LAUSR

Significance Translation initiation is tightly coupled with late-stage ribosome biogenesis in yeast and mammals by eIF5B. However, the global effects of eIF5B at single-nucleotide resolution have not been studied in any organism, and little is known about 18S ribosomal RNA (rRNA) maturation in plants. Here, by developing the quantitative 18S-ENDseq method, we defined the role of Arabidopsis HOT3/eIF5B1 in 18S rRNA 3′ end maturation. We uncovered the genome-wide role of HOT3 in gating the transition from translation initiation to elongation. Aberrant 18S rRNA maturation in hot3 was accompanied by the production of rRNA-derived siRNAs in an RDR1- and DCL2/DCL4-depedent manner. Our findings highlight the regulatory crosstalk among mRNA translation initiation, ribosome biogenesis, and siRNA biogenesis in plants.