Ribosome binding of phasiRNA precursors accelerates the 24-nt phasiRNA burst in meiotic maize anthers.

Reproductive phasiRNAs (phased, secondary, small interfering RNAs), produced from numerous PHAS loci, are essential for plant anther development. PHAS transcripts are enriched on endoplasmic reticulum-bound ribosomes in maize (Zea mays), but the impact of ribosome binding on phasiRNA biogenesis remains elusive. Through ribosome profiling of maize anthers at 10 developmental stages, we demonstrated that 24-PHAS transcripts are 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 maize inbred lines, with ribosomes enriched upstream of the miR2275 target sites. We detected short open reading frames (sORFs) in the ribosome-binding regions of some 24-PHAS transcripts and observed a 3-nt periodicity in most sORFs, but mass spectrometry failed to detect peptides corresponding to the sORFs. Deletion of the entire ribosome-binding region of 24PHAS_NO296 locus eliminated ribosome binding and decreased 24-nt phasiRNA production, without affecting 24PHAS_NO296 transcript levels. In contrast, disrupting only the sORFs in 24PHAS_NO296 did not substantially affect the generation of 24-nt phasiRNAs. A newly formed sORF in these mutants may have re-directed ribosome binding to its transcripts. Overall, these findings demonstrate that sORFs facilitate ribosome binding to 24-PHAS transcripts, thereby promoting phasiRNA biogenesis in meiotic anthers.