The RNAi Natural Process View image detail

The naturally occurring RNA gene silencing pathway in humans

As research progressed, it became evident that the reason siRNAs, but not long dsRNAs, are able to induce efficient gene silencing in humans is because they feed into an endogenous RNA silencing pathway. This pathway is also known to process microRNAs. MicroRNAs are a large class of small RNAs, typically 19-24 nucleotides in length, and are believed to regulate a significant portion of our genes with diverse roles ranging from development to cell signaling.

While this RNA gene silencing pathway is used by both siRNAs and microRNAs, there exist some important differences. The siRNA pathway begins with cytoplasmic cleavage of long double-stranded RNA by the Dicer enzyme that results into short double-stranded RNA duplexes. The short double-stranded RNAs (called siRNAs) are then incorporated into the RNA-induced silencing complex (RISC). If the siRNA strand loaded into RISC has perfect sequence complementarity with its target mRNA sequence, then site-specific mRNA cleavage takes place. This cleavage is a catalytic event, involving repeated cleavage of multiple copies of the target mRNA, leading to reduced copies of target mRNA and ultimately to inhibition of the target protein. Therefore, RNAi therapeutics developed to harness the siRNA pathway typically involves delivery of sufficient synthetic siRNA into the cell cytoplasm to trigger this catalytic process.

In contrast to the siRNA pathway, the mRNA pathway begins with endogenously encoded primary miRNA transcripts in the cell nucleus that are processed into precursor microRNA and then exported from the nucleus into the cytoplasm. Here, the Dicer enzyme complex further processes them for RISC loading. Unlike siRNAs, mature miRNAs typically have imperfect sequence complementarity to their RNA target sites which are typically limited to the 3’ untranslated regions of the mRNA. Furthermore, interaction of a microRNA with its RNA target usually leads to direct translational inhibition rather than specific site-directed cleavage of the mRNA. Regardless of translational inhibition or cleavage, both microRNAs and siRNAs are able to regulate protein production at the RNA level in a very specific sequence-based manner.