Regulation of gene activity by microRNAs is critical to myriad aspects of eukaryotic development and physiology. Amidst an extensive regulatory web that is predicted to involve thousands of transcripts, emergent themes are now beginning to illustrate how microRNAs have been incorporated into diverse settings. These include potent inhibition of individual key targets, fine-tuning of target activity, the coordinated regulation of target batteries, and the reversibility of some aspects of microRNA-mediated repression. Such themes may reflect some of the inherent advantages of exploiting microRNA control in biological circuits, and provide insight into the consequences of microRNA dysfunction in disease.MicroRNAs (miRNAs) are ~21-24 nucleotide regulatory RNAs derived from hairpin transcripts that are abundant in animals, plants and even viruses. Although miRNAs were formally codified as a gene class in late 2001 (REFS 1-3), their study was rooted in genetic analyses of worm4-7 and fly8,9 development during the 1990s. These early works revealed key features of miRNA transcripts, some of the significant biological uses of miRNAs and insights into how ~7-nucleotide 3′ UTR elements mediate miRNA function through target deadenylation and destabilization, as well as translational inhibition. The subsequent explosion of miRNA research in the current decade has yielded breathtaking advances in our understanding of the mechanism and biology of miRNA control10,11. Our goal in this Review is to rationalize how and why miRNAs have been incorporated into biological networks. In particular, some of the emerging principles that are shared across unrelated developmental or physiological settings seem to reflect the particular utilities of miRNAs in gene regulation. Overview of miRNA biogenesis and activitymiRNAs derive from longer primary transcripts bearing one or more local hairpins, which are cleaved by RNase III enzymes to yield ~21-24 nucleotide RNA duplexes. Single strands from the duplexes are selected for association with Argonaute proteins, which guide them to complementary targets for regulation10.In plants, most miRNAs exhibit extended complementarity to one or a few predicted targets12, and directed tests have validated ~100 mRNAs that are cleaved by an endogenous miRNA and an Argonaute protein with Slicer activity13-15. High-throughput sequencing of RNAs with 5′ monophosphates (which excludes capped mRNAs) provides an opportunity to identify miRNA-cleaved transcripts genome-wide16,17. Application of this unbiased approach to Arabidopsis thaliana uncovered some novel targets, but most of the high-confidence targets

Numerous microRNAs (miRNAs) have been discovered in the genomes of higher eukaryotes, and functional studies indicate that they are important during development. However, little is known concerning the function of individual miRNAs. We approached this problem in zebrafish by combining identification of miRNA expression, functional analyses and experimental validation of potential targets. We show that miR-214 is expressed during early segmentation stages in somites and that varying its expression alters the expression of genes regulated by Hedgehog signaling. Inhibition of miR-214 results in a reduction or loss of slow-muscle cell types. We show that su(fu) mRNA, encoding a negative regulator of Hedgehog signaling, is targeted by miR-214. Through regulation of su(fu), miR-214 enables precise specification of muscle cell types by sharpening cellular responses to Hedgehog.Multicellular organisms such as zebrafish use miRNAs to regulate gene expression in a tissue-or time-specific manner, guiding developmental decisions 1,2 . To identify target genes regulated by miRNAs, we first developed a microarray to examine temporal miRNA expression patterns during the first 5 d post-fertilization (dpf) of zebrafish development (unpublished data). To understand the function of a subset of these miRNAs, we performed loss-of-function experiments using antisense morpholino oligonucleotides complementary to mature miRNAs. Morpholinos have been used extensively in zebrafish as antisense inhibitors of mRNA translation and splicing 3 but are also capable of interfering with miRNA function ( Supplementary Fig. 1 online). Injection of morpholinos designed to block the function of miR-214 (214 MO ) yielded embryos with U-shaped somites at 1dpf (1 dpf) ( Fig. 1a-d). Expression of miR-214 begins during early somitogenesis and continues throughout embryogenesis (Fig. 1e). In situ hybridization showed that miR-214 is expressed in somites at 1 dpf ( Fig. 1f,g; see also ref. 2).Somites are transient embryonic structures derived from paraxial mesoderm that give rise to muscle and skeleton 4 . Presomitic mesodermal cells immediately adjacent to the notochord (adaxial cells) are highly influenced by Hedgehog and give rise to the slow-twitch muscle lineage 5,6 . Lateral presomitic cells give rise to fast-twitch muscle fibers and experience little stimulation by Hedgehog initially, whereas later-developing fast-muscle fates are dependent on Hedgehog signaling 7 . There are two slow muscle cell types that require precise COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests. Hedgehog signals for proper development: superficial slow fibers (SSFs), which migrate from the midline to populate the surface of the myotome, and slow muscle pioneers that remain close to the midline 6,8 . Muscle pioneers require higher levels of and longer exposure to Hedgehog for proper specification than SSFs and can be distinguished from slow muscle fibers by the expression of the transcription factor Engrailed (Eng) 4,...

