Poster abstracts
Poster number 113 submitted by Lucas Serdar
The use of tethered function analysis to investigate the role of Upf1 in targeting substrates to NMD
Lucas D. Serdar (Center for RNA Molecular Biology, Case Western Reserve University), Kristian E. Baker (Center for RNA Molecular Biology, Case Western Reserve University)
Abstract:
Cells employ multiple quality control mechanisms to ensure the accurate flow of genetic information. Nonsense-mediated mRNA decay (NMD) is a post-transcriptional quality control mechanism responsible for the detection and destruction of transcripts that contain nonsense mutations or other features which cause translation termination to occur in a premature context. The superfamily I RNA helicase Upf1 is a central component of the NMD machinery. Upf1 has been well characterized in vitro. However, relatively little is known about how Upf1 functions to induce rapid mRNA decay in vivo. Here, we use tethered function analysis as an inroad to evaluate the specific requirements for Upf1 to target substrates to NMD.
Initial studies indicate that tethering Upf1 to the 3’ UTR of a reporter mRNA lacking a nonsense codon results in reduction of both steady state mRNA abundance and half-life. Destabilizing activity is lost when Upf1 alleles which contain mutations that result in loss of ATPase and helicase activity are tethered to the mRNA. Interestingly, tethered Upf1 is able to induce reporter destabilization in the absence of Upf2 and Upf3, as well as under conditions where translation is strongly repressed. These initial results indicate that tethered function analysis is a robust and effective assay for examining the requirements for Upf1 to induce rapid mRNA decay during NMD.
Keywords: quality control, NMD, Upf1