Safeguard DCL2-Dependent 22-nt siRNA Generation by DCL1 data
In plants, miRNAs and siRNAs are crucial for their growth and development via mediating post-transcriptional gene silencing. In wild-type Arabidopsis, DCL2-dependent 22-nt siRNAs are rare, whereas DCL1- and DCL4-dependent 21-nt miRNAs and siRNAs are highly abundant. DCL4 naturally inhibits DCL2 in producing abundant 22-nt siRNAs from endogenous transcripts, but whether DCL1 suppresses endogenous 22-nt siRNA production and the extent of the DCL1-mediated repression of DCL2 activity are still unknown. To address these issues, we used small sequencing technologies to investigate the potential mechanism for the critical roles of DCL1 and DCL4 in protecting plant growth from the disturbing of DCL2-dependent 22-nt siRNAs. Arabidopsis mutants used in this study, dcl1-9 (CS3828) is in the Ler background, and the others are all in the Col-0 background, including dcl2-1 (SALK_064627.42.50.x), dcl4-2 (GABI_160G05), and dcl2-1 dcl4-2t (N66078; CS66078). Other double and triple mutants including dcl1-9 dcl2-1, dcl1-9 dcl4-2, and dcl1-9 dcl2-1 dcl4-2t were generated by genetic crossing. Our data showed that DCL1 and DCL2 cleaved both miRNA precursors and coding transcript-derived double-stranded RNAs in Arabidopsis defective for both DCL1 and DCL4. In the dcl1 dcl4 double mutant, massive 22-nt siRNAs broke the RNA-decay safeguard were produced by endogenous protein-coding genes (genic siRNAs), of which NIA1, NIA2, DGAT3, and both SMXL4 and SMXL5 accumulated the abundance of 22-nt siRNAs up to 95%. These 22-nt genic siRNAs were likely loaded into AGO1 or AGO2 and further mediated gene translation repression. Thus, our results demonstrated that both DCL1 and DCL4 safeguard post-transcriptional gene silencing, preventing the production of DCL2-dependent 22-nt genic siRNAs from disturbing plant growth and development.