RNA interference

RNA interference (also RNAi) is the sequence-specific down-regulation of mRNAs, either by degradation or translation initiation, by short dsRNA molecules. It is a biological mechanism, first discovered in plants and then later in the nematode worm C. elegans, that now has applications in genetic engineering and medicine. The sequence-specificity of RNAi is in contrast to other mechanisms of mRNA down-regulation such as:

  • De-capping and 5'-3' exonucleolytic decay
  • Deadenylation and 3'-5' exonucleolytic decay
  • Deadenylation, then de-capping and 5'-3' exonucleolytic decay
  • Endonucleolytic decay and then 3'-5' exonucleolytic decay by the exosome

All of these can be performed universally, since all eukaryotic mRNAs possess these features.

RNA interference involves the recognition of specific sequences within the mRNA. Small, complementary dsRNA molecules (called siRNAs, see below) target these sequences.

The mechanism:

The initial dsRNA which triggers RNAi can be either endogenous or exogenous in origin. The dsRNA is cleaved into smaller fragments by an enzyme called Dicer (an ATP-dependent cytoplasmic RNaseIII), and the products of cleavage are small interfering RNA (siRNA) molecules with a 2 nucleotide overhang at the 3' end of each strand. The siRNAs are then separated into single strands, and the anti-sense strand of siRNA interacts with the catalytic component of the RISC (RNA-induced silencing complex). This catalytic component is called argonaut (another RNase III enzyme). After integration into RISC, the siRNAs base-pair to their complementary regions in the targeted mRNA and proceed with one of two silencing mechanisms:

  • Where there is complete complementarity, the siRNAs induce mRNA cleavage, resulting in a fragmented mRNA that is not competent for protein translation.
  • Where there is only partial complementarity, the siRNAs induce translation inhibition simply by RISC binding and RNA storage

As said above, the original dsRNA can be either endogenous or exogenous. Endogenous dsRNA is typically derived from a small regulatory, hairpin-loop RNA called microRNA (or miRNA) that is synthesised from one of the 1000s of miRNA genes in the human genome (miRNAs are synthesised in tandem by RNA polymerase II and then processed in the nucleus by a Drosha/Posha complex before export to the cytoplasm). The miRNAs are 21-23 nucleotides in length, and can be processed by Dicer to produce siRNAs in much the same way as any other dsRNA source. MicroRNAs are typically expressed in a tissue-specific manner and may have important roles in developmental biology. Exogenous dsRNA is typically introduced to the cell by viruses or by medical intervention.

It was originally believed that a single-stranded antisense RNA was the medium of RNAi; it has only recently been known that dsRNA (that is later processed to ssRNA) is the mediator (although pure ssRNA has been reported to have a 'moderate' effect). Only a small amount of dsRNA is required per affected cell, arguing that RNAi is not caused by stochiometric interference of mRNA, but is rather a catalytic or amplification process. This is because one siRNA is involved in many rounds of mRNA cleavage; this could not be performed with only single-stranded anti-sense RNA.

Exploiting RNAi in genetic engineering and medicine

In genetic engineering, RNAi has an important role in functional genomics: knocking out genes in order to ascertain their function.

In medicine, RNAi-based therapeutics have proven useful in treating viral infection, cancers and neurodegenerative disease with much higher specificity than drugs by down-regulating offending mRNAs. Nevertheless, there are barriers to overcome in the development of siRNA as a 'drug': sequence specificity (to avoid off-target recognition), chemical stabilisation of otherwise-unstable RNA, packaging and targeting to the appropriate cell (using liposomes with antibodies specific to cellular antigens is one possible solution here), and problems with immune reactions, cytotoxicity and accumulation in renal and liver tissues, to name only a few of these hurdles.