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

基因表达是所有生物体的基本过程，需要精细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可能干扰细胞正常功能及其分子机制的错误蛋白质产物的潜在有害影响。一种被广泛研究的降解途径和细胞监测机制，被称为无义介导的mRNA衰变（NMD），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，并使用一组保守的核心因子来消除不能在适当位置终止翻译的异常转录本。NMD的中心蛋白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的磷酸化残基是NMD特异性降解因子SMG5/7和SMG6的结合位点，它们分别通过死基化、脱冠和核内溶裂解启动降解。尽管有人认为NMD的关键成分UPF1在翻译终止和mRNA衰变之间起着分子链接作用，UPF1在NMD过程不同阶段的确切分子功能尚不完全清楚，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相关特征联系起来，例如内切位点或终止密码子处的核糖体暂停位点。具体来说，我们将使用PAR-CLIP来确定UPF1在转染的NMD报告mrna以及内源性mrna上的位置。UPF1的这些mRNA结合位点将与SMG6执行的内切位点相关，我们将通过改进的5测序方法进行鉴定。同时，我们将通过核糖体分析分析翻译速率和停止密码子处的核糖体堆积，并通过蛋白质占用分析表征终止密码子附近的mRNP结构。对于缺乏特定分子活性的UPF1的不同突变体，将产生相似的数据集。我们期望这些高通量数据将通过揭示upf1依赖的mRNA底物识别和降解的分子原理来深入了解NMD的机制。结合UPF1突变体的生化特征及其对mRNA结合和mRNP组成的影响，将有助于我们了解NMD中心因子UPF1的分子功能。UPF1结合和活性的衍生特征将在报告者分析中进行检查。作为我们的最终目标，我们的目标是开发一个通用的NMD模型，适当地集成已知的NMD特性，并正确地预测NMD基板的行为。

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.