In vitro study of aberrant translation termination in the context of NMD-sensitive mRNAs

Nonsense-mediated mRNA decay (NMD) is one of the best studied eukaryotic mRNA degradation pathways but the initial steps of its activation in mammalian cells remain unclear. According to the current working model, NMD ensues when a ribosome fails to terminate translation properly. Since poly(A)-binding protein (PABP) suppresses NMD in vivo, the absence of PABP from the vicinity of the termination codon is thought to cause aberrant termination and NMD activation (1). However, due to technical limitations, little is known about the exact function of PABP in translation termination and the difference between aberrant and proper termination. Here we present the establishment of a robust toeprinting protocol allowing the detection of ribosomes at stop codons of reporter transcripts during translation in rabbit reticulocyte lysate. This approach revealed a prolonged ribosome occupancy at NMD-eliciting stop codons as well as a crucial role of PABP for efficient translation termination. These results are consistent with the current NMD model and the NMD antagonizing role of PABP observed in vivo. Moreover the assay provides an excellent tool to study mammalian translation termination. In parallel we present an approach that allows the identification of stalled ribosomes at termination codons in HeLa translation-competent extracts. Since NMD is well characterized in HeLa cells, we aspire to deplete and sequester NMD and translation factors to address their role in termination. An in vitro assay that faithfully recapitulates well-defined in vivo observations has great potential to help us gaining novel insights into the mechanism of aberrant translation termination and its connection to NMD