Today is a biotechnology-based day on Microbial Mondays. Lately, I have been doing a lot of experiments using "RNA interference". This is a technology that I can use in the lab to suppress the production of any specific protein within a cell that I'm interested in. This is an interesting undertaking because, well, all of the important things that happen in cells, happen thanks to proteins. And, when many individual proteins come together, they can create quasi-production lines, which carry out all of the cellular processes necessary to keep us all alive, from autophagy to metabolism to immune defense.
One of the major reasons why scientists use RNA interference technology is to pick apart which proteins are parts of which production lines. RNA interference is used to "knock-down" one or two proteins at a time - sort of like pulling out a single worker in a production line - to see what happens in the overall picture. The idea is that, if you pull out a key worker, you should see an effect on the eventual product that is produced. Or, alternatively, if you pull out a worker who toils solely on production line A, you won't see any impact on production line B. In biological terms, if you knock down a protein that is only important in immune defense against bacteria, you won't see any effect on fungal or viral infections upon knocking down that protein. However, if that particular protein is important for antimicrobial defense in general - if it has a more central role to play - then you would observe that if you knock it down in a population of cells, for example in some immune cells isolated from a blood donation (thank you donors who allow their blood donations to be used for science!), those cells might become more susceptible to infection with any microbe you throw at it. In other words, RNA interference helps scientists to identify "molecular mechanisms" behind our biology - i.e. map out the protein-based production lines. The plan to "unravel molecular mechanisms" has kind of become a tagline that is included on every single scientific grant these days - although, as it was recently pointed out by Barry Fitzgerald at a Science Communication workshop that I attended, you don't unravel a mechanism, it's not a ball of wool. But, anyways… whether we are unraveling or deciphering or simply "picking apart" a "molecular mechanism" - how would RNA interference technology help us scientists with that?
RNA interference takes advantage of a theory often referred to as "The Central Dogma" in biology. This central dogma holds that, 1) DNA is 'transcribed' (a sort of translation from one type of biological molecule to another) into RNA, and then 2) that RNA is respectively used as a blueprint to build proteins, the machines that permit the dynamism of life. What RNA interference does, is throw a crowbar into the second stage of the Central Dogma, by taking advantage of a sort of inherent stickiness of RNA. RNA interference actually involves introducing a new piece of RNA that will stick specifically to the RNA required to make the protein that you want to knock down. When the new piece of RNA you've introduced has stuck to your target RNA, that target RNA will no longer be readable as a blueprint. In other words, you stick a wad of RNA-gum onto the RNA-blueprint required to make the protein, and the cells you're working with won't be able to make more of that protein in question until they yank the gum off of the blueprint. As this gum analogy suggests, RNA interference isn't permanent, but it is definitely effective at temporarily blocking protein production.
At the moment, I'm using RNA interference to identify specific proteins that are involved in both autophagy, and immune defense against viruses and other microbes, to see where these two areas that I'm interested in cross over. However, in my opinion, the coolest bit of this whole story is that when I use RNA interference to study defense systems against viruses, I am actually using a defense system against viruses to study a defense system against viruses! RNA interference naturally evolved across many different domains of life, including fungi, plants, and animals, most likely to serve as a defense mechanism against viruses that use RNA as their genetic material. Just like how it is used in the lab, this "natural" RNA interference would basically stick a wad of gum onto the blueprint for the virus - effectively stopping the virus in its tracks. While the virus is trying to get the gum off of itself, other defense systems in the cell can join in the fight, and try to dispose of the virus while it's still all stuck up in RNA-gum. Meta, eh?
Until next time - dispose of your gum carefully! You never know what it'll stick to.
The inspiration behind this Microbial Mondays post was this handy how-to guide on high-throughput RNA interference screening:
Falschlehner C, Steinbrink S, Erdmann G, Boutros M. High-throughput RNAi screening to dissect cellular pathways: a how-to guide. Biotechnol J. 2010 Apr;5(4):368-76. doi: 10.1002/biot.200900277. PMID: 20349460.