Cis-directed cleavage and nonstoichiometric abundances of 21-nt reproductive phasiRNAs in grasses

Abstract

SummaryPost-transcriptional gene silencing in plants results from independent activities of diverse small RNA types. In anthers of grasses, hundreds of loci yield non-coding RNAs that are processed into 21- and 24-nt phased small interfering RNAs (phasiRNAs); these are triggered by miR2118 and miR2275.We characterized these “reproductive phasiRNAs” from rice panicles and anthers across seven developmental stages. Our computational analysis identified characteristics of the 21-nt reproductive phasiRNAs that impact their biogenesis, stability, and potential functions.We demonstrate that 21-nt reproductive phasiRNAs can function in cis to target their own precursors. We observed evidence of this cis regulatory activity in both rice (Oryza sativa) and maize (Zea mays). We validated this activity with evidence of cleavage and a resulting shift in the pattern of phasiRNA production.We characterize biases in phasiRNA biogenesis, demonstrating that the Pol II-derived “top” strand phasiRNAs are consistently higher abundance than the bottom strand. The first phasiRNA from each precursor overlaps the miR2118 target site, and this impacts phasiRNA accumulation or stability, evident in the weak accumulation of this phasiRNA position. Additional influences on this first phasiRNA duplex include the sequence composition and length, and we show that these factors impact Argonaute loading.

Several phased siRNA annotation methods can frequently misidentify 24 nucleotide siRNA-dominated PHAS loci

“…We examined all available angiosperm genomes on Phytozome (ver 12.1) for potential homologs of Alignment of the MIR2275 loci in species for which potential homologs could be identified shows strong conservation of the mature miRNA and miRNA* sequences (Supporting information Figure S2), suggesting that these loci evolved from a common ancestor. Importantly, because of the high specificity of miR2275 expression in developing anthers (Tamim et al, 2018), it is possible that our analysis includes false negatives, especially in situations where no reproductive tissue small RNA libraries were available.…”

“…Aside from the combination of their size and biogenesis patterns, 24 nt-dominated PHAS loci are distinct in various ways. These loci as well as their triggering miRNA, miR2275, are very specifically expressed in the tapetum during early meiosis and quickly recede in expression in other stages of male gametogenesis in rice and maize (Tamim et al, 2018). The AGO protein that loads these phasiRNAs is unknown; however, in maize, AGO18b expression levels match those of the 24 nt-dominated PHAS loci quite closely and is therefore the most likely candidate to load 24 nt phasiRNAs (Komiya et al, 2014;Zhang, Xia, Meyers, & Walbot, 2015).…”

Several phased siRNA annotation methods can frequently misidentify 24 nucleotide siRNA‐dominated PHAS loci

“…Alignment of the MIR2275 loci in species for which potential homologs could be identified shows strong conservation of the mature miRNA and miRNA* sequences (Supporting information Figure ), suggesting that these loci evolved from a common ancestor. Importantly, because of the high specificity of miR2275 expression in developing anthers (Tamim et al., ), it is possible that our analysis includes false negatives, especially in situations where no reproductive tissue small RNA libraries were available. Altogether, our data suggest that miR2275 is not found in A. thaliana and that this loss apparently occurred before the last common ancestor for Brassicales.…”

“…Aside from the combination of their size and biogenesis patterns, 24 nt‐dominated PHAS loci are distinct in various ways. These loci as well as their triggering miRNA, miR2275, are very specifically expressed in the tapetum during early meiosis and quickly recede in expression in other stages of male gametogenesis in rice and maize (Tamim et al., ). The AGO protein that loads these phasiRNAs is unknown; however, in maize, AGO18b expression levels match those of the 24 nt‐dominated PHAS loci quite closely and is therefore the most likely candidate to load 24 nt phasiRNAs (Komiya et al., ; Zhang, Xia, Meyers, & Walbot, ).…”

Plant non-coding RNAs function in pollen development and male sterility

“…miR2275 is the unique miRNA known to trigger the production of 24-nt-dominated phasiRNAs, and all of these trigger events preferentially occurred in the tapetum and meiocytes. miR2275 is specifically expressed in the tapetum during the early meiosis stage, and its expression is quickly reduced in other stages of male gametogenesis in both rice and maize ( Tamim et al., 2018 ). In maize, the tapetum is the primary site of the 24-PHAS precursor and Dcl5 transcripts, as well as the resulting 24-nt phasiRNAs, and then the phasiRNAs are mobile from the tapetum to meiocytes and to other somatic cells ( Zhou et al., 2022 ).…”