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中的一个基本过程，需要详细的质量控制机制来限制RNA或蛋白质故障的合成。消除异常转录物可保护有机体免受可能干扰细胞及其分子机械功能的错误蛋白质产物的潜在有害作用。一个良好的降解途径和细胞监测机制，称为废话介导的mRNA衰变（NMD），降解了包含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，并采用了一组保守的核心因素来消除未能在适当位置终止翻译的异常转录本。 NMD中的中央蛋白RNA解旋酶UPF1在NMD的检测和降解阶段起着重要作用。 UPF1通过与真核释放因子ERF3的相互作用而募集以基板mRNA，并随后被其激酶SMG1磷酸化。 UPF1的磷酸化残基是NMD特异性降解因子SMG5/7和SMG6的结合位点，这些因子通过降解，分解和核核切解分别启动降解，尽管已经表明，NMD Compants upf1在降低了型号之间的构成和MRNNA在MRNNA之间的链接，但在降级上均能降级。 NMD过程的不同阶段尚未完全了解，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mRNA的NMD相关特征相关联，例如内接胚层的位点或在终止密码子处的核糖体暂停。具体而言，我们将使用PAR-CLIP来确定UPF1在转染的NMD报告基因mRNA和内源mRNA上的位置。 UPF1的这些mRNA结合位点将与SMG6执行的内漏的位点相关，我们将通过修改的5个测序方法识别。同时，我们将通过核糖体分析来分析终止密码子的翻译速率和核糖体堆积，并通过蛋白质占用分析在终止密码子附近表征MRNP结构。将为UPF1的不同突变体生成相似的数据集，UPF1的不同突变体在特定的分子活动中不足。我们预计，这些高通量数据将通过发现UPF1依赖性mRNA底物识别和降解的分子原理来洞悉NMD机制。将UPF1突变体的生化特性与对mRNA结合和MRNP组成的影响相结合，将使我们能够了解中央NMD因子UPF1的分子功能。在记者测定中将检查UPF1结合和活动的派生特征。作为我们的最终目标，我们旨在开发NMD的一般模型，该模型正确整合已知的NMD特征并正确预测NMD底物的行为。