The molecular controls that govern the differentiation of embryonic stem (ES) cells remain poorly understood. DGCR8 is an RNA-binding protein that assists the RNase III enzyme Drosha in the processing of microRNAs (miRNAs), a subclass of small RNAs. Here we study the role of miRNAs in ES cell differentiation by generating a Dgcr8 knockout model. Analysis of mouse knockout ES cells shows that DGCR8 is essential for biogenesis of miRNAs. On the induction of differentiation, DGCR8-deficient ES cells do not fully downregulate pluripotency markers and retain the ability to produce ES cell colonies; however, they do express some markers of differentiation. This phenotype differs from that reported for Dicer1 knockout cells, suggesting that Dicer has miRNA-independent roles in ES cell function. Our findings indicate that miRNAs function in the silencing of ES cell self-renewal that normally occurs with the induction of differentiation.

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...

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.

In vitro selection was used to identify deoxyribozymes that ligate two RNA substrates. In the ligation reaction, a 2'-5' RNA phosphodiester linkage is created from a 2',3'-cyclic phosphate and a 5'-hydroxyl group. The new Mg(2+)-dependent deoxyribozymes provide 50-60% yield of ligated RNA in overnight incubations at pH 7.5 and 37 degrees C, and they afford 40-50% yield in 1 h at pH 9.0 and 37 degrees C. Various RNA substrate sequences may be joined by simple Watson-Crick covaration of the DNA binding arms that interact with the two RNA substrates. The current deoxyribozymes have some RNA substrate sequence requirements at the nucleotides immediately surrounding the ligation junction (either UAUA GGAA or UAUN GGAA, where the arrow denotes the ligation site and N equals any nucleotide). One of the new deoxyribozymes was used to prepare by ligation the Tetrahymena group I intron RNA P4-P6 domain, a representative structured RNA. Nondenaturing gel electrophoresis revealed that a 2'-5' linkage between nucleotides A233 and G234 of P4-P6 does not disrupt its Mg(2+)-dependent folding (DeltaDeltaG degrees ' < 0.2 kcal/mol). This demonstrates that a 2'-5' linkage does not necessarily interfere with structure in a folded RNA. Therefore, these non-native linkages may be acceptable in modified RNAs when structure/function relationships are investigated. Deoxyribozymes that ligate RNA should be particularly useful for preparing site-specifically modified RNAs for studies of RNA structure, folding, and catalysis.

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