Argonaute (AGO) proteins recruit small RNAs to form the core of RNAi effector complexes. Arabidopsis encodes ten AGO proteins and a large network of small RNAs. How these small RNAs are sorted into specific AGO complexes remains largely unknown. We have cataloged small RNAs resident in four AGO complexes. We found that AGO2 and AGO4 preferentially recruit small RNAs with a 5' terminal adenosine, whereas AGO1 harbors microRNAs (miRNAs) that favor a 5' terminal uridine. AGO5 predominantly binds small RNAs that initiate with cytosine. Changing the 5' terminal nucleotide of an miRNA predictably redirected it into a different AGO complex and alters its biological activity. These results reveal a role for small RNA sequences in assorting among AGO complexes. This suggests that specialization of AGO complexes might involve remodeling the 5' end-binding pocket to accept certain small RNA sequences, perhaps explaining the evolutionary drive for miRNAs to initiate with uridine.

In plants, the known microRNAs (miRNAs) are produced as approximately 21 nucleotide (nt) duplexes from their precursors by Dicer-like 1 (DCL1). They are incorporated into Argonaute 1 (AGO1) protein to regulate target gene expression primarily through mRNA cleavage. We report here the discovery of a class of miRNAs in the model monocot rice (Oryza sativa). These are 24 nt in length and require another member of the Dicer family, DCL3, for their biogenesis. The 24 nt long miRNAs (lmiRNAs) are loaded into AGO4 clade proteins according to hierarchical rules, depending on the upstream biogenesis machinery and the 5'-terminal nucleotide. We demonstrated that lmiRNAs direct DNA methylation at loci from which they are produced as well as in trans at their target genes and play roles in gene regulation. Considered together, our findings define a miRNA pathway that mediates DNA methylation.

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.

CRISPR-mediated genome editing efficiently corrected cardiac abnormalities associated with DMD in human engineered heart tissue.