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，需要复杂的质量控制机制来限制错误RNA或蛋白质的合成。消除异常转录物有助于保护生物体免受可能干扰细胞及其分子机制的正常功能的错误蛋白质产物的潜在有害影响。一个被充分研究的降解途径和细胞监视机制，称为无义介导的mRNA衰变（NMD），降解含有提前翻译终止密码子（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未能在适当位置终止翻译的异常转录物。RNA解旋酶UPF 1是NMD的中心蛋白，在NMD的检测和降解阶段起着重要作用。UPF 1通过与真核释放因子eRF 3相互作用被募集到底物mRNA，随后被其激酶SMG 1磷酸化。UPF 1的磷酸化残基作为NMD特异性降解因子SMG 5/7和SMG 6的结合位点，它们分别通过去腺苷化和去帽以及内切核酸裂解来启动降解。尽管已经提出关键的NMD组分UPF 1作为翻译终止和mRNA衰变之间的分子连接，UPF 1在NMD过程的不同阶段的精确分子功能还没有完全了解，因此需要进一步的研究。为此，我们建议将UPF 1的结合位点与其结合mRNA的NMD相关特征相关联，例如内切酶切位点或终止密码子处的核糖体暂停位点。具体而言，我们将使用PAR-CLIP来确定UPF 1在转染的NMD报告基因mRNA以及内源性mRNA上的位置。UPF 1的这些mRNA结合位点将与SMG 6执行的内切酶切位点相关，我们将通过改良的5测序方法鉴定SMG 6。同时，我们将通过核糖体分析分析终止密码子处的翻译速率和核糖体堆积，并通过蛋白质占有率分析表征终止密码子附近的mRNP结构。将为UPF 1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将通过揭示UPF 1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UPF 1突变体的生物化学特性及其对mRNA结合和mRNP组成的影响，将使我们能够了解中央NMD因子UPF 1的分子功能。将在报告基因测定中检查UPF 1结合和活性的衍生特征。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，适当地整合已知的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.