PT  - JOURNAL ARTICLE
AU  - Paul J. Russell
AU  - Jacob A. Slivka
AU  - Elaina P. Boyle
AU  - Arthur H. M. Burghes
AU  - Michael G. Kearse
TI  - Small uORFs favor translation re-initiation but do not protect mRNAs from nonsense-mediated decay
AID  - 10.1101/2022.01.10.475702
DP  - 2022 Jan 01
TA  - bioRxiv
PG  - 2022.01.10.475702
4099  - http://biorxiv.org/content/early/2022/01/10/2022.01.10.475702.short
4100  - http://biorxiv.org/content/early/2022/01/10/2022.01.10.475702.full
AB  - 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 5′ 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 5′ 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.Competing Interest StatementThe authors have declared no competing interest.