UC Riverside

Ribosome biogenesis is essential for protein synthesis in gene expression. Yeast eIF5B has been shown biochemically to facilitate 18S ribosomal RNA (rRNA) 3 end maturation during late-stage 40S ribosomal subunit assembly and gate the transition from translation initiation to elongation. But the genome-wide effects of eIF5B have not been studied at the single-nucleotide resolution in any organism, and 18S rRNA 3 end maturation is poorly understood in plants. Arabidopsis HOT3/eIF5B1 was found to promote development and heat stress acclimation by translational regulation, but its molecular function remained unknown. Here, we show that HOT3 is a late-stage ribosome biogenesis factor that facilitates 18S rRNA 3 end processing and is a translation initiation factor that globally impacts the transition from initiation to elongation. By developing and implementing 18S-ENDseq, we revealed previously unknown events in 18S rRNA 3 end maturation or metabolism. We quantitatively defined processing hotspots and identified adenylation as the prevalent nontemplated RNA addition at the 3 ends of pre-18S rRNAs. Aberrant 18S rRNA maturation in hot3 further activated RNA interference to generate RDR1- and DCL2/4-dependent risiRNAs mainly from a 3 portion of 18S rRNA. We further showed that risiRNAs in hot3 were predominantly localized in ribosome-free fractions and were not responsible for the 18S rRNA maturation or translation initiation defects in hot3. Our study uncovered the molecular function of HOT3/eIF5B1 in 18S rRNA maturation at the late 40S assembly stage and revealed the regulatory crosstalk among ribosome biogenesis, messenger RNA (mRNA) translation initiation, and siRNA biogenesis in plants.