title: Roles of DCL4 and DCL3b in rice phased small RNA biogenesis.
abstract: Higher 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.
authors.affiliation: State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Xianwei
authors.initials: X
authors.lastname: Song
authors.forename: Pingchuan
authors.initials: P
authors.lastname: Li
authors.forename: Jixian
authors.initials: J
authors.lastname: Zhai
authors.forename: Ming
authors.initials: M
authors.lastname: Zhou
authors.forename: Lijia
authors.initials: L
authors.lastname: Ma
authors.forename: Bin
authors.initials: B
authors.lastname: Liu
authors.forename: Dong-Hoon
authors.initials: DH
authors.lastname: Jeong
authors.forename: Mayumi
authors.initials: M
authors.lastname: Nakano
authors.forename: Shouyun
authors.initials: S
authors.lastname: Cao
authors.forename: Chunyan
authors.initials: C
authors.lastname: Liu
authors.forename: Chengcai
authors.initials: C
authors.lastname: Chu
authors.forename: Xiu-Jie
authors.initials: XJ
authors.lastname: Wang
authors.forename: Pamela J
authors.initials: PJ
authors.lastname: Green
authors.forename: Blake C
authors.initials: BC
authors.lastname: Meyers
authors.forename: Xiaofeng
authors.initials: X
authors.lastname: Cao
mesh_terms: D001483:Base Sequence; D018506:Gene Expression Regulation, Plant; D015723:Gene Library; D035683:MicroRNAs / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D012275:Oryza / Q000201:enzymology / Q000235:genetics*; D010802:Phylogeny; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D030821:Plants, Genetically Modified / Q000201:enzymology / Q000235:genetics; D018749:RNA, Plant / Q000096:biosynthesis*; D034741:RNA, Small Interfering / Q000096:biosynthesis*; D043244:Ribonuclease III / Q000235:genetics / Q000378:metabolism*; D017423:Sequence Analysis, RNA
chemical_list: D035683:MicroRNAs; D010940:Plant Proteins; D018749:RNA, Plant; D034741:RNA, Small Interfering; D043244:Ribonuclease III