Plant nonsense-mediated mRNA decay is controlled by different autoregulatory circuits and can be induced by an EJC-like complex.

Nonsense-mediated mRNA decay (NMD) is a eukaryotic quality control system that recognizes and degrades transcripts containing NMD cis elements in their 3'untranslated region (UTR). In yeasts, unusually long 3'UTRs act as NMD cis elements, whereas in vertebrates, NMD is induced by introns located >50 nt downstream from the stop codon. In vertebrates, splicing leads to deposition of exon junction complex (EJC) onto the mRNA, and then 3'UTR-bound EJCs trigger NMD. It is proposed that this intron-based NMD is vertebrate specific, and it evolved to eliminate the misproducts of alternative splicing. Here, we provide evidence that similar EJC-mediated intron-based NMD functions in plants, suggesting that this type of NMD is evolutionary conserved. We demonstrate that in plants, like in vertebrates, introns located >50 nt from the stop induces NMD. We show that orthologs of all core EJC components are essential for intron-based plant NMD and that plant Partner of Y14 and mago (PYM) also acts as EJC disassembly factor. Moreover, we found that complex autoregulatory circuits control the activity of plant NMD. We demonstrate that expression of suppressor with morphogenic effect on genitalia (SMG)7, which is essential for long 3'UTR- and intron-based NMD, is regulated by both types of NMD, whereas expression of Barentsz EJC component is downregulated by intron-based NMD.

Expression and purification of active protein kinases from wheat germ extracts.

In vitro functional studies of eukaryotic kinases are often constrained by the availability of pure and -enzymatically active kinase of interest. Though numerous proteins have been synthesized by cell-based systems, in vivo production of properly folded, eukaryotic proteins remains a challenging task. Current wheat-germ-based cell-free in vitro translation systems present a plausible alternative for protein synthesis since majority of eukaryotic proteins could be obtained in their native folded form with general protocols. The use of special in vitro translation vectors with ligation-independent cloning sites and cleavable affinity tags eliminates further bottlenecks of the protein producing procedure and makes this system a reasonable method for simultaneous generation of active kinases.

Plant upstream ORFs can trigger nonsense-mediated mRNA decay in a size-dependent manner.

Nonsense-mediated decay (NMD) is a quality control mechanism that identifies and degrades aberrant mRNAs containing premature termination codons (PTC). NMD also regulates the expression of many wild-type genes. In plants, NMD identifies a stop codon as a PTC and initiates the rapid degradation of the transcript if the 3'untranslated region (UTR) is unusually long or if it harbors an intron. Approximately 20% of plant transcripts have an upstream ORF (uORF) in the 5'UTR. In theory, if a uORF is translated, the 3'UTR downstream of the uORF will be long and harbor introns, thus these transcripts might be degraded by NMD. Therefore, if uORFs can trigger NMD, uORF containing transcripts would be a major group of NMD regulated wild-type plant mRNAs. The aim of this study was to clarify whether plant uORFs could activate NMD. Here we demonstrate that plant uORFs induce NMD in a size-dependent manner, a 50 amino acid (aa) long uORF triggered NMD efficiently, whereas similar but shorter (31 and 15 aa long) uORFs failed to activate NMD response. We have found that only ~2% of annotated Arabidopsis genes contain a first uORF that is longer than 35 aa, thus we propose that NMD regulates only a small fraction of uORF containing transcripts. However, as mRNAs having uORF that is longer than the critical size are strongly overrepresented within the up-regulated transcripts of NMD deficient plants, it is likely that this subset of natural NMD targets induces NMD because of containing a relatively long translatable uORF.