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 are 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. Here we show that NSD is required for the elimination of 5′ cleavage product of mi- or siRNA-guided silencing complex when the cleavage occurs in the coding region. We also show that NSD and nonsense-mediated decay (NMD) quality control systems operate independently in plants.

RNA quality control systems identify and degrade aberrant mRNAs, thereby preventing the accumulation of faulty proteins. Non-stop decay (NSD) and No-go decay (NGD) are closely related RNA quality control systems that act during translation. NSD degrades mRNAs lacking a stop codon, while NGD recognizes and decays mRNAs that contain translation elongation inhibitory structures. NGD has been intensively studied in yeast and animals but it has not been described in plants yet. In yeast, NGD is induced if the elongating ribosome is stalled by a strong inhibitory structure. Then, the mRNA is cleaved by an unknown nuclease and the cleavage fragments are degraded. Here we show that NGD also operates in plant. We tested several potential NGD cis-elements and found that in plants, unlike in yeast, only long A-stretches induce NGD. These long A-stretches trigger endonucleolytic cleavage, and then the 5' fragments are degraded in a Pelota-, HBS1- and SKI2- dependent manner, while XRN4 eliminates the 3' fragment. We also show that plant NGD operates gradually, the longer the A-stretch, the more efficient the cleavage. Our data suggest that mechanistically NGD is conserved in eukaryotes, although the NGD inducing cis-elements could be different. Moreover, we found that Arabidopsis AtPelota1 functions in both NGD and NSD, while AtPelota2 represses these quality control systems. The function of plant NGD will be discussed.

Elongation factor TFIIS (transcription factor IIS) is structurally and biochemically probably the best characterized elongation cofactor of RNA polymerase II. However, little is known about TFIIS regulation or its roles during stress responses. Here, we show that, although TFIIS seems unnecessary under optimal conditions in Arabidopsis, its absence renders plants supersensitive to heat; tfIIs mutants die even when exposed to sublethal high temperature. TFIIS activity is required for thermal adaptation throughout the whole life cycle of plants, ensuring both survival and reproductive success. By employing a transcriptome analysis, we unravel that the absence of TFIIS makes transcriptional reprogramming sluggish, and affects expression and alternative splicing pattern of hundreds of heat-regulated transcripts. Transcriptome changes indirectly cause proteotoxic stress and deterioration of cellular pathways, including photosynthesis, which finally leads to lethality. Contrary to expectations of being constantly present to support transcription, we show that TFIIS is dynamically regulated. TFIIS accumulation during heat occurs in evolutionary distant species, including the unicellular alga Chlamydomonas reinhardtii, dicot Brassica napus and monocot Hordeum vulgare, suggesting that the vital role of TFIIS in stress adaptation of plants is conserved.