Introduction

RNA interference (RNAi) is the process by which expression of a target gene is effectively silenced or knocked down by the selective inactivation of its corresponding mRNA by double-stranded RNA (dsRNA). RNAi is activated by dsRNA species delivered to the cytoplasm of cells. The slicing mechanisms can either lead to the degradation of a target mRNA, as induced by small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs).

shRNAs are synthesized in the nucleus of transfected/transduced cells and form hairpin structures that consist of a stem region of paired antisense and sense strands connected by unpaired nucleotides that make up a loop. They are converted into siRNAs by the same RNAi machinery that processes miRNAs.  shRNAs are introduced into the nuclei of target cells using either bacterial or viral vectors that, in some cases, can stably integrate into the genome. shRNAs are transcribed by either RNA polymerase II or III, depending on the promoter driving their expression. These initial precursors are processed by Drosha and its dsRNA-binding partner DGCR8, resulting in species known as pre-shRNAs, before being exported to the cytoplasm by Exportin-5. The pre-shRNA is then cleaved by Dicer and TRBP/PACT, removing the hairpin and creating a 20–25 nt double-stranded siRNA with 2 nt 3’ overhangs at each end. This active siRNA is then loaded onto the RISC complex.

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