Upon heat stress processing of ribosomal RNA precursors into mature rRNAs is compromised after cleavage at primary P site in Arabidopsis thaliana.

Transcription and processing of 45S rRNAs in the nucleolus are keystones of ribosome biogenesis. While these processes are severely impacted by stress conditions in multiple species, primarily upon heat exposure, we lack information about the molecular mechanisms allowing sessile organisms without a temperature-control system, like plants, to cope with such circumstances. We show that heat stress disturbs nucleolar structure, inhibits pre-rRNA processing and provokes imbalanced ribosome profiles in Arabidopsis thaliana plants. Notably, the 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 35S transcripts are, respectively, increased and reduced. In contrast, precursors of 18S, 5.8S and 25S RNAs are rapidly undetectable upon heat stress. Remarkably, nucleolar structure, pre-rRNAs from major ITS1 processing pathway 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 control towards a productive pre-rRNA processing pathway.

Mapping rRNA 2'-O-methylations and identification of C/D snoRNAs in Arabidopsis thaliana plants.

In all eukaryotic cells, the most abundant modification of ribosomal RNA (rRNA) is methylation at the ribose moiety (2'-O-methylation). Ribose methylation at specific rRNA sites is guided by small nucleolar RNAs (snoRNAs) of C/D-box type (C/D snoRNA) and achieved by the methyltransferase Fibrillarin (FIB). Here we used the Illumina-based RiboMethSeq approach for mapping rRNA 2'-O-methylation sites in A. thaliana Col-0 (WT) plants. This analysis detected novel C/D snoRNA-guided rRNA 2'-O-methylation positions and also some orphan sites without a matching C/D snoRNA. Furthermore, immunoprecipitation of Arabidopsis FIB2 identified and demonstrated expression of C/D snoRNAs corresponding to majority of mapped rRNA sites. On the other hand, we show that disruption of Arabidopsis Nucleolin 1 gene (NUC1), encoding a major nucleolar protein, decreases 2'-O-methylation at specific rRNA sites suggesting functional/structural interconnections of 2'-O-methylation with nucleolus organization and plant development. Finally, based on our findings and existent database sets, we introduce a new nomenclature system for C/D snoRNA in Arabidopsis plants.