<scp>RNA</scp> processing/modifying enzymes play key roles in the response to thermospermine in <i>Arabidopsis thaliana</i>

Thermospermine is involved in negative regulation of xylem differentiation by enhancing the translation of mRNAs of the SAC51 gene family in Arabidopsis (Arabidopsis thaliana). These mRNAs contain conserved upstream open reading frames (uORFs) that interfere with the translation of the main ORF. To investigate the mechanism by which thermospermine acts in this process, we isolated mutants insensitive to thermospermine, named 'its'. We show that the four genes responsible for these mutants, its1 to its4, encode: (i) a homolog of SPOUT RNA methyltransferase, (ii) an rRNA pseudouridine synthase CBF5/NAP57, (iii) a putative spliceosome disassembly factor STIPL1/NTR1, and (iv) a plant-specific RNA-binding protein PHIP1. These four mutants were found to have much higher levels of thermospermine than the wild-type. While all these mutants except its1 appear almost normal, they enhance the dwarf phenotype of a mutant of ACL5, which encodes thermospermine synthase, resulting in tiny plants resembling a double knockout of ACL5 and SACL3, a member of the SAC51 family. Reporter assays revealed that GUS activity from the CaMV 35S promoter-SAC51 5'-GUS fusion construct was significantly reduced in its1 and its4 or not affected in its2 and its3, while it was slightly increased in its1, its3, and its4, or not changed in its2 by thermospermine. These findings underscore the critical role of RNA processing and modification in the thermospermine-dependent translational regulation of uORF-containing transcripts.