Expression of a Cell Cycle Regulator from a Bicistronic Transcript in Plants

Abstract

Eukaryotic mRNAs frequently contain upstream open reading frames (uORFs), encoding small peptides which may control the translation of the main ORF (mORF) by various mechanisms. We report the identification of a bicistronic transcript in Arabidopsis, in which the uORF encodes an ortholog of a cell cycle regulator previously thought to be absent from the plant lineage. While disruption of the mORF does not cause any major phenotype under our tested conditions, knock-out mutants of the uORF are embryo lethal. Immunoprecipitation assays followed by mass-spectrometry identified the uORF-encoded protein as a member of an E3-ubiquin ligase complex required for cell cycle progression. The two ORFs are translated together from a single transcript in all the plant lineage. Generation of specific antibodies against the two proteins allowed us to follow protein expression throughout the plant’s life cycle. Even though there is no apparent biochemical or physiological connection between the two gene products, concerted transcription and translation occurs in different organs. Indeed, downstream sequences of the bicistronic transcript (mORF and 3’UTR) mediate the translation of the uORF. Also, translation of the uORF promotes the degradation of its own transcript by nonsense-mediated decay (NMD), adding an additional layer of regulation. Our work reveals that uORFs may code for functional proteins not involved in the translational regulation of the mORF