Rice MicroRNA Effector Complexes and Targets

MicroRNAs (miRNAs) are small silencing RNAs with regulatory roles in gene expression. miRNAs interact with Argonaute (AGO) proteins to form effector complexes that cleave target mRNAs or repress translation. Rice (Oryza sativa) encodes four AGO1 homologs (AGO1a, AGO1b, AGO1c, and AGO1d). We used RNA interference (RNAi) to knock down the four AGO1s. The RNAi lines displayed pleiotropic developmental phenotypes and had increased accumulation of miRNA targets. AGO1a, AGO1b, and AGO1c complexes were purified and further characterized. The three AGO1s all have a strong preference for binding small RNAs (sRNAs) with 59 U and have Slicer activity. We cataloged the sRNAs in each AGO1 complex by deep sequencing and found that all three AGO1s predominantly bound known miRNAs. Most of the miRNAs were evenly distributed in the three AGO1 complexes, suggesting a redundant role for the AGO1s. Intriguingly, a subset of miRNAs were specifically incorporated into or excluded from one of the AGO1s, suggesting functional specialization among the AGO1s. Furthermore, we identified rice miRNA targets at a global level. The validated targets include transcription factors that control major stages of development and also genes involved in a variety of physiological processes, indicating a broad regulatory role for miRNAs in rice.