Identification of miR-29b targets using 3-cyanovinylcarbazole containing mimics - human

MicroRNAs (miRNAs) are highly conserved ∼22 nt small noncoding RNAs that bind partially complementary sequences in target transcripts. MicroRNAs regulate both translation and transcript stability, and play important roles in development, cellular homeostasis, and disease. There are limited approaches available to agnostically identify microRNA targets transcriptome-wide, and methods using miRNA mimics, which in principle identify direct miRNA:transcript pairs, have low sensitivity and specificity. Here, we describe a novel method to identify microRNA targets using miR-29b mimics containing 3cyanovinylcarbazole ( CNV K), a photolabile nucleoside analog. We demonstrate that biotin-tagged, CNV K-containing miR-29b ( CNV K-miR-29b) mimics are nontoxic in cell culture, associate with endogenous mammalian Argonaute2, arehighly sensitive for known targets and recapitulate endogenous transcript destabilization. Partnering CNV K-miR-29b with ultra-low-input RNA sequencing, we recover ∼40% of known miR-29b targets and findrobust conservation of the focal adhesion and apoptotic target pathways in mouse and human. We also identify hundreds of novel targets, including NRAS, HOXA10, and KLF11, with a validation rate of 71% for a subset of 73 novel target transcripts interrogated using a high-throughput luciferase assay. Consistent with previous reports, we show that both endogenous miR-29b and CNV K-miR-29b are trafficked to the nucleus, but find no evidence of nuclear-specific miR-29b transcript binding. This may indicate that miR-29b nuclear sequestration is a regulatory mechanism in itself. We suggest that CNV K-containing small RNA mimics may find applicability in other experimental models pulldown assay with 3x3x3x3 design: miRNA mimic and scrambled miRNA mimic x input and pulldown conditions