Regulation of programmed -1 ribosomal frameshifting by micro-RNAs

项目摘要/摘要 自从20世纪60年代初S阐明遗传密码以来，人们一直认为mRNAs总是 被解码为三个基本密码子，任何对这一基本规则的偏离肯定是错误的，因此， 有害的。然而，在过去的十年里，我们已经表明，相当一部分细胞mRNAs 港湾顺式作用序列元件，指示细长的核糖体将阅读框架移动一个碱基 5‘(-1)方向。在细胞mRNAs中，这种程序化的核糖体移码(-1\f25 PrF)信号直接 核糖体到提前终止密码子，在那里它们成为通过 无意义介导的信使核糖核酸衰退(NMD)途径，导致编码蛋白表达减少 这些信使核糖核酸。重要的是，信使核糖核酸的降解速率与-1\f25 PRF-1的速率成正比，这种关系 这在从酵母到人类的真核生物中都是保守的。从酵母到人类的观察表明，全球变化 -1\f25 PRF-1\f6率对细胞功能有害建议-1\f25 PRF-1\f6的调节必须按以下顺序进行-1\f25-1\f6-1\f6 具体的。最近，我们发现这是通过-1\f25 PRF-1信号和-1\f25 PRF-1\f6之间的相互作用实现的 MiRNAs。通过在细胞上建立-1\f25 PRF-1\f6，这些发现开启了一条全新的研究途径 MRNAs，以及miRNAs对其的调控，作为基因表达的一种新的基本范式。这项建议 旨在加深我们对人类-1PRF调控的分子机制的理解 细胞。明确定义的Jurkat人类T细胞系并关注嵌入mRNAs编码的-1\f25 PRF-1信号 细胞因子受体和关键的细胞因子反应性酪氨酸激酶提供了一个模型系统来解决 一系列问题，从基础分子和结构生物学到对病毒的调控 获得性免疫反应。该建议的目标1试图确认已识别的序列所促进的-1\f25 PRF 在编码IL2Rγ、IL7Rα和JAK2的mRNAs中，表征SNP对-1PRF的影响，并开发 下一代PRF技术。AIM 2将识别和验证与自然相互作用的miRNAs 这些-1\f25 PRF-1\f6信号，并测试-1\f25 PRF-1\f6的自动调节反馈回路模型。目标3是定向的 研究miRNAs对基因表达和RNA结构的影响。到年底的时候 提出的研究，我们将1)加深我们对这一新的基因表达调控范式的理解， 2)确定T细胞用来控制其对重要细胞因子的反应的特定miRNAs，以及3) 建立了描述mRNA/miRNA原子级结构相互作用的新规则。这些研究将 立即影响到许多领域，包括基础分子和细胞生物学，以及更多应用领域，包括 免疫学和艾滋病毒/艾滋病，并将为小分子的设计和发现奠定基础 针对特定-1 PRF信号的治疗。

PROJECT SUMMARY/ABSTRACT Since the genetic code was elucidated in the early 1960's, it has been assumed that mRNAs are always decoded in three base codons, that any deviation from this fundamental rule must be erroneous and thus, deleterious. However, over the past decade, we have shown that a significant fraction of cellular mRNAs harbor cis-acting sequence elements that direct elongating ribosomes to shift reading frame by one base in the 5' (-1) direction. In cellular mRNAs, such Programmed -1 Ribosomal Frameshift (-1 PRF) signals direct ribosomes to premature termination codons where they become substrates for rapid degradation through the Nonsense-Mediated mRNA Decay (NMD) pathway, resulting in decreased expression of the proteins encoded by these mRNAs. Importantly, rates of mRNA degradation are proportional to rates of -1 PRF, a relationship that is conserved in eukaryotes from yeast to humans. Observations from yeast to humans that global changes in -1 PRF rates are deleterious to cellular function suggested that regulation of -1 PRF must be sequence- specific. Recently, we discovered that this is achieved through the interactions between -1 PRF signals and miRNAs. These findings have initiated a completely new avenue of research by establishing -1 PRF on cellular mRNAs, and its regulation by miRNAs as a new, fundamental paradigm in gene expression. This proposal seeks to deepen our understanding of the molecular mechanisms underlying regulation of -1 PRF in human cells. The well-defined Jurkat human T-cell line and a focus on -1 PRF signals embedded in mRNAs encoding cytokine receptors and a critical cytokine-responsive tyrosine kinase provides a model system to address a series of questions ranging from basic molecular and structural biology to regulation and control of the acquired immune response. Aim 1 of this proposal seeks to confirm -1 PRF promoted by sequences identified in the mRNAs encoding IL2Rγ, IL7Rα, and JAK2, characterize the effects of SNPs on -1 PRF, and develop the next generation PRF technology. Aim 2 will identify and validate miRNAs that naturally interact with these -1 PRF signals, and will test an autoregulatory feedback loop model of -1 PRF. Aim 3 is oriented towards characterizing the effects of miRNAs on gene expression and RNA structure. By the end of the proposed studies, we will have 1) deepened our understanding of this new paradigm gene expression control, 2) identified specific miRNAs that are used by T-cells to control their responses to important cytokines, and 3) established new rules describing mRNA/miRNA atomic scale structural interactions. These studies will immediately impact many fields including basic molecular and cell biology, and more applied fields including immunology and HIV/AIDS, and will lay the foundation for the design and discovery of small molecule therapeutics targeted to specific -1 PRF signals.