Poster abstracts

Poster number 106 submitted by Sarah Nock

Insight into the requirements for UPF1 association with translating mRNA

Sarah L. Nock (Center for RNA Molecular Biology, Case Western Reserve University), Kristian E. Baker (Center for RNA Molecular Biology, Case Western Reserve University)

Abstract:
Nonsense-mediated mRNA decay (NMD) is a cellular process in eukaryotes that acts primarily in the recognition of mRNAs containing premature termination codons (PTCs) and the targeting of these aberrant transcripts for rapid degradation. The core components of the NMD machinery are highly conserved and include the Up-frameshift proteins, UPF1, UPF2, and UPF3. UPF1 is an RNA-dependent ATPase (i.e. ‘RNA helicase’) with known RNA binding activity. We, and others, have demonstrated that UPF1 interacts with both normal and PTC-containing mRNAs, and are interested in better understanding the requirements for the association of UPF1 with mRNA.

In this study, we use sucrose gradient centrifugation (i.e. polysome analysis) to monitor the co-sedimentation of yeast UPF1 with translating mRNA, and to evaluate a role for UPF2, UPF3, and biochemical activities intrinsic to UPF1 for their ability to influence UPF1 association with polysomes. We find that the majority of cellular UPF1 is found on polysomes and that this association of UPF1 with translating RNA does not require either UPF2 or UPF3. We also show that mutations that disrupt the ability of UPF1 to either bind or hydrolyze ATP result in an increase in the amount of UPF1 associated with polysomes. Continuing work will be presented on the analysis of additional UPF1 mutants. Overall, our results provide insight into the requirements for UPF1 to interact with translating mRNA and contribute to our overall understanding of how the NMD machinery surveys and targets substrates to this important quality control pathway.

Keywords: nonsense-mediated mRNA decay, NMD, UPF1, polysomes