Two decades of miRNA biology: lessons and challenges.

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The celebration of 20 years of RNA is opportune to reflect on what has been learned during this time regarding miRNA biology, and challenges that lay ahead. I focus this perspective on genetics, which arguably laid the foundation for the miRNA field. Indeed, genetic studies revealed critical miRNA:target interactions, the fundamental logic of miRNA target recognition, and phenotypically vital usages of miRNA regulation, all prior to formal recognition of the miRNA pathway. Ironically then, deciphering miRNA biology remains as challenging as ever, despite extensive mechanistic insights gained from biochemical and structural approaches, and a wealth of data garnered from genomewide approaches. Nevertheless, accumulating knowledge on miRNA function and ever-expanding genetic resources for manipulating miRNAs and their targets promise an exciting future in understanding in vivo necessities of these tiny RNAs. The miRNA era exploded in 2001 with the revelation that myriad hairpin-encoding loci generate ∼22 nucleotide (nt) RNAs. However, miRNA studies were already well on their way at that point, through developmental analyses in invertebrates. As is evident from their low-numbered gene names, mutants of the founding miRNAs lin-4 and let-7 were amongst the earliest C. elegans mutants recovered. Notably, the stage for comprehending their cloned sequences was set by genetic inquiries, initially by Chalfie and Ambros, which revealed antagonistic control of heterochronic identity by lin-4 and its major target lin-14. Ruvkun and colleagues studied gain-of-function lin-14 3′ UTR deletion mutants that phenocopied lin-4 loss-of-function, and these were critical to establish the cis-regulatory domain of lin-4 and its impact on lin-14 activity. However, there are older precedents for which loss of miRNA-mediated target regulation has phenotypic consequences. In 1923, Bridges isolated the dominant Drosophila wing serration mutant Beadex. Its eventual cloning showed that recurrent 3′ UTR disruptions underlie gain-of-function of dLMO. Interestingly, mir-9a knockouts exhibit similar wing serration, and miR-9a directly represses dLMO. Strikingly, removal of one dLMO allele fully restores mir-9a mutant wings, defining it as a critical miR-9a target. Thus, genetics revealed a miRNA-related phenotype nearly a century ago.