The behaviour of genes can be affected by other genes and other elements that interact with genes, ranging from transcription factors to antisense transcripts that operate post-transcriptionally, and today the focus of my post will be something called RNA Interference (RNAi in short)
If one were to look at what happens to genes in terms of the central dogma of molecular biology, you will find that the biochemistry of genes can be written down as follows.
DNA ————-> RNA (Transcription) ———–> Protein (Translation)
RNAi acts on genes following transcription but before translation, and it does so by breaking down mRNA that would have been translated otherwise, and this is mediated by the RNA Interference pathway, something that is conserved in Eukaryotes (with the exception of certain fungal lineages, the interesting thing is that the loss appears to be independent in fungal lineages) and is, as I will elaborate later in this post, of much practical utility.
The next part of my post will deal with the RNAi pathway per se. But before we venture there, it would be prudent to have a brief outline of the components required, to put it extremely simply, we will need double stranded RNA and a set of enzymes to use double stranded RNA as the reference for destroying target RNA and then something to cleave the target RNA.
Double stranded RNA does the trick well, and basically long chains of double stranded RNA are cut into smaller double stranded RNA fragments of a length that is 20-25 base pairs long by an enzyme called, funnily enough, Dicer. These shorter double stranded fragments, that come with a two base overhang, are called siRNA (short interfering RNA).
siRNA then binds to Argonaute proteins to form what is called an RISC, a multi-protein complex that is involved in the final part of RNAi, the identification of complementary mRNA and the breakdown thereof. RISC stands for RNA Induced Silencing Complex, by the way.
A very good video of the process can be found below.
Organisms may use RNAi for gene regulation through the function of miRNA (microRNA) , here a long noncoding chain of RNA (pre-miRNA to be precise) is produced first by transcription, and this undergoes self-base pairing within the ssRNA strand to form a structure with loops and stems, further processing by enzymes like Drosha and Pasha help in the conversion of the long transcript into a double stranded form, which is mature miRNA, after which the RNAi pathway functions as described above.
That is about the jist of what RNAi is, but what about the implications? And what empirical standing do the proposed applications of this system have?
Well, we already know that RNAi can turn genes off, which means it is great when we are looking to target the mRNA of viruses et cetera, identify target sequence, find sites that are unique only to the viral mRNA, design dsRNA, introduce that into cells, and watch as the RNAi apparatus goes about marmalizing the viral genome. The very fact that some regions of viral genomes are conserved, even in highly variable strains like Influenza viruses means that broad spectrum therapies could be developed utilizing this process.
Want to study gene function and what happens if you knock a gene out? Same principle more or less, put in your dsRNA sequence, watch as the gene is silenced, observe for phenotypic changes, the very fact that one is now able to silence genes directly instead of generating knockout organisms by traditional mutagenesis and breeding is certainly very attractive.
Want to study or control gene regulation? Not a problem, you can just add in extra copies of miRNA or target miRNA for cleavage, either using other antisense approaches (antagomirs) or using complementary dsRNA. I hope you are now able to appreciate the scope of this technique.
There have been recent advances, too, such as the development of a technique called dicer substrate RNAi , wherein, instead of using long dsRNA which has to be processed by dicer, it is possible to introduce 25-28 nucleotide long double stranded RNA directly for integration with Argonaute, and this method, according to reports in the scientific literature, can greatly increase the efficacy of RNAi.
I am not sure that we have many RNAi therapies in active clinical use at the moment, but it sure does look promising. You may want to peruse the following resources for further information.
Wikipedia article on RNA interference.
RNAi Therapeutics: Principles,Prospects & Challenges (peer reviewed paper)
RNAi as a means of controlling lipid levels (peer reviewed paper)
Science : RNAi Collection (A collection of peer reviewed research papers)
Science: miRNA Collection (A collection of peer reviewed research papers)
Antagomirs et cetera and case studies of RNAi application will be parts of future posts somewhere down the line, until then, have a happy Interfering time :P.
These are always entertaining to watch because they give you an opportunity to ‘connect’ with these researchers in a way that research papers themselves do not. Happy viewing.