Plant transcripts with long or structured upstream open reading frames in the NDL2 5' UTR can escape nonsense-mediated mRNA decay in a reinitiation-independent manner.

Many eukaryotic transcripts contain upstream open reading frames (uORFs). Translated uORFs can inhibit the translation of main ORFs by imposing the need for reinitiation of translation. Translated uORFs can also lead to transcript degradation by the nonsense-mediated mRNA decay (NMD) pathway. In mammalian cells, translated uORFs were shown to target their transcripts to NMD if the uORFs were long (>23-32 amino acids), structured, or inhibit reinitiation. Reinitiation was shown to rescue uORF-containing mammalian transcripts from NMD. Much less is known about the significance of the length, structure, and reinitiation efficiency of translated uORFs for NMD targeting in plants. Although high-throughput studies suggested that uORFs do not globally reduce plant transcript abundance, it was not clear whether this was due to NMD-escape-permitting parameters of uORF recognition, length, structure, or reinitiation efficiency. We expressed in Arabidopsis reporter genes that included NDL2 5' untranslated region and various uORFs with modulation of the above parameters. We found that transcripts can escape NMD in plants even when they include efficiently translated uORFs up to 70 amino acids long, or structured uORFs, in the absence of reinitiation. These data highlight an apparent difference between the rules that govern the exposure of uORF-containing transcripts to NMD in mammalian and plant cells.

The Arabidopsis NMD Factor UPF3 Is Feedback-Regulated at Multiple Levels and Plays a Role in Plant Response to Salt Stress.

Nonsense-mediated mRNA decay (NMD) is a eukaryotic RNA surveillance mechanism that degrades aberrant transcripts and controls the levels of many normal mRNAs. It was shown that balanced expression of the NMD factor UPF3 is essential for the maintenance of proper NMD homeostasis in Arabidopsis. UPF3 expression is controlled by a negative feedback loop that exposes UPF3 transcript to NMD. It was shown that the long 3' untranslated region (3' UTR) of UPF3 exposes its transcript to NMD. Long 3' UTRs that subject their transcripts to NMD were identified in several eukaryotic NMD factors. Interestingly, we show here that a construct that contains all the regulatory regions of the UPF3 gene except this long 3' UTR is also feedback-regulated by NMD. This indicates that UPF3 expression is feedback-regulated at multiple levels. UPF3 is constitutively expressed in different plant tissues, and its expression is equal in leaves of plants of different ages. This finding is in agreement with the possibility that UPF3 is ubiquitously operative in the Arabidopsis NMD pathway. Expression mediated by the regulatory regions of UPF3 is significantly induced by salt stress. We found that both a deficiency and a strong excess of UPF3 expression are detrimental to plant resistance to salt stress. This indicates that UPF3 plays a role in plant response to salt stress, and that balanced expression of the UPF3 gene is essential for coping with this stress.