Project Summary/Abstract A substantial fraction of human inherited disease-causing mutations introduce an early stop codon that truncates protein production and elicits mRNA decay in a process called Nonsense-Mediated mRNA Decay (NMD). Despite much work, it is still unclear how early stop codons are recognized and how they bring about mRNA decay. The long-term goal of the work is to illuminate how cells recognize and repress mRNAs with early stop codons in animals. Work over the last several decades has highlighted many of the factors involved as well as some of their biochemical capabilities, but the steps and structure/function of the molecules involved remains unclear. In this proposal, the PI and his lab will dissect the pathway of protein synthesis and degradation of early stop codon-containing mRNAs in vivo. The specific aims of the proposed work are to: [Aim 1]: characterize the mRNA cleavage reaction, its products, and its dependencies on protein factors. Results from this aim will provide information about the biochemistry of the RNA decay reaction underlying NMD. [Aim 2]: study the role the factor UPF1 has in licensing mRNAs for decay. Results from this aim will illuminate the factors underlying the timing, recruitment, and commitment of mRNAs to decay. [Aim 3]: characterize the role ribosomes have in the NMD pathway. Results from this aim will showcase how ribosomes signal to cellular machinery to bring about RNA decay during NMD. Experiments will: (a) analyze the phenotype of NMD mutant C. elegans strains, (b) profile the RNA species produced during NMD and in particular mutant backgrounds via both short (Illumina) and long-read (Oxford Nanopore) sequencing, and (c) biochemically analyze purified NMD complexes. Results from this work will illuminate the molecular details of the pathway by which cells recognize and repress early stop codon mutations, relevant to many human disease-causing alleles.