Mammalian microRNAs: Experimental evaluation of novel and previously annotated genes

MicroRNAs (miRNAs) are small regulatory RNAs that derive from distinctive hairpin transcripts. To learn more about the miRNAs of mammals, we sequenced 60 million small RNAs from mouse brain, ovary, testes, embryonic stem cells, three embryonic stages, and whole newborns. Analysis of these sequences confirmed 387 annotated miRNA genes and identified 110 novel miRNA genes. Over 150 previously annotated miRNAs and hundreds of candidates failed to yield sequenced RNAs with miRNA-like features. Ectopically expressing these previously proposed miRNA hairpins also did not yield small RNAs, whereas ectopically expressing the confirmed and newly identified hairpins usually did yield small RNAs with the classical miRNA features, including dependence on the Drosha endonuclease for processing. These experiments, which suggest that previous estimates of conserved mammalian miRNAs were inflated, provide a substantially revised list of confidently identified mammalian miRNAs from which to infer the general features of mammalian miRNAs. Our analyses also revealed new aspects of miRNA biogenesis and modification, including tissue-specific strand preferences, sequential Dicer cleavage of a metazoan pre-miRNA, newly identified instances of miRNA editing, and evidence for widespread Lin28-like miRNA regulation. Overall design: For miRNA discovery, small RNAs were sequenced from mouse brain, ovary, testes, three embryonic stages, and whole newborns; for ectopic over-expression assays, pre-miRNA hairpins and the surrounding regions were transfected into HEK293T, and the small RNA were sequenced from the transfected cells 39-48 hours after transfection.