Ribosome binding is an accelerator to the burst of 24-nt phasiRNAs in meiotic anthers

Phased secondary small interfering RNAs (phasiRNAs) are essential for anther development in the grass family. Many phasiRNAs are generated on endoplasmic reticulum (ER)-bound ribosomes in maize (Zea mays), but the general patterns and functions of ribosome binding remain elusive. Through ribosome profiling using maize anthers at 5 developmental stages, we demonstrated that 24-nt phasiRNA precursors (24-PHAS) are widely bound by ribosomes, with patterns corresponding to the timing and abundance of 24-PHAS transcripts. Ribosome binding to 24-PHAS transcripts is conserved among different inbred lines. Deletion of the entire ribosome binding region of 24PHAS_NO296 eliminated ribosome binding and decreased 24-nt phasiRNA production, without affecting 24PHAS_NO296 transcript levels. Deletion of the sORFs of 24PHAS_NO296 and 24PHAS_NO196 impacted generation of 24-nt phasiRNAs less severely, while a 1-bp frameshift causing premature termination produced no significant changes in 24-nt phasiRNA levels.