Understanding the molecular principles of UPF1-dependent mRNA substrate recognition and degradation

了解 UPF1 依赖性 mRNA 底物识别和降解的分子原理

• 批准号: 276820253
• 负责人: Professor Dr. Niels H. Gehring
• 金额: --
• 依托单位: Max-Delbrück-Centrum für Molekulare Medizin (MDC) in der Helmholtz-Gemeinschaft
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2015
• 资助国家: 德国
• 起止时间: 2014-12-31 至 2019-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/276820253?language=en
• 关键词: Understanding; molecular; principles; UPF1; dependent

Gene expression is a fundamental process in all living organisms and requires elaborate quality control mechanisms to restrict the synthesis of faulty RNAs or proteins. The elimination of aberrant transcripts serves to protect the organism from the potentially harmful effects of erroneous protein products that may interfere with the normal function of cells and their molecular machinery. A well-studied degradation pathway and cellular surveillance mechanism, referred to as nonsense mediated mRNA decay (NMD), degrades transcripts containing premature translation termination codons (PTC). NMD exists in all eukaryotic organisms and employs a conserved set of core factors to eliminate aberrant transcripts that fail to terminate translation at a proper position. The central protein in NMD, the RNA helicase UPF1, plays an important role during the detection and degradation phases of NMD. UPF1 is recruited to substrate mRNAs by its interaction with the eukaryotic release factor eRF3 and subsequently becomes phosphorylated by its kinase SMG1. Phosphorylated residues of UPF1 serve as binding sites for the NMD-specific degradation factors SMG5/7 and SMG6, which initiate degradation via deadenylation and decapping and endonucleolytic cleavage, respectively Although it has been suggested that the key NMD component UPF1 acts as a molecular link between translation termination and mRNA decay, the precise molecular function of UPF1 during the different phases of the NMD process is not fully understood and therefore requires further investigation. To this end, we propose to correlate binding sites of UPF1 with NMD-related features of its bound mRNAs, such as sites of endocleavage or ribosomal pausing at termination codons. Specifically, we will use PAR-CLIP to determine positions of UPF1 on transfected NMD reporter mRNAs as well as endogenous mRNAs. These mRNA binding sites of UPF1 will be correlated with sites of endocleavage executed by SMG6, which we will identify by a modified 5 sequencing approach. In parallel, we will analyze translation rates and ribosome pile-up at stop codons by ribosome profiling and characterize the mRNP architecture in the vicinity of termination codons by protein occupancy profiling. Similar data sets will be generated for different mutants of UPF1, which are deficient in specific molecular activities. We expect that these high-throughput data will provide insight into the mechanism of NMD by uncovering the molecular principles of UPF1-dependent mRNA substrate recognition and degradation. Combining biochemical characteristics of UPF1 mutants with their effects on mRNA binding and mRNP composition will enable us to understand the molecular function of the central NMD factor UPF1. Derived features of UPF1 binding and activity will be examined in reporter assays. As our ultimate goal we aim to develop a general model of NMD that properly integrates known NMD characteristics and correctly predicts the behavior of NMD substrates.

基因表达是所有生物体的基本过程，需要复杂的质量控制机制来限制有缺陷的 RNA 或蛋白质的合成。消除异常转录本可以保护生物体免受错误蛋白质产物的潜在有害影响，这些蛋白质产物可能会干扰细胞及其分子机制的正常功能。一种经过充分研究的降解途径和细胞监视机制，称为无义介导的 mRNA 衰减 (NMD)，可降解含有过早翻译终止密码子 (PTC) 的转录本。 NMD 存在于所有真核生物中，并利用一组保守的核心因子来消除无法在正确位置终止翻译的异常转录本。 NMD 的核心蛋白 RNA 解旋酶 UPF1 在 NMD 的检测和降解阶段发挥着重要作用。 UPF1 通过与真核释放因子 eRF3 相互作用而被招募到底物 mRNA 上，随后被其激酶 SMG1 磷酸化。 UPF1 的磷酸化残基充当 NMD 特异性降解因子 SMG5/7 和 SMG6 的结合位点，分别通过脱腺苷化、脱帽和核酸内切裂解启动降解。翻译终止和 mRNA 衰减，UPF1 在 NMD 过程不同阶段的精确分子功能尚未完全了解，因此需要进一步研究。为此，我们建议将 UPF1 的结合位点与其结合的 mRNA 的 NMD 相关特征相关联，例如终止密码子处的内切或核糖体暂停位点。具体来说，我们将使用 PAR-CLIP 来确定 UPF1 在转染的 NMD 报告基因 mRNA 以及内源 mRNA 上的位置。 UPF1 的这些 mRNA 结合位点将与 SMG6 执行的内切酶位点相关，我们将通过改进的 5 测序方法对其进行识别。同时，我们将通过核糖体分析来分析终止密码子处的翻译率和核糖体堆积，并通过蛋白质占据分析来表征终止密码子附近的 mRNP 结构。将为 UPF1 的不同突变体生成类似的数据集，这些突变体缺乏特定的分子活性。我们期望这些高通量数据能够通过揭示 UPF1 依赖性 mRNA 底物识别和降解的分子原理来深入了解 NMD 的机制。将 UPF1 突变体的生化特征与其对 mRNA 结合和 mRNP 组成的影响相结合，将使我们能够了解中心 NMD 因子 UPF1 的分子功能。 UPF1 结合和活性的衍生特征将在报告基因检测中进行检查。作为我们的最终目标，我们的目标是开发一个 NMD 的通用模型，正确集成已知的 NMD 特性并正确预测 NMD 基质的行为。