Canonical microRNAs (miRNAs) require two processing steps: the first by the Microprocessor, a complex of DGCR8 and Drosha, and the second by a complex of TRBP and Dicer. dgcr8⌬/⌬ mouse embryonic stem cells (mESCs) have less severe phenotypes than dicer1⌬/⌬ mESCs, suggesting a physiological role for Microprocessor-independent, Dicer-dependent small RNAs. To identify these small RNAs with unusual biogenesis, we performed high-throughput sequencing from wild-type, dgcr8⌬/⌬, and dicer1⌬/⌬ mESCs. Several of the resulting DGCR8-independent, Dicer-dependent RNAs were noncanonical miRNAs. These derived from mirtrons and a newly identified subclass of miRNA precursors, which appears to be the endogenous counterpart of shRNAs. Our analyses also revealed endogenous siRNAs resulting from Dicer cleavage of long hairpins, the vast majority of which originated from one genomic locus with tandem, inverted short interspersed nuclear elements (SINEs). Our results extend the known diversity of mammalian small RNA-generating pathways and show that mammalian siRNAs exist in cell types other than oocytes.[Keywords: ES cells; small RNAs; high throughput sequencing] Supplemental material is available at http://www.genesdev.org. Small RNAs that mediate RNAi-related processes are classified by their biogenesis pathways. Central to the processing of most small RNAs is the RNase III-containing enzyme, Dicer, which forms a complex with TRBP in mammals and cleaves dsRNA precursors into the characteristic ∼22-nucleotide (nt) final product (Hammond 2005;Maniataki and Mourelatos 2005). Dicer products can be classified into two main categories: siRNAs and microRNAs (miRNAs). siRNAs are generated from multiple Dicer cleavages along a long precursor dsRNA, whereas miRNAs are generated from a single Dicer cleavage of a short hairpin pre-miRNA (Bartel 2004). miRNAs require additional upstream processing to convert a longer pol II expressed pri-miRNA transcript to the short pre-miRNA hairpin. For canonical miRNAs, this processing event is performed by the Microprocessor complex, which consists of the RNase III enzyme Drosha ) and the dsRNA-binding protein DGCR8 (Denli et al. 2004;Gregory et al. 2004;Han et al. 2004Han et al. , 2006Landthaler et al. 2004). A subclass of pre-miRNAs, the mirtrons, bypass the Microprocessor; for these noncanonical miRNAs, the upstream processing is performed by the spliceosome and debranching enzyme, which produce a short hairpin directly suitable for Dicer cleavage without further processing (Okamura et al. 2007;Ruby et al. 2007a).A central role for miRNAs in metazoan development is well established (Bartel 2004). Endogenous siRNAs play important roles in plants (Poethig et al. 2006), Schizosaccharomyces pombe (Verdel and Moazed 2005) and Tetrayhymena (Lee and Collins 2006). They also have been observed in metazoa including Caenorhabditis elegans (Ambros et al. 2003), Drosophila melanogaster (Czech et al. 2008;Ghildiyal et al. 2008;Kawamura et al. 2008;Okamura et al. 2008), and mouse oocytes (Watanabe et al. 2006(Watana...

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 398 annotated miRNA genes and identified 108 novel miRNA genes. More than 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 murine 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 precursor miRNA (pre-miRNA), consequential 59 heterogeneity, newly identified instances of miRNA editing, and evidence for widespread pre-miRNA uridylation reminiscent of miRNA regulation by Lin28.[Keywords: MicroRNA; miRNA biogenesis; noncoding RNA genes; high-throughput sequencing] Supplemental material is available at http://www.genesdev.org.

Dgcr8 knockout embryonic stem (ES) cells lack microprocessor activity and hence all canonical microRNAs (miRNAs). These cells proliferate slowly and accumulate in G1 phase of the cell cycle1. Here, by screening a comprehensive library of individual miRNAs in the background of the Dgcr8 knockout ES cells, we report that multiple ES cell-specific miRNAs, members of the miR-290 family, rescue the ES cell proliferation defect. Furthermore, rescued cells no longer accumulate in the G1 phase of the cell cycle. These miRNAs function by suppressing several key regulators of the G1/S transition. These results show that post-transcriptional regulation by miRNAs promotes the G1/S transition of the ES cell cycle enabling their rapid proliferation. Furthermore, our screening strategy provides an alternative and powerful approach for uncovering the role of individual miRNAs in biological processes as it overcomes the common problem of redundancy and saturation in the miRNA system.

This report demonstrates that introduction of physiologically relevant miRNAs can enhance somatic cell reprogramming. The mouse embryonic stem (ES) cell specific microRNAs (miRNA) miR-291-3p, miR-294, and miR-295 enhanced the efficiency of Klf4, Oct4 and Sox2 induced pluripotency. These miRNAs did not further enhance reprogramming in the presence of cMyc. cMyc binds the promoter of these miRNAs, suggesting that they are downstream effectors of cMyc promoted pluripotency. However, unlike exogenous cMyc, these miRNAs induced a homogeneous population of reprogrammed colonies suggesting overlapping and independent functions of cMyc and the miRNAs.The miR-290 cluster constitutes over 70% of the entire miRNA population in mouse ES cells1. Its expression is rapidly down-regulated upon ES cell differentiation2. A subset of the miR-290 cluster, called the embryonic stem cell cycle (ESCC) regulating miRNAs, enhances the unique stem cell cell cycle3. This subset includes miR-291-3p, miR-294, and miR-295. To test whether ESCC miRNAs could promote the induction of pluripotency, we introduced these miRNAs along with retroviruses4 expressing Oct4, Sox2, and Klf4 (OSK) into mouse embryonic fibroblasts (MEFs). The MEFs carried two reporters: an Oct4-GFP reporter that activates GFP with the induction of pluripotency and ubiquitous expression of a β-galactosidase/neo fusion from the Rosa26 locus5. MiRNAs were introduced on days 0 and 6 post-infection by transfection of synthesized double-stranded RNAs that mimic their mature endogenous counterparts. This method transiently recapitulates ES-like levels of the miR-290 cluster miRNAs ( Supplementary Fig. 1).OSK plus miR-291-3p, miR-294, or miR-295 consistently increased the number of Oct4-GFP+ colonies as compared to controls transduced with OSK plus transfection reagent (Fig. 1a). The miR-294 mimic showed the greatest effects, increasing efficiency from 0.01-0.05% to 0.1-0.3% of transduced MEFs. Introduction of a chemically synthesized miR-294 premiRNA similarly enhanced reprogramming (Supplementary Figure 2). Two other members of the miR-290 cluster that are not ESCC miRNAs, miR-292-3p and miR-293, did not increase colony number (Fig. 1a). The ESCC miRNAs share a conserved seed sequence, which largely specifies target mRNAs (Fig. 1b). MiR-302d, a member of another miRNA cluster that has the same seed sequence also enhanced reprogramming (Fig. 1b&c). Mutation of the seed sequence in miR-294 blocked the increase in colony number (Fig. 1b&c). In summary, together with Oct4, Sox2, and Klf4, the ESCC miRNAs and related