Translation reinitiation after uORFs does not fully protect mRNAs from nonsense-mediated decay

It is estimated that nearly 50% of mammalian transcripts contain at least one upstream open reading frame (uORF), which are typically one to two orders of magnitude smaller than the downstream main ORF. Most uORFs are thought to be inhibitory as they sequester the scanning ribosome, but in some cases allow for translation reinitiation. However, termination in the 5' UTR at the end of uORFs resembles premature termination that is normally sensed by the nonsense-mediated mRNA decay (NMD) pathway. Translation reinitiation has been proposed as a method for mRNAs to prevent NMD. Here, we test how uORF length influences translation reinitiation and mRNA stability in HeLa cells. Using custom 5' UTRs and uORF sequences, we show that reinitiation can occur on heterologous mRNA sequences, favors small uORFs, and is supported when initiation occurs with more initiation factors. After determining reporter mRNA half-lives in HeLa cells and mining available mRNA half-life data sets for cumulative predicted uORF length, we conclude that translation reinitiation after uORFs is not a robust method for mRNAs to prevent NMD. Together, these data suggest that the decision of whether NMD ensues after translating uORFs occurs before reinitiation in mammalian cells.