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

Abstract

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 re-initiation. However, termination in the 5ʹ UTR at the end of uORFs resembles pre-mature termination that is normally sensed by the nonsense-mediated mRNA decay (NMD) pathway. Translation re-initiation has been proposed as a method for mRNAs to prevent NMD. Here we test how uORF length influences translation re-initiation and mRNA stability. Using custom 5ʹ UTRs and uORF sequences, we show that re-initiation 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 and mining available mRNA half-life datasets for cumulative uORF length, we conclude that translation re-initiation after uORFs is not a robust method for mRNAs to evade NMD. Together, these data support a model where uORFs have evolved to balance coding capacity, translational control, and mRNA stability.

The Emerging Role of uORF-Encoded uPeptides and HLA uLigands in Cellular and Tumor Biology

“…Besides nascent protein degradation and ribosome recycling [ 96 , 103 , 104 , 105 , 106 , 107 ], the stalled ribosomes also trigger the nonsense-mediated mRNA decay (NMD) pathway, leading to degradation of the entire mRNA in most cases [ 97 , 108 , 109 , 110 ]. Even ribosomal re-initiation at the CDS start after uORF termination does not necessarily protect the mRNAs from NMD [ 111 ]. Determination of reporter mRNA half-life time and mining available mRNA stability datasets [ 112 , 113 ] revealed that neither uORF length nor re-initiation efficiency, but rather pausing translation is the main cause of TLS-stimulated mRNA decay [ 111 ].…”

“…Even ribosomal re-initiation at the CDS start after uORF termination does not necessarily protect the mRNAs from NMD [ 111 ]. Determination of reporter mRNA half-life time and mining available mRNA stability datasets [ 112 , 113 ] revealed that neither uORF length nor re-initiation efficiency, but rather pausing translation is the main cause of TLS-stimulated mRNA decay [ 111 ].…”