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

• Applicants: Professor Dr. Niels H. Gehring; Professor Dr. Markus Landthaler
• Subject Area: General Genetics and Functional Genome Biology ; Cell Biology
• Term: from 2015 to 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 276820253

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.

• DFG Programme: Research Grants