Roles of DCL4 and DCL3b in rice phased small RNA biogenesis

Abstract

SUMMARYHigher plants have evolved multiple proteins in the RNase III family to produce and regulate different classes of small RNAs with specialized molecular functions. In rice (Oryza sativa), numerous genomic clusters are targeted by one of two microRNAs (miRNAs), miR2118 and miR2275, to produce secondary small interfering RNAs (siRNAs) of either 21 or 24 nucleotides in a phased manner. The biogenesis requirements or the functions of the phased small RNAs are completely unknown. Here we examine the rice Dicer-Like (DCL) family, including OsDCL1, -3a, -3b and -4. By deep sequencing of small RNAs from different tissues of the wild type and osdcl4-1, we revealed that the processing of 21-nucleotide siRNAs, including trans-acting siRNAs (tasiRNA) and over 1000 phased small RNA loci, was largely dependent on OsDCL4. Surprisingly, the processing of 24-nucleotide phased small RNA requires the DCL3 homolog OsDCL3b rather than OsDCL3a, suggesting functional divergence within DCL3 family. RNA ligase-mediated 5¢ rapid amplification of cDNA ends and parallel analysis of RNA ends (PARE)/degradome analysis confirmed that most of the 21-and 24-nucleotide phased small RNA clusters were initiated from the target sites of miR2118 and miR2275, respectively. Furthermore, the accumulation of the two triggering miRNAs requires OsDCL1 activity. Finally, we show that phased small RNAs are preferentially produced in the male reproductive organs and are likely to be conserved in monocots. Our results revealed significant roles of OsDCL4, OsDCL3b and OsDCL1 in the 21-and 24-nucleotide phased small RNA biogenesis pathway in rice.

Spatiotemporally dynamic, cell-type–dependent premeiotic and meiotic phasiRNAs in maize anthers

“…For the gene targets that showed PARE signals, GO analysis did not yield significant enrichment in any particular pathway or process (Dataset S4). These results are consistent with an earlier conclusion from rice that 21-nt premeiotic phasiRNAs lack obvious targets (9). Furthermore, an absence of premeiotic or meiotic phasiRNAs in ocl4, mac1, and ms23 does not result in TE transcript accumulation (Dataset S5).…”

“…One of the two major subgroups with the NB-LRR gene family, the TIR-NBLRRs, is not found in grass genomes, perhaps hinting at an origin for the miR2118-targeted 21-PHAS precursors. The origin of miR2275 is unknown, but DCL5 is most similar to DCL3, and was earlier named DCL3b (9,36). Both miR2275 and DCL5 are absent from dicot genomes, suggesting their recent derivation within the grasses or within related monocots.…”

“…Although both miR2275 and meiotic phasiRNAs have only been reported in grass species (8)(9)(10), miR2118 is present in dicots. The primary miR2118 targets in dicots are NB-LRR pathogen-defense genes; the 21-nt phasiRNAs produced from the NB-LRR mRNAs function in trans and in cis, and they are expressed constitutively (16).…”

“…In rice and putatively in maize, RDR6 recognizes the cleaved, uncapped 3′ fragments of these transcripts and synthesizes a second strand, forming double-stranded RNA (11). Subsequent processing by DCL4 and DCL5 generates 21-and 24-nt phasiRNAs, respectively (9). Both dicers exhibit sequential slicing activity, starting precisely at the 11th nucleotide of the miRNA binding…”

“…In Arabidopsis pollen, TE-derived small-interfering RNAs (siRNAs) expressed in the vegetative nuclei reinforce silencing after transfer to sperm nuclei (7). Additionally, rice inflorescences produce 21-and 24-nt phased, secondary siRNAs (phasiRNAs) from nonrepeat regions (8,9).…”