Nonsense‐mediated mRNA decay (NMD) is a quality control system that degrades mRNAs containing premature termination codons. Although NMD is well characterized in yeast and mammals, plant NMD is poorly understood. We have undertaken the functional dissection of NMD pathways in plants. Using an approach that allows rapid identification of plant NMD trans factors, we demonstrated that two plant NMD pathways coexist, one eliminates mRNAs with long 3′UTRs, whereas a distinct pathway degrades mRNAs harbouring 3′UTR‐located introns. We showed that UPF1, UPF2 and SMG‐7 are involved in both plant NMD pathways, whereas Mago and Y14 are required only for intron‐based NMD. The molecular mechanism of long 3′UTR‐based plant NMD resembled yeast NMD, whereas the intron‐based NMD was similar to mammalian NMD, suggesting that both pathways are evolutionarily conserved. Interestingly, the SMG‐7 NMD component is targeted by NMD, suggesting that plant NMD is autoregulated. We propose that a complex, autoregulated NMD mechanism operated in stem eukaryotes, and that despite aspect of the mechanism being simplified in different lineages, feedback regulation was retained in all kingdoms.

Germline transgenesis is an important procedure for functional investigation of biological pathways, as well as for animal biotechnology. We have established a simple, nonviral protocol in three important biomedical model organisms frequently used in physiological studies. The protocol is based on the hyperactive Sleeping Beauty transposon system, SB100X, which reproducibly promoted generation of transgenic founders at frequencies of 50–64, 14–72, and 15% in mice, rats, and rabbits, respectively. The SB100X‐mediated transgene integrations are less prone to genetic mosaicism and gene silencing as compared to either the classical pronuclear injection or to lentivirus‐mediated transgenesis. The method was successfully applied to a variety of transgenes and animal models, and can be used to generate founders with single‐copy integrations. The transposon vector also allows the generation of transgenic lines with tissue‐specific expression patterns specified by promoter elements of choice, exemplified by a rat reporter strain useful for tracking serotonergic neurons. As a proof of principle, we rescued an inborn genetic defect in the fawn‐hooded hypertensive rat by SB100X transgenesis. A side‐by‐side comparison of the SB100X‐ and piggyBac ‐based protocols revealed that the two systems are complementary, offering new opportunities in genome manipulation.—Katter, K., Geurts, A. M., Hoffmann, O., Mátés, L., Landa, V., Hiripi, L., Moreno, C., Lazar, J., Bashir, S., Zidek, V., Popova, E., Jerchow, B., Becker, K., Devaraj, A., Walter, I., Grzybowksi, M., Corbett, M., Rangel Filho, A., Hodges, M. R., Bader, M., Ivics, Z., Jacob, H. J., Pravenec, M., Bősze, Z., Rülicke, T., Izsvák, Z. Transposon‐mediated transgenesis, transgenic rescue, and tissue‐specific gene expression in rodents and rabbits. FASEB J. 27, 930–941 (2013). www.fasebj.org

Summary: Nonsense‐mediated mRNA decay (NMD) is a eukaryotic quality control system that identifies and degrades mRNAs containing premature termination codons (PTCs). If translation terminates at a PTC, the UPF1 NMD factor binds the terminating ribosome and recruits UPF2 and UPF3 to form a functional NMD complex, which triggers the rapid decay of the PTC‐containing transcript. Although NMD deficiency is seedling lethal in plants, the mechanism of plant NMD remains poorly understood. To understand how the formation of the NMD complex leads to transcript decay we functionally mapped the UPF1 and SMG7 plant NMD factors, the putative key players of NMD target degradation. Our data indicate that the cysteine–histidine‐rich (CH) and helicase domains of UPF1 are only essential for the early steps of NMD, whereas the heavily phosphorylated N‐ and C–terminal regions play a redundant but essential role in the target transcript degradation steps of NMD. We also show that both the N‐ and the C–terminal regions of SMG7 are essential for NMD. The N terminus contains a phosphoserine‐binding domain that is required for the early steps of NMD, whereas the C terminus is required to trigger the degradation of NMD target transcripts. Moreover, SMG7 is a P–body component that can also remobilize UPF1 from the cytoplasm into processing bodies (P bodies). We propose that the N‐ and C–terminal phosphorylated regions of UPF1 recruit SMG7 to the functional NMD complex, and then SMG7 transports the PTC‐containing transcripts into P bodies for degradation.