Microprocessor knockdown CAGE

MicroRNA (miRNA) is a class of functional small RNAs that play crucial roles by regulating gene expressions in a wide range of biological processes. In the miRNA biogenesis, hairpin-structured RNAs are cleaved out from long primary miRNAs co-transcriptionally in the nucleus by a microprocessor complex comprising Drosha and Dgcr8, double-stranded ~22nt RNAs are cleaved out from the hairpins by Dicer in the cytosol, and single stranded small RNAs are loaded in a RNA-induced silencing complex (RISC) to repress their target genes. While these maturation and functional processes have been extensively investigated to date, their transcription have been poorly understood. Here we tackled to identify transcription initiation of primary miRNAs by combining a genome-wide survey of transcription starting sites (TSSs) based on a single molecule sequencer and perturbation of the microprocessor complex. We depleted the microprocessor proteins by siRNA knock down in HeLa cells and isolated their nuclei to enrich primary miRNAs (long RNA) in the RNA extracts.