Poster abstracts
Poster number 1 submitted by Fuad Abbas
Developing an optimal AID-degron system to elucidate UPF protein function in human cells
Fuad Abbas (Department of Molecular Genetics, Center for RNA Biology, The Ohio State University, Columbus), Zhongxia Yi (Department of Molecular Genetics, Center for RNA Biology, The Ohio State University, Columbus), Guramrit Singh (Department of Molecular Genetics, Center for RNA Biology, The Ohio State University, Columbus)
Abstract:
The Nonsense Mediated mRNA Decay (NMD) pathway is of critical importance as a watchdog in the regulation of gene expression and selective degradation of erroneous mRNA across all eukaryotic species. NMD operates via several pathways in order to selectively degrade mRNA transcripts containing a premature termination codon (PTC), also known as a nonsense mutation. Implicated in the NMD pathway are several proteins, with the core being composed of the up-frameshift proteins UPF1, UPF2, UPF3A and UPF3B, which help mediate the degradation of mRNAs bearing a premature stop codon by recruiting and interacting with downstream exon junction complexes (EJCs). UPF2 has been observed to have a diverse array of functions across different cells, and at different stages of cell growth and differentiation. To help elucidate these functions, UPF2 proteins will be constitutively degraded in HCT116 human cancer cells and the resulting phenotype will be observed. Protein destabilization will be carried out using an auxin-inducible degron (AID) system. Upon addition of auxin, OsTIR1 expressed in the HCT116-OsTIR1 cell line will ubiquitinate the AID-domain on translated UPF2 proteins, thereby targeting them for destruction. This project aims to investigate parallel, alternative NMD pathways associated with the UPF2 gene and their potential mechanisms via knockout of UPF2 using an auxin-inducible degron (AID) system, and analysis of the knockout phenotype.
Keywords: NMD, UPF proteins, auxin-inducible degron