GSE99805
The Nonstop decay and the RNA silencing systems operate cooperatively in plants
Translation-dependent mRNA quality control systems protect the protein homeostasis of eukaryotic cells by eliminating aberrant transcripts and stimulating the decay of their protein products. Although these systems are intensively studied in animals, little is known about the translation-dependent quality control systems in plants. Here we characterize the mechanism of nonstop decay (NSD) system in Nicotiana benthamiana model plant. We show that plant NSD efficiently degrades nonstop mRNAs, which can be generated by premature polyadenylation, and stop codon-less transcripts, which can be produced by endonucleolytic cleavage. We demonstrate that in plants, like in animals, Pelota, Hbs1 and SKI2 proteins are required for NSD, supporting that NSD is an ancient and conserved eukaryotic quality control system. Relevantly, we found that NSD and RNA silencing systems cooperate in plants. Plant silencing predominantly represses target mRNAs through endonucleolytic cleavage in the coding region. The cleavage products are degraded by general decay systems or subjected to silencing amplification. The balance between decay and silencing amplification might be finely regulated by unknown mechanisms. Here we show that NSD is required for the elimination of 5'cleavage product when mi- or siRNA-guided silencing complex cleaves in the coding region. Thus NSD might regulate the frequency of silencing amplification. Overall design: To verify if NSD is also involved in the degradation of 5' cleavage products of endogenous miRNA targets, RNA-seq libraries (in triplicates) were prepared with random primers from ribo-depleted RNA samples, which were isolated from Pelota- and Control silenced N. benthamiana leaves (RNA-seq1 and RNA-seq2 experiments) and from wild type (Columbia) and pelota1 and hbs1 T-DNA mutant Arabidopsis thaliana leaves (Arabidopsis RNA-seq). N. benthamiana sequencing was repeated twice.