No Dicer for Me: MicroRNAs (miRNAs) are small noncoding RNAs found in most eukaryotes. Most are processed from primary transcripts in the nucleus by the microprocessor enzyme complex, which includes the nuclease Drosha, with a small number being generated by the messenger RNA splicing machinery. All pre-miRNAs are then exported into the cytoplasm where they are cleaved further by a second nuclease, Dicer, into the mature, functional miRNA. Cifuentes et al. (p. 1694 , published online 6 May), now show that in a Dicer mutant fish at least one miRNA, miR-451, is still formed from pre-miR-451. The processing of pre-miR-451 requires the slicing activity of another protein in the miRNA pathway, Argonaute2. The unusual secondary structure of the pre-miR-451 determines its noncanonical processing pathway, which suggests that other miRNAs might also be processed in this way.

Context-dependent assembly (CoDA) of zinc finger nucleases is reported. Starting from an archive of zinc finger modules known to function well together, effective multifinger arrays can be constructed using standard techniques. Also in this issue, Doyon et al. report rational design of nucleases with improved cleavage activity. Engineered zinc-finger nucleases (ZFNs) enable targeted genome modification. Here we describe context-dependent assembly (CoDA), a platform for engineering ZFNs using only standard cloning techniques or custom DNA synthesis. Using CoDA-generated ZFNs, we rapidly altered 20 genes in Danio rerio, Arabidopsis thaliana and Glycine max. The simplicity and efficacy of CoDA will enable broad adoption of ZFN technology and make possible large-scale projects focused on multigene pathways or genome-wide alterations.

When oxygen becomes limiting, cells reduce mitochondrial respiration and increase ATP production through anaerobic fermentation of glucose. The Hypoxia Inducible Factors (HIFs) play a key role in this metabolic shift by regulating the transcription of key enzymes of glucose metabolism. Here we show that oxygen regulates the expression of the muscle glycogen synthase (GYS1). Hypoxic GYS1 induction requires HIF activity and a Hypoxia Response Element within its promoter. GYS1 gene induction correlated with a significant increase in glycogen synthase activity and glycogen accumulation in cells exposed to hypoxia. Significantly, knockdown of either HIF1α or GYS1 attenuated hypoxia-induced glycogen accumulation, while GYS1 overexpression was sufficient to mimic this effect. Altogether, these results indicate that GYS1 regulation by HIF plays a central role in the hypoxic accumulation of glycogen. Importantly, we found that hypoxia also upregulates the expression of UTP:glucose-1-phosphate urydylyltransferase (UGP2) and 1,4-α glucan branching enzyme (GBE1), two enzymes involved in the biosynthesis of glycogen. Therefore, hypoxia regulates almost all the enzymes involved in glycogen metabolism in a coordinated fashion, leading to its accumulation. Finally, we demonstrated that abrogation of glycogen synthesis, by knock-down of GYS1 expression, impairs hypoxic preconditioning, suggesting a physiological role for the glycogen accumulated during chronic hypoxia. In summary, our results uncover a novel effect of hypoxia on glucose metabolism, further supporting the central importance of metabolic reprogramming in the cellular adaptation to hypoxia.

MicroRNAs (miRNAs) are typically generated as ~22-nucleotide double-stranded RNAs via the processing of precursor hairpins by the ribonuclease III enzyme Dicer, after which they are loaded into Argonaute (Ago) proteins to form an RNA-induced silencing complex (RISC). However, the biogenesis of miR-451, an erythropoietic miRNA conserved in vertebrates, occurs independently of Dicer and instead requires cleavage of the 3' arm of the pre-miR-451 precursor hairpin by Ago2. The 3' end of the Ago2-cleaved pre-miR-451 intermediate is then trimmed to the mature length by an unknown nuclease. Here, using a classical chromatographic approach, we identified poly(A)-specific ribonuclease (PARN) as the enzyme responsible for the 3'-5' exonucleolytic trimming of Ago2-cleaved pre-miR-451. Surprisingly, our data show that trimming of Ago2-cleaved precursor miRNAs is not essential for target silencing, indicating that RISC is functional with miRNAs longer than the mature length. Our findings define the maturation step in the miRNA biogenesis pathway that depends on Ago2-mediated cleavage.