Post-transcriptional gene regulation by the exon junction complex

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Project Summary Nonsense mutations pose a serious challenge to fitness and survival of cells and organisms. To suppress mRNAs carrying such nonsense mutations, all eukaryotes possess a conserved mRNA surveillance pathway called Nonsense-Mediated mRNA Decay (NMD). NMD is also an essential post-transcriptional regulator of normal mRNAs that shapes processes such as stem cell maintenance, neurogenesis, germ cell development and anti-viral response. In all eukaryotes, NMD is governed by three UPF proteins, UPF1, UPF2 and UPF3. In multicellular organisms, NMD is also regulated by a conserved multi-protein exon junction complex (EJC), which binds upstream of mRNA exon-exon junctions. During translation, if at least one EJC remains present downstream of a terminating ribosome, it can signal premature termination and trigger NMD. Understanding NMD mechanism and its regulation by EJC is crucial for betterment of human health as mutations in EJC and NMD proteins cause developmental defects, intellectual disability and mental retardation. The overarching goal of this research program is to understand how the remarkable variation in composition and function of EJC/UPF machinery regulates NMD to dictate cellular function and fate in animal cells. To achieve this goal, we are using a combination of genetic, genomic, molecular, biochemical and cellular approaches in cultured human cells and in zebrafish embryos to pursue four main directions. (1) We will identify the mechanism of a switch in EJC composition that we recently discovered and define the role of distinct EJC compositions in gene expression. (2) Our recent discovery that mammalian UPF3 paralogs and their interaction with EJC are non-essential for NMD challenges a decades old model of EJC-dependent NMD in eukaryotes. We will apply new genomic technologies that probe in vivo ribosome function to identify the role of UPF3 and other UPF proteins in premature termination complex assembly and activity on hundreds of human mRNAs. We will also identify the factors and features that govern signaling between the termination complex and the EJC. (3) We and others have previously shown that EJCs are often detected at unexpected locations on RNAs. By exploiting a new step in EJC recycling that we have uncovered, we will define the assembly mechanisms and functions of EJCs at such unexpected sites. (4) We have developed zebrafish mutants that lack one of the EJC or its NMD adapter proteins, which will be used to identify the genetic and cellular processes controlled by these factors during motor neuron and muscle development. Overall, our work will advance the knowledge of NMD mechanisms and how they regulate post- transcriptional gene regulation to control cellular function and organismal development. This progress will also elevate our ability to target NMD for therapeutics.