uORF-mediated post-transcriptional control of stem cell niche activity in plants – ReinitiaTOR

The recent development of mRNA sequencing techniques for obtaining ribosome footprints (RFs) in plants has changed protein synthesis studies. Using this method, we performed precise mapping of ribosome positions on mRNAs to follow their dynamics in different TOR contrasting conditions. Polyribosomes were obtained by immunopurification from transgenic Arabidopsis seedlings expressing tagged ribosomal protein L18 integrated into functional ribosomes and placed under specific promoters marking the shoot apical meristem, SAM, and SAM regions. We compared the number and position of ribosomes on total and polysomal mRNAs. RF profiling revealed an inhibitory effect of uORF on mRNA translation in TOR inactivation conditions. The effect of TOR on translation was further confirmed by several techniques—analysis of endogenous mRNA levels in polyribosomes, mRNA translation levels in mesophyll protoplasts in different TOR contrasting conditions, confocal microscopy image analysis of protein levels and SAM activity. Bringing together, we present an experimental approach that highlights the regulation of protein synthesis under conditions of energy limitation.

We used the uORF mRNA library to select uORFs with an inhibitory effect on mRNA translation that is attenuated by TOR activation. Analysis of the reinitiation mechanism allowed us to identify the RACK1 protein as a key player in translation reinitiation. We have discovered a new upstream TOR effector, spermidine, which activates TOR and enhances translation of uORF mRNAs. Moreover, TOR negatively regulates spermidine metabolism during the establishment of maize and Arabidopsis seedlings. TOR suppresses stress responses through global regulation of H3K27 trimethylation deposited on chromatin by CURLY LEAF (CLF), encoded by uORF mRNA

We have shown for the first time that TOR, by sensing spermidine sufficiency, can regulate spermidine homeostasis by facilitating reinitiation of translation of uORF-containing mRNAs encoding enzymes that degrade polyamines. In a future, combining our approach with direct analysis of endogenous levels of spermidine and other polyamines will expand our understanding of how polyamine homeostasis is regulated by TOR.
We demonstrated that TOR regulates translation reinitiation by interacting with factors of the cellular mechanism of translation. The new reinitiation factor, RACK1, was identified. The mechanism of action of translation reinitiation factors in this study is not fully understood, and we continue to study interactions and functions of these proteins in order to control them. Combining biochemical approaches with direct global profiling mRNA analysis will allow for a complete set of mRNAs under the control of TOR.

Dong Y, Ryabova LA.* Do plants drive translation reinitiation to dodge nonsense-mediated decay? 2023. J Exp Bot. 74(1):7-11.
Martin-Arevalillo R,Vernoux T. Decoding the auxin matrix. Auxin biology through the eye of the computer (2023) Annu Rev Plant Biol. 74:387-413
Dong Y., Uslu V., Berr A., Singh G., Papdi C., Steffens V., Heitz T., Ryabova L.* (2021) TOR represses stress responses through global regulation of H3K27 trimethylation in plants. Journal of Experimental Botany J Exp Bot. 74(5):1420-1431.
Salazar-Díaz K., Dong Y., Papdi C., Ferruzca-Rubio E., Olea-Badillo G., Ryabova L.*, Dinkova T.* 2021. TOR senses and regulates spermidine metabolism during seedling establishment and growth in maize and Arabidopsis. iScience, 24 : 103260.
Mancera-Martínez E, Dong Y, Makarian J, Srour O, Thiébeauld O, Jamsheer M, Chicher J, Hammann P, Schepetilnikov M, Ryabova LA.* 2021. Phosphorylation of a reinitiation supporting protein, RISP, determines its function in translation reinitiation. Nucleic Acids Res. 49 : 6908-6924.
Galvan-Ampudia CS, Cerutti G, Legrand J, Brunoud G, Martin-Arevalillo R, Azais R, Bayle V, Moussu S, Wenzl C, Jaillais Y, Lohmann JU, Godin C, Vernoux T (2020) Elife 9: e55832.

Growth and regeneration in all organisms depends on a proper balance between stem cell division and differentiation. Despite extensive study of the transcriptional and epigenetic networks that control stem cell populations, mechanisms of post-transcriptional regulation in the stem cell niche remain unknown. Translation is a critical step in controlling gene activity; upstream ORFs (uORFs) located within the leaders of many mRNAs act as prevalent translation repressors that, in plants, are under control of TOR (target of rapamycin)—a central controller of growth. Plant stem cell mRNAs are heavily enriched in uORFs, and we aim to study uORF-responsive translation mechanisms and their control by TOR in the shoot apical meristem—a specialized tissue containing a stem cell niche and responsible for building shoots. TOR is also controlled by the plant hormone auxin, suggesting a TOR-dependent function in controlling protein levels of stem cell regulators that will be tested in this project. We predict that TOR by de-repressing translation of pre-existing mRNAs of key meristematic factors downstream of auxin would allow rapid modulation of the stem cell niche activity during development, a key hypothesis that will be tested in this project.
The main objectives of the project are four-fold :
1. Screening for meristematic mRNAs that are under translation repression by uORFs, and determining the role of TOR in activation of their translation
2. Deciphering the mechanism of eIF3h function in TOR-responsive reinitiation of translation
3. Understanding the impact of uORF suppressors on SAM size, structure, growth and organogenesis in different TOR contrasting conditions
4. Analyzing the TOR-dependent function of auxin in regulation of stem cell niche regulator translation
We expect to discover and analyse meristem-related genes with translatable uORFs under post-transcriptional control of the auxin-TOR signaling axis. This project is expected to provide fundamental knowledge on emerging translation reinitiation mechanisms in plants that would help to understand these translation strategies in animals. Indeed, given the high conservation of TOR, understanding how TOR impacts stem cell development would provide us with potential tools to manipulate stem cell niche activity in plant and also in animal cells. In humans, this could prove to be essential for fighting tumorigenesis, and, in plants, recent data have shown that regulation of stem cell niche activity has a strong potential for the manipulation of yield.