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

基因表达是所有生物的基本过程，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。消除异常转录本有助于保护生物体免受错误蛋白质产物的潜在有害影响，这些错误蛋白质产物可能干扰细胞及其分子机制的正常功能。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，它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本。NMD的核心蛋白是RNA解旋酶UPF1，它在NMD的检测和降解过程中起着重要的作用。UPF1通过与真核释放因子eRF3的相互作用被招募到底物mRNAs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，这两个因子分别通过去烯化、去帽和内切启动降解，尽管有人认为NMD的关键成分UPF1在翻译终止和mRNA衰退之间起着分子联系，但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mRNAs的NMD相关特征关联起来，例如内切或核糖体在终止密码子上暂停的位置。具体地说，我们将使用PAR-CLIP来确定UPF1在转染的NMD报告mRNAs和内源性mRNAs上的位置。这些UPF1的mRNA结合位点将与SMG6执行的内切位点相关联，我们将通过改进的5测序方法进行鉴定。同时，我们将通过核糖体图谱分析终止密码子的翻译速率和核糖体堆积，并通过蛋白质占据图谱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。我们预计，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，为NMD的机制提供洞察力。结合UPF1突变体的生化特征及其对mRNA结合和mRNP组成的影响，将有助于我们了解中枢NMD因子UPF1的分子功能。UPF1结合和活性的衍生特征将在报告分析中进行检查。作为我们的最终目标，我们的目标是开发一种NMD的通用模型，该模型能够恰当地整合已知的NMD特性，并正确地预测NMD衬底的行为。

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.