Recognition and degradation of mRNA by nonsense-mediated decay

描述（申请人提供）：mRNA的降解在调节基因表达中起重要作用。在真核细胞中，已经进化出一种特殊的途径来刺激含有过早无义密码子（PTC）的异常mrna的降解，PTC是一种导致翻译早期终止的信号，如果不加控制，则会导致截短多肽的积累。这种途径被称为无义介导的mRNA衰变（NMD），代表了一种保守的质量控制机制，可以维持保真度并保护细胞免受错误基因表达的影响。广泛接受的NMD模型假设ptc - mRNA被认为是异常的，这是由于翻译终止发生在太远的地方，导致终止核糖体和mRNA poly(A)尾部结合蛋白PAB1之间的相互作用受损。我们的观察结果挑战了这一范式，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。本研究的长期目标是阐明NMD途径对mRNA的识别和快速降解的分子机制。我们将在三个特定的目标下解决包含无义mRNA如何被识别和靶向快速降解的重要问题。首先，我们将识别和表征mRNP元件，这些元件对于将ptc - mRNA靶向NMD至关重要。此外，我们提出了两种创新和公正的方法来识别NMD的顺式和反式拮抗剂。其次，我们将研究NMD蛋白与mRNA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还是两者兼而有之，以及它们在NMD mRNP组装中的重要性的特定mRNA特征。最后，我们还将评估独立于RNA结合能力的单个NMD蛋白的功能能力。第三，我们将阐明对含有PTC的mRNA的异常识别如何传达给细胞衰变机制，并导致mRNA的加速降解。我们的初步数据反驳了一种普遍的观点，即NMD机制通过保守的蛋白质-蛋白质相互作用直接将衰变机制招募到mRNA。我们计划进一步挑战这一模型，并验证我们的假设，即mRNA的快速衰减是限制翻译起始因子与mRNA 52帽结合的间接后果。

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.