Atypical miRNA substrates do not fit criteria often used to annotate canonical miRNAs, and can escape the notice of miRNA genefinders. Recent analyses expanded the catalogs of invertebrate splicing-derived miRNAs (''mirtrons''), but only a few tens of mammalian mirtrons have been recognized to date. We performed meta-analysis of 737 mouse and human small RNA data sets comprising 2.83 billion raw reads. Using strict and conservative criteria, we provide confident annotation for 237 mouse and 240 human splicing-derived miRNAs, the vast majority of which are novel genes. These comprise three classes of splicing-derived miRNAs in mammals: conventional mirtrons, 59-tailed mirtrons, and 39-tailed mirtrons. In addition, we segregated several hundred additional human and mouse loci with candidate (and often compelling) evidence. Most of these loci arose relatively recently in their respective lineages. Nevertheless, some members in each of the three mirtron classes are conserved, indicating their incorporation into beneficial regulatory networks. We also provide the first Northern validation for mammalian mirtrons, and demonstrate Dicer-dependent association of mature miRNAs from all three classes of mirtrons with Ago2. The recognition of hundreds of mammalian mirtrons provides a new foundation for understanding the scope and evolutionary dynamics of Dicer substrates in mammals.[Supplemental material is available for this article.]Diverse pathways of conserved post-transcriptional gene regulation are mediated by Argonaute proteins and their guide, short RNAs. Among Argonaute-mediated small RNA pathways, the beststudied are the microRNAs (miRNAs). Generally speaking, miRNAs are ;21 to 24-nucleotide (nt) RNAs whose termini are precisely defined, and derive from precursor transcripts bearing one or more inverted repeats or hairpins (Axtell et al. 2011). The first miRNAs emerged from genetic studies of Caenorhabditis elegans developmental mutants (Lee et al. 1993;Reinhart et al. 2000), and were only recognized as noncoding loci upon their cloning. This set the stage for the directed identification of miRNA genes from cloned short RNAs (Lagos-Quintana et al. 2001;Lau et al. 2001;Lee and Ambros 2001). In animals, most miRNAs are generated by stepwise cleavage of primary miRNA transcripts ). These are processed in the nucleus by the Drosha RNase III enzyme to release an ;50-to 80-nt pre-miRNA hairpin, and again in the cytoplasm by a Dicer-class RNase III enzyme to yield a small RNA duplex. One of the strands is preferentially stably incorporated as a singlestranded RNA in an Argonaute (Ago) complex, and guides it to target transcripts (Czech and Hannon 2010).Although bioinformatic strategies have been used to identify miRNA genes Lim et al. 2003;Huang et al. 2007; van der Burgt et al. 2009), these have mostly been superseded by deep sequencing. This is in large part due to the fact that effective computational methods rely on comparative genomics and are ill-suited to identify species-specific miRNAs with reasonable specificit...

RNA interference (RNAi) is now an umbrella term referring to post-transcriptional gene silencing mediated by either degradation or translation arrest of target RNA. This process is initiated by double-stranded RNA with sequence homology driving specificity. The discovery that 21-23 nucleotide RNA duplexes (small-interfering RNAs, siRNAs) mediate RNAi in mammalian cells opened the door to the therapeutic use of siRNAs. While much work remains to optimize delivery and maintain specificity, the therapeutic advantages of siRNAs for treatment of viral infection, dominant disorders, cancer, and neurological disorders show great promise. Gene Therapy (2005) 12, 5-11.

Next-generation sequencing technologies generate vast catalogs of short RNA sequences from which to mine microRNAs. However, such data must be vetted to appropriately categorize microRNA precursors and interpret their evolution. A recent study annotated hundreds of microRNAs in three Drosophila species on the basis of singleton reads of heterogeneous length1. Our multi-million read datasets indicated that most of these were not substrates of RNAse III cleavage, and comprised many mRNA degradation fragments. We instead identified a distinct and smaller set of novel microRNAs supported by confident cloning signatures, including a high proportion of evolutionarily nascent mirtrons. Our data support a much lower rate in the emergence of lineage-specific microRNAs than previously inferred1, with a net flux of ~1 microRNA/million years of Drosophilid evolution.