Cis‐directed cleavage and nonstoichiometric abundances of 21‐nucleotide reproductive phased small interfering RNAs in grasses

Abstract

Post-transcriptional gene silencing in plants results from independent activities of diverse small RNA types. In anthers of grasses, hundreds of loci yield noncoding RNAs that are processed into 21- and 24-nucleotide (nt) phased small interfering RNAs (phasiRNAs); these are triggered by miR2118 and miR2275. We characterized these 'reproductive phasiRNAs' from rice (Oryza sativa) 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 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 in 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.

Reproductive phasiRNAs in grasses are compositionally distinct from other classes of small RNAs

“…As observed for animal miRNAs (Chatterjee et al ., ; Tamim et al ., ), nonstoichiometric abundances at a PHAS locus may result from AGO loading and subsequent stabilization of functional siRNAs. At a given PHAS locus, some phasiRNAs are never observed in the sequenced sRNAs; we could extract these computationally.…”

“…Dukowic-Schulze et al (2016) reported that DNA methylation increased at 24-PHAS loci in maize meiocytes, and described a potential role in the regulation of chromosomal structure to promote normal meiosis. As reproductive phasiRNAs are extraordinarily abundant, cis methylation or even cis cleavage could result from nonspecific misloading of a small proportion into the 'wrong' AGO (Tamim et al, 2018). The pairing rules for 24-nt sRNAs and their targets is still unknown, that is, whether standard target prediction tools can be used that were built for 21-and 22-nt sRNAs.…”

sRNAanno --- a database repository of uniformly-annotated small RNAs in plants

“…For instance, phasiRNAs are profusely produced during the reproductive stage, especially in the monocots and their subgroups of grasses (Xia et al, 2017)(Zhai et al, 2015). In rice there are >2000 PHAS loci generating 21-nt phasiRNAs and ∼400 loci 24-nt phasiRNAs (Tamim et al, 2018). Although phasiRNAs, including the well-known tasiRNAs, have been characterized in more and more plant species, there is no public online repository collecting reported or annotated phasiRNAs and provide convenient and quick access to this information.…”

Evolution of PHAS loci in the young spike of Allohexaploid wheat

“…In rice inflorescence, 828 and 35 of 21-and 24-PHAS were identified by Johnson et al [3], which could produce 21-and 24-nt phasiRNAs, respectively. In addition, 1136 and 1540 of 21-PHAS were identified in 93-11 and Nipponbare, respectively [9]. 70 and 34 of 24-PHAS were identified in 93-11 and Nipponbare by Song et al [16], respectively.…”

“…The genes encoding PIWI-interacting RNAs (piRNAs), which are a type of phasiRNAs, are required for producing mature sperm in animals [4]. In plants, phased siRNAs play a series of roles in abiotic and biotic stresses [5,6], seed germination [7] and reproductive development [3,[8][9][10]. Trans-acting siRNAs (ta-siRNAs) are a special class of phasiRNAs generated from TAS precursors (noncoding or coding transcripts), which silence targets in trans.…”