Understanding the mechanics of RNA silencing

The introduction of double-stranded RNA (dsRNA) can elicit a gene-specific RNA interference response in a variety of organisms and cell types, effectively preventing the transcription, or “silencing” the target genes. This formidable phenomenon has lead to the development of a whole new research field, with unbounded potential for treatment of various autoimmune and infectious diseases. Once inside cells, long dsRNA molecules are cleaved into double-stranded small interfering RNAs (siRNAs) by a Dicer enzyme. This is the earliest step in the RNAi silencing mechanism. siRNAs are long enough to mediate sequence-specific mRNA cleavage, silencing the transcription of the corresponding gene.

http://www.molbiolcell.org/cgi/content/abstract/14/7/2972

The following article expands upon the basic findings of RNA interference by Craig Mello and Andrew Fire. Although we have a general model for cellular uptake of dsRNA responsible for silencing, the specific mechanisms involved are unclear. The model does not account for the variability in efficacy of RNA silencing between different cells type. Also, the mechanisms behind the systemic nature of the interference response, meaning how RNA silencing spreads to regions away from the site of dsRNA delivery, are unknown. Incidentally, genetic variability can influence the extent of RNA silencing in organisms, and more specifically, the trafficking of RNA silencing signals. This has an impact when considering RNA interference as a treatment method. Indeed, the variability of response to RNAi therapy within a population would mean implementing more personalized and expensive treatments down the line.

To illustrate the versatility of this RNA interference pathway, the article points out four distinct methods for delivery of dsRNA into an organism: injection into a given site, feeding the organism with bacteria engineered to express dsRNA, soaking the organism in dsRNA, and finally in vivo transcription of dsRNA from transgene promoters. This article specifically assessed the ability of in vivo-delivered dsRNA to elicit systemic (global) RNA silencing. This was done by introducing transgenes that expressed dsRNA under the direction of tissue-specific promoters into C. elegans worms. Promoters are short sequences of the DNA strand that facilitate the transcription of the gene in that discrete region. Loss of fluorescence through the GFP gene was the marker for successful RNAi.

Several interesting mechanics were observed in the interference of GFP expression. Firstly, only those tissues that expressed the interference trigger (gfp hairpin, promoter for the GFP dsRNA transcription) exhibited loss of fluorescence, while the other tissues were not substantially silenced for GFP. This suggests cell specificity of dsRNA action through the in vivo transcription pathway, and a potential for tissue-specific interference of dsRNA. Secondly, and in the continuity of the observed specificity, the extent of silencing was dependent on the method of delivery. Exposure to extracellular nonspecific dsRNA by soaking or injection lead to RNA silencing in many more cells and their progeny, provided these cells had proper endogenous dissemination of RNA silencing signals. Thus, dsRNA introduced from exogenous sources can spread to distant tissues. This systemic silencing is thought to be related to a protective response from circulating virus, where exogenous viral dsRNA triggers the silencing of viral DNA in infected cells. Essentially, this shows that the action of RNAi is strongly related to the mode of delivery of dsRNA into an organism.

These findings are of paramount important for RNAi therapy. Doctors and researchers will have to take into account factors such as mode of delivery of dsRNA and genetic variability of the patients in trying to find the best therapeutic applications. Also, we will need methods to control for the extent and specificity of RNA silencing, especially for treatment of cancers and viral diseases, where accurate cell-targeting for gene silencing is primordial (in order to avoid silencing the genes of “healthy” cells). RNAi therapy is still at its dawn, and with much potential to be elucidated, but may bring the reward of powerful treatment against otherwise uncurable diseases.