Talk abstracts
Talk on Saturday 09:15-09:30am submitted by Sarah Nock
Characterization of the UPF1 protein interactome in yeast provides mechanistic insight into mRNP transitions during nonsense-mediated mRNA decay
Sarah LH Nock (Department of Genetics and Genome Sciences, Case Western Reserve University), Kristian E Baker (Department of Genetics and Genome Sciences, Case Western Reserve University)
Abstract:
Nonsense-mediated mRNA decay (NMD) is a highly conserved cellular RNA quality control process recognizing aberrant RNAs and targeting them to rapid degradation. Typical substrates of the NMD pathway include transcripts harboring nonsense mutations which invoke premature translation termination and the production of potentially deleterious, C-terminally truncated polypeptides. NMD is mediated through interactions between the mRNA substrate, the prematurely terminating ribosome, and the NMD machinery – consisting of core proteins UPF1, UPF2, and UPF3. The central factor, UPF1, exhibits essential RNA binding and ATPase activities and is thought to coordinate interaction between the NMD and translation machineries, subsequently promoting accelerated decay of substrates. Despite considerable investigation into the molecular events underlying NMD, how UPF1 mediates its function on terminating ribosomes and promotes substrate degradation, and the precise roles for UPF2 and UPF3 in this process remain unknown.
To identify UPF1 protein interaction partners key for mediating NMD in vivo, we have applied proximity-based protein labeling (employing a fusion of UPF1 to the catalytically-enhanced biotin ligase, TurboID) in a yeast strain engineered for removal of abundant naturally-occurring biotinylated proteins from our samples. In addition to monitoring functional UPF1, we have analyzed a number of NMD-deficient UPF1 mutants that stall NMD at various stages of the pathway, thereby enabling us to monitor protein dynamics throughout the NMD process. Data demonstrating enhanced sensitivity in our assay and the detection of novel UPF1 protein partners will be presented.
Keywords: UPF1, proximity labeling, nonsense-mediated mRNA decay (NMD)