RNAi – From Discovery to Nobel Prize in Record Time?

RNAi is Discovered!

Small, non-coding RNA caught the attention of the scientific community in 1993 in a paper published by Victor Ambros' research group, who was working with the model organism Caenorhabditis elegans (C. elegans) (1). The small non-coding RNA was later named microRNA (miRNA), and this is now known to be an endogenous product from the organism's own genome (if exogenous, it´s instead termed small-interfering RNA, siRNA) (Reviewed in 2). Some years later, American scientists Andrew Fire and Craig Mello discovered that simultaneous injection of sense and antisense dsRNA of the unc-22 gene (which codes for myofilament protein) into the gonads of C. elegans led to pronounced twitching in the subsequent generation (3). 

How Does RNAi Work?

So, how come the progeny of injected parent nematodes became strong twitchers upon injection of unc-22 sense and antisense RNA, and not when either sense or antisense was injected?

The explanation is RNA interference (RNAi) and the mechanism behind this is quite simple (Figure 1):

  • Double-stranded RNA (dsRNA) binds to the cytoplasmic enzyme Dicer, a member of the RNase III family (2).
  • Once loaded, the Dicer enzyme exercises its ribonuclease activity and cleaves the dsRNA into smaller fragments, typically 21-23 nucleotides in length. These smaller dsRNA fragments are now termed small interfering RNA (siRNA) (2).
  • At this stage, double-stranded siRNA is bound onto the RNA-activated silencing complex (RISC). The two RNA strands are separated, leaving the antisense strand bound to the corresponding mRNA whilst the sense strand dissociates (2).
  • The mRNA is then cleaved, thus resulting in mRNA degradation and suppressed protein synthesis (2).

Thus, when Andrew Fire and Craig Mello injected sense and antisense dsRNA homologues of the unc-22 muscle protein gene into the gonads of C. elegans, synthesis of the myofilament protein was suppressed. This caused progeny nematodes to twitch, similarly to individuals bearing a defective unc-22 gene (3).

Figure 1. The mechanism of RNA interference. In the cytoplasm of the cell, dsRNA attaches to the enzyme Dicer digesting the dsRNA to smaller fragments, typically 21-23 nucleotides. The antisense strand is transferred and associates onto the RISC protein. This enables the homologous mRNA transcript to bind the antisense strand. This results in cleavage and degradation of the mRNA thus suppressing protein synthesis.  Image adapted from (4).
Figure 1. The mechanism of RNA interference. In the cytoplasm of the cell, dsRNA attaches to the enzyme Dicer digesting the dsRNA to smaller fragments, typically 21-23 nucleotides. The antisense strand is transferred and associates onto the RISC protein. This enables the homologous mRNA transcript to bind the antisense strand. This results in cleavage and degradation of the mRNA thus suppressing protein synthesis. Image adapted from (4).

What Can RNAi Be Used For?

Since its discovery, RNAi has quickly risen to an all-star in genetic research and the first RNAi-based therapeutic was approved in the US and Europe last year. The significance of its discovery is further highlighted by the awarding of the Nobel prize in Physiology or Medicine to Andrew Fire and Craig Mello in 2006, just 8 years after their seminal publication (4).

Extensive efforts are underway to explore and develop RNAi-based treatments for cancer, with the goal of silencing oncogenes. Other candidate diseases for RNAi-based therapy include Huntington's disease, chronic liver diseases, eye diseases such as glaucoma and retinopathy in diabetes (reviewed in 2). However, the development of RNAi-based treatments isn't without challenges! To mention a few, the small RNA molecule is effectively cleared via the renal system, and that's if it "survives" the high amounts of nucleases present in the body. Furthermore, siRNA can exert a toxic immunological response by binding certain receptors of the innate immune system, for example, Toll-Like Receptors (2).

RNAi as a research tool is used extensively in labs all over the world and has gained the appreciation of many scientists. It has become easy to silence a specific gene with high success rate thanks to target-specific plasmids packed in viral vectors. This tailor-made genetic material often includes a fluorescent reporter such as Green Fluorescent Protein and various selection markers for use in both bacterial and mammalian hosts alongside the RNA sequence corresponding to the specific gene of interest.

Harness the Potential of RNAi

Perhaps it was the dual potential of RNAi in disease treatment as well as being a powerful research tool that led to the remarkably fast awarding of the Nobel Prize to Mello and Fire. When it comes to using RNAi as a tool in medical research, Nordic BioSite cooperates with the most knowledgeable suppliers and we´re always happy to help with your questions. Big or small!

References

  • Lee RC, Feinbaum RL, Ambros V. The C. elegans Heterochronic Gene lin-4 Encodes Small RNAs with Antisense Complementarity to lin-14. Cell. 1993;75: 843-854.
  • Liu F, Wang C, Gao Y, Li X, Tian F, Zhang Y, Fu M, Li P, Wang Y, Wang F. Current Transport Systems and Clinical Applications for Small Interfering (siRNA) Drugs. Mol Diagn Ther. 2018;22:551 https://doi.org/10.1007/s40291-018-0338-8.
  • Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent and Specific Genetic Interference by Double-Strand RNA in Caenorhabditis elegans. Nature. 1998;39: 806-811.
  • NobelPrize.org. Nobel Media AB (2018). The Nobel Prize in Physiology or Medicine 2006. Available at: https://www.nobelprize.org/prizes/medicine/2006/press-release/. [Accessed December 19, 2018].