Final Report Summary - NMDPATHS (Functional and structural analysis of the mammalian nonsense-mediated mRNA decay pathway) In eukaryotic cells such as human cells, the genomic information that encodes all the proteins a cell needs to function, is stored in the nucleus in the form of DNA. Before a specific protein is produced, this information is copied (transcribed) into messenger RNA (mRNA), which is then exported from the nucleus and translated into the encoded protein. To ensure that they can adapt to changes in their environment, cells regulate both production and degradation of mRNAs and proteins. Furthermore, since the quality of the mRNA determines the quality of the protein it encodes, cells need to make sure that mRNAs are error free. One important pathway that cells employ to both check the fidelity of mRNAs and also control the abundance of certain mRNAs is termed nonsense-mediated mRNA decay (NMD). It detects and destroys mRNAs haboring premature translation stop codons (PTCs or nonsense-codons) in order to prevent production of truncated proteins that might have deleterious effects on the cell. NMD not only protects cells from faulty mRNAs but also regulates the expression of ~10% of all naturally occurring mRNAs containing features recognized by the NMD machinery.The large number of high-profile publications on NMD in recent years shows the considerable interest this pathway has engendered in the scientific community. This is due to the considerable biological and therapeutic significance of NMD, which is demonstrated by its essential role in embryo development, genetic disorders and tumor formation. However, to tap its therapeutic potential several gaps in our strutural and mechanistic understanding of the NMD pathway have to be closed. In our project we collected important data on the mechanism of NMD and its connection to general mRNA decay that closed several of these knowledge gaps. This kind of insight will be valuable for the development of drugs or cancer therapies in the future. Especially the molecular structures of protein complexes obtained during this project are important to allow structure based drug design.In particular we made important contributions to the elucidation of the NMD mechanism and its connection with the general mRNA deadenylation machinery. To address the first two objectives of the project we mapped the interacting domains and solved the structure of the Smg5-Smg7 protein complex, which connects recognition of the mRNA target to the degradation machineries. This structure allowed us to test and verify the importance of this complex for the function of NMD in human cells. We found that only the intact Smg5-Smg7 complex has enough affinity for Upf1, the central effector protein that marks mRNAs for NMD, to elicit decay of the mRNA. To adress the third objective of our project, we identified a direct interaction between a catalytic subunit of the deadenylation complex CCR4-NOT and the NMD protein Smg7. Deadenylation is usually the first step in mRNA degradation. This direct interaction shows that NMD employs several cellular decay mechanisms (deadenylation in addition to endonucleolytic cleavage and decapping) to ensure robust and efficient decay of the target mRNAs. Since the lack of structural information on the general mRNA degradation machinery proteins also made mapping of interactions with the NMD machinery difficult, we proceeded to obtain structures of degradation machinery complexes in an extension of the third project objective. We solved structures of important parts of the two cytoplasmic deadenylase complexes in eukaryotes: the CCR4-NOT complex and the PAN2-PAN3 complex. In particular we elucidated the structure of the human CCR4-NOT subcomplex made up by the proteins CNOT1-CNOT2-CNOT3. These three proteins are frequently used as docking platforms by many degradation inducing proteins to bring the CCR4-NOT complex to mRNAs and thereby lead to deadenylation. This structure shows that regions in CNOT2 and CNOT3 that were previously thought to be flexible and unstructured co-fold for complex formation and mediate most of the contacts between the three proteins. Analysis of the sequence conservation of the complex surface indicates patches that are potentially bound by factors, which recruit the CCR4-NOT complex. Furthermore, we solved the crystal structure of the complex formed by the PAN2-PAN3 proteins. The structure and biochemical assays revealed the unusual stoichiometric and structural assymmetry of this complex with one PAN2 bound to a PAN3 dimer. This asymmetry is mediated by an unstructured linker of PAN2 that wraps around the surface of PAN3. Apart from their involvement as downstream effectors of NMD these two deadenylase complexes are of general importance for the regulation of the abundance of all mRNAs in eukayotic cells. In summary the structural and functional data obtained in this project has important implications for our understanding of general post-transcriptional gene regulation as well as for the understanding of mRNA quality control and will be useful towards the development of drugs to modify mRNA abundance in the future.