Polyamine-Responsive Ribosomal Arrest at the Stop Codon of an Upstream Open Reading Frame of the AdoMetDC1 Gene Triggers Nonsense-Mediated mRNA Decay in Arabidopsis thaliana

Abstract

During mRNA translation, nascent peptides with certain specific sequences cause arrest of ribosomes that have synthesized themselves. In some cases, such ribosomal arrest is coupled with mRNA decay. In yeast, mRNA quality control systems have been shown to be involved in mRNA decay associated with ribosomal arrest. However, a link between ribosomal arrest and mRNA quality control systems has not been found in multicellular organisms. In this study, we aimed to explore the relationship between ribosomal arrest and mRNA decay in plants. For this purpose, we used an upstream open reading frame (uORF) of the Arabidopsis thaliana AdoMetDC1 gene, in which the uORF-encoded peptide is involved in polyamine-responsive translational repression of the main coding sequence. Our in vitro analyses revealed that the AdoMetDC1 uORF-encoded peptide caused ribosomal arrest at the uORF stop codon in response to polyamine. Using transgenic calli harboring an AdoMetDC1 uORF-containing reporter gene, we showed that polyamine promoted mRNA decay in a uORF sequence-dependent manner. These results suggest that the polyamine-responsive ribosomal arrest mediated by the uORF-encoded peptide is coupled with mRNA decay. Our results also showed that the polyamine-responsive acceleration of mRNA decay was compromised by defects in factors that are essential for nonsense-mediated mRNA decay (NMD), an mRNA quality control system that degrades mRNAs with premature stop codons, suggesting that NMD is involved in AdoMetDC1 uORF peptide-mediated mRNA decay. Collectively, these findings suggest that AdoMetDC1 uORF peptide-mediated ribosomal arrest at the uORF stop codon induces NMD.

Unique Aspects of Plant Nonsense-Mediated mRNA Decay

“…The conservation of CPuORFs peptides suggests that they are translated and, hence, may lead to NMD [51]. NMD is also triggered in rare cases in which the uORF peptide stalls the ribosome at its TC, as seen for example in the Arabidopsis AdoMetDC1 gene [54], and as previously identified in yeast (Saccharomyces cerevisiae) [55] and other eukaryotes. Although long uORFs are more likely to elicit NMD, even a short uORF of only 13 aa mediated efficient NMD of the Arabidopsis MHX gene [50], possibly because the efficiency of reinitiation following its translation was low [56].…”

“…Indeed, mammalian NMD targets were shown to be involved in various processes, including stem cell development, genomic stability, amino acid homeostasis, cell cycle, splicing, brain development, and stress responses (reviewed in [7,[76][77][78]). Similarly, NMD controls the transcript levels of many genes that play important roles in plant development (e.g., [11,54,[79][80][81], see also [82,83]). …”

Global Analysis of Truncated RNA Ends Reveals New Insights into Ribosome Stalling in Plants

“…Among them, the conserved peptide uORFs (CPuORFs) of yeast CPA1 and an Arabidopsis gene producing S-ADENOSYLMETHIONINE DECARBOXYLASE (SAMDC/ AdoMetDC1) have been demonstrated to induce mRNA decay through the NMD pathway (Gaba et al, 2005;Uchiyama-Kadokura et al, 2014). To provide additional evidence that the RNA degradome contains ribosome footprints, we examined the positional distribution of 59-truncated mRNA ends (PARE reads) derived from several CPuORFs of Arabidopsis.…”

“…The stop codon of a translated upstream open reading frame (uORF) might be recognized as a PTC and thus initiate NMD. Some uORFs encoding peptides conserved across species have been shown to stall ribosomes at uORF stop codons, resulting in the repression of downstream main ORF translation and acceleration of RNA degradation in a few cases (Gaba et al, 2005;Uchiyama-Kadokura et al, 2014).…”

An upstream open reading frame represses expression of a tomato homologue of Arabidopsis <i>ANAC096</i>, a NAC domain transcription factor gene, in a peptide sequence-dependent manner

“…However, some uORFs have been shown to repress translation of the main ORF in a peptide sequence-dependent manner (Ito and Chiba 2013;Morris and Geballe 2000). In this case, the nascent peptide encoded by a uORF acts inside the ribosome that had synthesized it to cause ribosome stalling, which results in translational repression of the main ORF (Cao and Geballe 1996;Law et al 2001;Uchiyama-Kadokura et al 2014;Wang and Sachs 1997;Wang et al 1999).…”