Multiple distinct small RNAs originate from the same microRNA precursors

Background MicroRNAs (miRNAs), which originate from precursor transcripts with stem-loop structures, are essential gene expression regulators in eukaryotes. Results We report 19 miRNA precursors in Arabidopsis that can yield multiple distinct miRNA-like RNAs in addition to miRNAs and miRNA*s. These miRNA precursor-derived miRNA-like RNAs are often arranged in phase and form duplexes with an approximately two-nucleotide 3'-end overhang. Their production depends on the same biogenesis pathway as their sibling miRNAs and does not require RNA-dependent RNA polymerases or RNA polymerase IV. These miRNA-like RNAs are methylated, and many of them are associated with Argonaute proteins. Some of the miRNA-like RNAs are differentially expressed in response to bacterial challenges, and some are more abundant than the cognate miRNAs. Computational and expression analyses demonstrate that some of these miRNA-like RNAs are potentially functional and they target protein-coding genes for silencing. The function of some of these miRNA-like RNAs was further supported by their target cleavage products from the published small RNA degradome data. Our systematic examination of public small-RNA deep sequencing data from four additional plant species ( Oryza sativa , Physcomitrella patens , Medicago truncatula and Populus trichocarpa ) and four animals ( Homo sapiens , Mus musculus , Caenorhabditis elegans and Drosophila ) shows that such miRNA-like RNAs exist broadly in eukaryotes. Conclusions We demonstrate that multiple miRNAs could derive from miRNA precursors by sequential processing of Dicer or Dicer-like proteins. Our results suggest that the pool of miRNAs is larger than was previously recognized, and miRNA-mediated gene regulation may be broader and more complex than previously thought.