollins
• 金额: $ 2.89万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-25 至 2021-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10216958
• 关键词: 5' Untranslated Regions; Address; Affect; Architecture; Behavior; Biochemical; Bioinformatics; Biological; CRISPR/Cas technology; Cell Line; Cells; Charge; Complement; Coupling; Cryoelectron Microscopy; Custom; DNA Viruses; Dermal; Electrostatics; Elements; Enhancers; Event; Fibroblasts; Foundations; Future; Gene Expression; Genes; Genetic Transcription; Global Change; Goals; Haploid Cells; Head; Human; Infection; Internal Ribosome Entry Site; Knock-out; Knowledge; Mediating; Messenger RNA; Modeling; Molecular; Output; Pathologic; Phosphorylation; Phosphotransferases; Play; Polyribosomes; Positioning Attribute; Poxviridae; Poxviridae Infections; Protein Biosynthesis; Protein Subunits; RNA Viruses; Regulation; Regulatory Element; Research; Ribosomal RNA; Ribosomes; Serine; Solid; Specificity; Structural Models; Structure; Study models; System; Testing; Transcript; Translations; Vaccinia virus; Vertebral column; Viral; Viral Proteins; Virus; Virus Replication; base; functional plasticity; inorganic phosphate; polyadenosine; polysome profiling; programs; receptors for activated C kinase; transcriptional reprogramming; transcriptome sequencing

ABSTRACT Viruses have evolved mechanisms to use 5’ untranslated regions (UTRs) to control viral protein synthesis during infection. For example, many RNA viruses use internal ribosome entry sites in the 5’ UTRs. By contrast, poxviruses, such as Vaccinia virus, encode polyadenosine (polyA) repeats in the 5’ UTR of post-replicative mRNA that enhance the translation of poxviral transcripts. The functionality of the polyA enhancer effect in human cells is dependent on poxvirus-mediated phosphorylation of an extended loop of the small subunit protein receptor of activated C kinase 1 (RACK1). Although we know ribosome specification plays a key role in selectively regulating poxviral protein synthesis, it is unclear how negative charge in the loop regulates mRNA selectivity of poxvirus-customized ribosomes and how this selectivity is influenced by global changes in cellular transcriptional and translational activity during infection. Using structure modeling and biochemical approaches, we have previously shown that charge in the RACK1 loop increases repulsive electrostatic interactions with the negatively charged backbone of 18S rRNA near the mRNA exit channel. Therefore, we hypothesize that introduction of negative charge remodels the loop or mRNA exit channel to more broadly regulate translation by altering recognition of specific 5’ UTR elements, such as polyA leaders. We will address this hypothesis by developing two complementary cell systems that we will use to study RACK1-loop mediated mRNA selectivity under different conditions that mimic aspects of poxvirus infection. We will use polysome profiling and RNA-Seq to analyze global changes in translational efficiency to gain a broader understanding of transcript-specific translation modulated by the RACK1 loop. The findings from the proposed research will lay a solid foundation for our understanding of how poxviruses control translation, as well as future studies of ribosome-centric modes of translation regulation in diverse biological and pathological contexts.

摘要 病毒已经进化出使用5'非翻译区（UTR）来控制病毒蛋白质合成的机制， 感染例如，许多RNA病毒在5'UTR中使用内部核糖体进入位点。相比之下， 痘病毒，如牛痘病毒，在复制后的DNA的5' UTR中编码聚腺苷（polyA）重复序列。 增强痘病毒转录物翻译的mRNA。polyA增强子在细胞中的功能性 人类细胞依赖于痘病毒介导的小亚基蛋白延伸环的磷酸化 活化的C激酶1受体（RACK 1）。虽然我们知道核糖体特化在 选择性调节痘病毒蛋白质合成，目前还不清楚环中的负电荷如何调节mRNA 痘病毒定制核糖体的选择性以及这种选择性如何受到细胞内 在感染期间的转录和翻译活性。使用结构建模和生物化学方法， 我们先前已经表明，RACK 1环中的电荷增加了与 mRNA出口通道附近的18S rRNA带负电荷的骨架。因此，我们假设 负电荷的引入重塑环或mRNA出口通道，以更广泛地调节翻译， 改变特异性5 ′ UTR元件的识别，例如polyA前导序列。我们将通过以下方式来解决这个假设 开发两个互补的细胞系统，我们将使用它们来研究RACK1环介导的mRNA选择性 在不同的条件下模拟痘病毒感染。我们将使用多核糖体分析和RNA测序 分析翻译效率的全球变化，以更广泛地了解转录特异性 翻译由RACK1循环调制。拟议研究的结果将奠定坚实的基础 对于我们理解痘病毒如何控制翻译，以及未来对核糖体中心模式的研究， 在不同的生物学和病理学背景下的翻译调节。