Investigation of aberrant vs. normal translation termination in HeLa extracts

Nonsense-mediated mRNA decay (NMD) is a translation-dependent mechanism in eukaryotes that degrades mRNAs harboring a termination codon which is not in the correct environment for proper translation termination. NMD affects mRNAs with a premature termination codon (PTC) as well as physiological transcripts. Like in D. melanogaster, C. elegans and S. cerevisiae, it has recently been found that the presence of an exon-junction-complex (EJC) located downstream of the stop codon is not an absolute requirement to trigger NMD in mammals (Bühler et al., NSMB 2006). Instead, the physical distance between the termination codon and the poly(A)-binding protein (PABPC1) seems to be crucial for PTC recognition (Eberle et al., PLoS Biology 2008).

The goal of this project is to understand the mechanistic differences between aberrant and proper translation termination. In particular we would like to examine if PABPC1 and the NMD factor UPF1 have antagonizing effects on translation termination by competing for binding to the eukaryotic release factor eRF3 as has been suggested previously (Singh et al., PLoS Biology 2008, Ivanov et al., EMBO J 2008). To test this directly, an in vitro system is required, in which we want to examine ribosome stalling on a termination codon of a particular mRNA and to modify the levels of PABPC1 and UPF1. For this approach, in vitro synthesized reporter mRNAs harboring different 3' UTR lengths will be translated in HeLa cell extracts depleted for UPF1 or PABPC1. Using toeprinting assays, we would like to examine if ribosomes stall longer at a termination codon in constructs with a long 3' UTR or under low PABPC1/high UPF1 protein levels.