Résumé
Ribosomal RNAs (rRNAs) are the structural and functional building blocks of ribosomes (reviewed in 1). In the nucleolus, rRNA 18S, 5.8S and 25S (28S in mammals) are transcribed by RNA polymerase I (Pol I) as polycistronic 35S, 45S or 47S pre-rRNAs respectively in yeast, plant and mammals. Therefore, these pre-rRNAs must be processed into mature 18S, 5.8S, and 25S/28S rRNAs. Processing consists on the one hand of exo- and endonucleolytic cleavages to remove Internal (ITS1 and ITS2) and External (5’ETS and 3’ETS) Transcribed Spacer sequences and on the other hand of sugar and base modifications of rRNA at specific positions 2-4. While these processes are severely impacted by stress conditions in multiple species, we lack information about the molecular mechanisms allowing sessile organisms without a temperature-control system, like plants, to cope with such circumstances. Here we show that heat stress disturbs nucleolar structure, inhibits pre-rRNA processing and provokes imbalanced ribosome profiles in Arabidopsis thaliana plants. Notably, accuracy of transcription initiation and cleavage at the primary P site in the 5’ETS (5’ External Transcribed Spacer) are not affected but the levels of primary 45S and pre-35S transcripts are respectively increased and reduced. In contrast, precursors of 18S, 5.8S and 25S RNAs are rapidly undetectable upon heat stress5. Concomitant, specific RNA methylation changes occur as well in response to heat stress, including associated rRNAs modifications m7G, m6A and A. Remarkably, nucleolar structure, pre-rRNAs processing and ribosome profiles are restored after returning to optimal conditions, shedding light on the extreme plasticity of nucleolar functions in plant cells. Further genetic and molecular analysis to identify molecular clues implicated in these nucleolar responses indicate that cleavage rate at P site and nucleolin protein expression can act as a checkpoint towards a productive pre-rRNA processing pathway. 1Sáez-Vásquez, J. & Delseny, M. Ribosome Biogenesis in Plants: From Functional 45S Ribosomal DNA Organization to Ribosome Assembly Factors. Plant Cell 31, 1945-1967, doi:10.1105/TPC.18.00874 (2019).2Azevedo-Favory, J. et al. Mapping rRNA 2'-O-methylations and identification of C/D snoRNAs in Arabidopsis thaliana plants. RNA Biol, 1-18, doi:10.1080/15476286.2020.1869892 (2021).3Sharma, S. & Lafontaine, D. L. 'View From A Bridge': A New Perspective on Eukaryotic rRNA Base Modification. Trends Biochem Sci 40, 560-575, doi:10.1016/j.tibs.2015.07.008 (2015).4Tomecki, R., Sikorski, P. J. & Zakrzewska-Placzek, M. Comparison of preribosomal RNA processing pathways in yeast, plant and human cells - focus on coordinated action of endo- and exoribonucleases. FEBS Lett 591, 1801-1850, doi:10.1002/1873-3468.12682 (2017).5- Darriere T., Jobet E., Zavala D., Escande M.L., Durut N., de Bures A., Blanco-Herrera F., Vidal E.A., Rompais M., Carapito C., Gourbiere S. and Sáez-Vásquez J. Upon heat stress processing of ribosomal RNA precursors into mature rRNAs is compromised after cleavage at primary P site in Arabidopsis thaliana. RNA Biology doi.org/10.1080/15476286.2022.2071517