RNA processing/modifying enzymes play key roles in the response to thermospermine in Arabidopsis thaliana

Thermospermine acts in negative regulation of xylem differentiation through enhancing mRNA translation of the members of the SAC51 gene family in Arabidopsis. These mRNAs contain conserved upstream open-reading-frames (uORFs) that are inhibitory to the main ORF translation. To address the mode of action of thermospermine in this process, we have isolated mutants that are insensitive to thermospermine, named its. We show here that four genes responsible for the mutants, its1 to its4, encode a homologue of SPOUT RNA methyl transferase, an rRNA pseudouridine synthase CBF5/NAP57, a putative spliceosome disassembly factor STIPL1/NTR1, and a plant-specific RNA-binding protein PHIP1, respectively. While these mutants except its1 are almost normal in appearance, they enhance the dwarf phenotype of a mutant of ACL5 defective in thermospermine biosynthesis, resulting in tiny-sized plants reminiscent of a double knockout of ACL5 and SACL3, a member of the SAC51 family. We confirmed that the GUS reporter activity from the SAC51 5-GUS fusion transcript was severely reduced in all of these mutants. These results unveil the importance of RNA processing and modification for the translation of transcripts containing regulatory uORFs.