Person:

Lieberman, Judy

Synthetic small interfering RNAs (siRNAs) are an indispensable tool to investigate gene function in eukaryotic cells1,2 and may be used for therapeutic purposes to knockdown genes implicated in disease3. Thus far, most synthetic siRNAs have been produced by chemical synthesis. Here we present a method to produce highly potent siRNAs in E. coli. This method relies on ectopic expression of p19, a siRNA-binding protein found in a plant RNA virus4, 5. When expressed in E. coli, p19 stabilizes ~21 nt siRNA-like species produced by bacterial RNase III. Transfection of mammalian cells with siRNAs, generated in bacteria expressing p19 and a hairpin RNA encoding 200 or more nucleotides of a target gene, at low nanomolar concentrations reproducibly knocks down gene expression by ~90% without immunogenicity or off-target effects. Because bacterially produced siRNAs contain multiple sequences against a target gene, they may be especially useful for suppressing polymorphic cellular or viral genes.

Identifying microRNA (miRNA)-regulated genes is key to understanding miRNA function. However, many miRNA recognition elements (MREs) do not follow canonical “seed” base-pairing rules, making identification of bona fide targets challenging. Here, we apply an unbiased sequencing-based systems approach to characterize miR-522, a member of the oncogenic primate-specific chromosome 19 miRNA cluster, highly expressed in poorly differentiated cancers. To identify miRNA targets, we sequenced full-length transcripts captured by a biotinylated miRNA mimic. Within these targets, mostly noncanonical MREs were identified by sequencing RNase-resistant fragments. miR-522 overexpression reduced mRNA, protein levels, and luciferase activity of >70% of a random list of candidate target genes and MREs. Bioinformatic analysis suggested that miR-522 regulates cell proliferation, detachment, migration, and epithelial-mesenchymal transition. miR-522 induces G1 cell-cycle arrest and causes cells to detach without anoikis, become invasive, and express mesenchymal genes. Thus, our method provides a simple but effective technique for identifying miRNA-regulated genes and biological function.