Size Distribution of Small Interfering RNAs in Various Organs at Different Developmental Stages is Primarily Determined by the Dicing Activity of Dicer-Like Proteins in Plants

RNA silencing is a fundamental mechanism to maintain plant growth and development, and regulation of the size distribution of small interfering RNAs (siRNAs) is critical in the control of normal gene expression throughout a plant's life cycle. However, the cause of organ- and developmental stage-specific accumulation of siRNAs has never been reported. Whereas 24 nt siRNAs accumulated about 5.3-fold more than 21 nt siRNAs in Arabidopsis rosette leaves, 21 and 24 nt siRNAs accumulated to similar levels in Arabidopsis pollen grains, rice spikelets and maize anthers. We successfully detected two distinct double-stranded RNA (dsRNA)-cleaving activities that produced 21 and 24 nt RNAs in cell-free extracts prepared from various organs at different developmental stages of A. thaliana, Brassica rapa, rice and maize. Although DCL4 transcript was expressed more than DCL3 transcript in most organs, the 21 nt RNA-producing activity of DCL4 or its orthologs was very low and was 5- to 10-fold lower than the 24 nt RNA-producing activity of DCL3 or its orthologs particularly in leaves, indicating that DCL4 activity is negatively regulated translationally or post-translationally in leaves. High dicing activity of DCL3 and DCL4 was detected in immature inflorescences, developing seeds, germinating embryos and callus, all of which contain actively dividing cells. In various organs at different developmental stages, the size distribution of siRNAs was positively correlated with the dicing activity of two Dicers, DCL3 and DCL4, or their orthologs. Taken together, the size distribution of siRNAs in most organs is primarily determined by the dicing activity of DCL3 and DCL4.