In vitro study of aberrant translation termination in the context of a long 3΄UTR

Although nonsense-mediated mRNA decay (NMD) is one of the best studied eukaryotic mRNA degradation pathways, the first 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 (1). Since poly(A)-binding protein (PABP) suppresses NMD in vivo (2), the absence of PABP interacting with eRF3 bound to the ribosome at the stop codon is thought to cause aberrant termination and to activate NMD. However, 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 in vitro system revealed prolonged ribosome stalling on a transcript with a long, NMD-inducing 3΄UTR or on a transcript that lacks a poly(A) tail. This is not only consistent with the current NMD model and the NMD antagonizing role of PABP observed in vivo, it furthermore provides the first in vitro evidence for a role of the poly(A) tail and PABP on mammalian translation 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. We use this assay to assess the function of NMD and translation-related factors in premature termination events