Bioinformatic prediction and experimental validation of a microRNA-directed tandem trans-acting siRNA cascade in Arabidopsis.
Small RNAs play pivotal roles in regulating gene expression in higher eukaryotes. Among them, trans-acting siRNAs (ta-siRNAs) are a class of small RNAs that regulate plant development. The biogenesis of ta-siRNA depends on microRNA-targeted cleavage followed by the DCL4-mediated production of small RNAs phased in 21-nt increments relative to the cleavage site on both strands. To find TAS genes, we have used these characteristics to develop the first computational algorithm that allows for a comprehensive search and statistical evaluation of putative TAS genes from any given small RNA database. A search in Arabidopsis small RNA massively parallel signature sequencing (MPSS) databases with this algorithm revealed both known and previously unknown ta-siRNA-producing loci. We experimentally validated the biogenesis of ta-siRNAs from two PPR genes and the trans-acting activity of one of the ta-siRNAs. The production of ta-siRNAs from the identified PPR genes was directed by the cleavage of a TAS2-derived ta-siRNA instead of by microRNAs as was reported previously for TAS1a, -b, -c, TAS2, and TAS3 genes. Our results indicate the existence of a small RNA regulatory cascade initiated by miR173-directed cleavage and followed by the consecutive production of ta-siRNAs from two TAS genes.
Chen
Ho-Ming
HM
Institute of Plant and Microbial Biology, Academia Sinica, Taipei 11529, Taiwan.
Li
Yi-Hang
YH
Wu
Shu-Hsing
SH
D000465:Algorithms; D017360:Arabidopsis / Q000235:genetics*; D015152:Blotting, Northern; D019295:Computational Biology / Q000379:methods*; D017931:DNA Primers; D030541:Databases, Genetic; D035683:MicroRNAs / Q000235:genetics*; D034741:RNA, Small Interfering / Q000096:biosynthesis* / Q000235:genetics*; D043244:Ribonuclease III; D012260:Ribonucleases / Q000235:genetics
D017931:DNA Primers; D035683:MicroRNAs; D034741:RNA, Small Interfering; C504982:DCL4 protein, Arabidopsis; D012260:Ribonucleases; D043244:Ribonuclease III