Poster abstracts
Poster number 85 submitted by Toby Frazer
Investigating the effect of UPF3 disease-linked mutations on nonsense-mediated mRNA decay
Toby Frazer (Ohio State University Department of Molecular Genetics), Rene Arvola (Ohio State University Department of Molecular Genetics), Debadrita Paul (Ohio State University Department of Molecular Genetics), Guramrit Singh (Ohio State University Department of Molecular Genetics)
Abstract:
Nonsense-mediated Decay (NMD) is an mRNA quality control pathway that is conserved throughout eukaryotes. NMD targets mRNAs containing Premature Termination Codons (PTCs) to mitigate the harmful effects of PTC-inducing mutations. Additionally, NMD can target normal mRNAs where translation stops resemble PTCs to serve as a general regulatory mechanism. UPF3 is a core NMD factor involved in the recognition of NMD substrates. In mammals, UPF3 has two paralogs: UPF3A and UPF3B. Mutations in UPF3B are associated with neurodevelopmental disorders in humans. To test the ability of these mutations to function in NMD, we expressed them in UPF3A/UPF3B double knockout HCT116 colorectal cancer cells. Surprisingly, these mutants rescue NMD as well as wild-type UPF3B, possibly due to overexpression. I hypothesize that mutants expressed at endogenous levels will have impaired NMD ability. To test this hypothesis, we developed constructs to reduce the expression of UPF3A and UPF3B via the inclusion of a hairpin in their 5′ UTR. Preliminary data suggests that the hairpin reduces expression of UPF3A between 1.5- and 5-fold, and for UPF3B between 2- and 20-fold. Hairpin constructs display mildly reduced NMD activity compared to constructs without the hairpin as tested by RT-qPCR of selected NMD targets. Ongoing work includes testing whether attenuated expression of UPF3B mutants impacts their NMD function. Furthermore, we also aim to determine whether altering UPF3 subcellular localization alters its NMD activity. To this end, we have fused UPF3A and UPF3B to a strong Nuclear Export Sequence (NES) and ongoing work includes testing their NMD function. Future work will determine whether UPF3B patient missense mutants have altered subcellular localization, as many of these mutations are located within predicted Nuclear Localization Sequences. Taken together, this work will help us understand how NMD activity is affected by UPF3B patient mutations and how NMD dysregulation contributes to disease.
Keywords: NMD, Hairpin, Expression