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Concerted expression of a cell cycle regulator and a metabolic enzyme from a bicistronic transcript in plants

Abstract

Eukaryotic mRNAs frequently contain upstream open reading frames (uORFs), encoding small peptides that may control translation of the main ORF (mORF). Here, we report the characterization of a distinct bicistronic transcript in Arabidopsis. We analysed loss-of-function phenotypes of the inorganic polyphosphatase TRIPHOSPHATE TUNNEL METALLOENZYME 3 (AtTTM3), and found that catalytically inactive versions of the enzyme could fully complement embryo and growth-related phenotypes. We could rationalize these puzzling findings by characterizing a uORF in the AtTTM3 locus encoding CELL DIVISION CYCLE PROTEIN 26 (CDC26), an orthologue of the cell cycle regulator. We demonstrate that AtCDC26 is part of the plant anaphase promoting complex/cyclosome (APC/C), regulates accumulation of APC/C target proteins and controls cell division, growth and embryo development. AtCDC26 and AtTTM3 are translated from a single transcript conserved across the plant lineage. While there is no apparent biochemical connection between the two gene products, AtTTM3 coordinates AtCDC26 translation by recruiting the transcript into polysomes. Our work highlights that uORFs may encode functional proteins in plant genomes.

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Fig. 1: Different ttm3 mutant alleles show inconsistent phenotypes related to embryo development and plant growth.
Fig. 2: Catalytically inactive TTM3 variants complement ttm3 phenotypes when expressed from the native promoter including the 5′ UTR.
Fig. 3: A uORF in TTM3 encodes the cell cycle regulator AtCDC26.
Fig. 4: CDC26 is expressed from a bicistronic transcript in plants.
Fig. 5: TTM3 translation recruits the bicistronic transcript to polysomes.
Fig. 6: The CDC26TTM3 transcript is a target of NMD.

Data availability

The authors confirm that all relevant data have been included in this paper and are available upon reasonable request. The Arabidopsis mutant accessions ttm3-1 (SALK_133625) and ttm3-4 (SALK_050319) are available at the Nottingham Arabidopsis Stock Center (NASC; http://arabidopsis.info/). ttm3-2 (FLAG_368E06, EMBL number AJ838411) is available at the Versailles Arabidopsis Stock Center (http://publiclines.versailles.inra.fr/).

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Acknowledgements

The authors thank J. M. Perez-Perez for sending the CYCB1;1-GFP line, A. Wachter for lba1 and upf3-1 seeds, and R. Ulm, A. Wachter and N. Geldner for critical reading of the manuscript. This project was supported by an ERC starting grant from the European Research Council under the European Union’s Seventh Framework Programme (FP/2007–2013)/ERC Grant Agreement no. 310856, the Max Planck Society, the European Molecular Biology Organisation (EMBO) Young Investigator Programme (to M.H.), the Howard Hughes Medical Institute (International Research Scholar Award to M.H.) and the Swiss National Fund Sinergia grant (CRSII3-154471 to Y.P.).

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L.L.-O. and M.H. designed the study, L.L.-O. performed the majority of the experiments and analysed data. J.W. and A.P. characterized the ttm3 insertion lines, the ttm3-2 embryo phenotype, performed localization experiments and produced the TTM3-specific antibody. J.D. and L.L.-O. designed and performed the polysome profile assays under the supervision of Y.P. J.M. together with L.L.-O. purified the AtCDC26 protein and antibody. S.L. quantified CYCB1;1 levels, Y.J. generated transgenic reporter lines and L.A.H. performed statistical analyses. L.L.-O., J.W., S.L., L.A.H. and M.H. analysed data, and M.H. supervised the study. L.L.-O. and M.H. drafted the manuscript and all authors discussed the results, edited and approved the final version of the manuscript.

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Correspondence to Laura Lorenzo-Orts or Michael Hothorn.

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Lorenzo-Orts, L., Witthoeft, J., Deforges, J. et al. Concerted expression of a cell cycle regulator and a metabolic enzyme from a bicistronic transcript in plants. Nature Plants 5, 184–193 (2019). https://doi.org/10.1038/s41477-019-0358-3

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