Recognition and degradation of mRNA by nonsense-mediated decay

描述(申请人提供)：信使核糖核酸的降解在基因表达调控中起着重要作用。在真核细胞中，已经进化出一条专门的途径来刺激含有早熟无意义密码子(PTC)的异常mRNAs的降解，PTC是一种导致翻译提前终止的信号，如果不加以抑制，还会导致截短多肽的积累。这一途径被称为无义介导的信使核糖核酸衰变(NMD)，代表了一种保守的质量控制机制，维持了保真度，并保护细胞免受错误基因表达的影响。被广泛接受的NMD模型认为，由于翻译终止发生得太远，终止核糖体与mRNA聚(A)尾结合蛋白PAB1之间的相互作用减弱，因此含有PTC的mRNA被认为是异常的。我们的观察对这一范式提出了挑战，并表明，将终止核糖体和PAB1之间的距离作为NMD底物识别的关键决定因素的模型不能充分解释细胞如何区分正常和过早的翻译终止。这一建议的长期目标是阐明NMD途径识别和快速降解mRNA的分子机制。我们将解决重要的问题，即如何在三个特定目标下识别并针对包含无义的信使核糖核酸快速降解。首先，我们将鉴定和鉴定mRNP元件，这些元件对于将含有PTC的mRNA靶向NMD具有重要作用。此外，我们提出了两种创新和公正的方法来识别NMD的顺式和反式作用拮抗剂。其次，我们将研究NMD蛋白与信使核糖核酸底物组装的要求。特别是，我们将研究蛋白质组装的顺序，结合目标是核糖体、mRNA还是两者，以及它们在NMD mRNP组装中的重要性的特定mRNA特征。最后，我们还将评估单个NMD蛋白的功能能力，而不是它们的RNA结合能力。第三，我们将阐明将含有PTC的mRNA识别为异常是如何传递到细胞衰退机制并导致mRNA加速降解的。我们的初步数据驳斥了一种普遍的看法，即NMD机制通过保守的蛋白质-蛋白质相互作用直接将衰退机制招募到mRNA上。我们计划进一步挑战这一模型，并测试我们的假设，即快速的mRNA衰退是限制翻译起始因子与mRNA52帽结合的间接结果。

DESCRIPTION (provided by applicant): Degradation of mRNA plays an important role in regulating gene expression. In eukaryotic cells a specialized pathway has evolved to stimulate the degradation of aberrant mRNAs containing a premature nonsense codon (PTC), a signal that causes early termination of translation and, if left unchecked, the accumulation of truncated polypeptides. This pathway, referred to as nonsense- mediated mRNA decay (NMD), represents a conserved quality control mechanism that upholds fidelity and protects cells from erroneous gene expression. The widely accepted model for NMD posits that PTC-containing mRNA are recognized as aberrant due an impaired interaction between the terminating ribosome and the mRNA poly(A) tail-bound protein, PAB1, as a consequence of translation termination occurring too far away. Our observations challenge this paradigm and indicate that a model invoking a distance between the terminating ribosome and PAB1 as the critical determinant in NMD substrate recognition cannot sufficiently explain how cells discriminate between normal and premature translation termination. The long term goal of this proposal is to elucidate the molecular mechanisms underlying both recognition and rapid degradation of mRNA by the NMD pathway. We will address the important questions of how nonsense-containing mRNA are both recognized and targeted for rapid degradation under three specific aims. First, we will identify and characterize mRNP elements important for targeting PTC-containing mRNA to NMD. In addition, we propose two innovative and unbiased approaches to identify cis- and trans-acting antagonists of NMD. Second, we will investigate the requirements for assembly of NMD proteins with mRNA substrates. In particular, we will examine the order for protein assembly, whether binding targets are ribosomes, mRNA, or both, and particular mRNA features for their importance in NMD mRNP assembly. Finally we will also assess the functional capabilities of individual NMD proteins independent of their RNA binding capacities. Third, we will elucidate how recognition of a PTC- containing mRNA as aberrant is communicated to the cellular decay machinery and leads to accelerated degradation of the mRNA. Our preliminary data dispute a common belief that the NMD machinery directly recruits the decay machinery to the mRNA through conserved protein- protein interactions. We plan to further challenge this model as well as test our hypothesis that rapid mRNA decay is an indirect consequence of restricting translation initiation factor binding to the mRNA 52 cap.