With the price of avocados rising, many restaurants are taking avocado-y items off the menu – the chefs still know how to make it, they just don’t because customers can’t order it. Just like a restaurant’s menu changes seasonally, our cells produce different proteins at different times but they still have the genetic recipes for making the proteins that aren’t “on the menu” at some point in time. If cells are like molecular restaurants, where the chefs are ribosomes and the dishes they make, how do your cells take proteins off the menu? I spend a lot of my time researching one such way – RNA interference (RNAi). This mechanism sends recipe destruction machinery on the hunt for specific recipes based on “key words” hidden in them. And at stakes is a lot more than a few bad Yelp reviews…
Your body contains billions of cells, each of which has to be able to respond “in real time” to what’s happening around (and inside) you. Responses often involve the use of molecules called proteins, whose original recipes (GENES) are written in DNA and locked up in a membrane-bound compartment called the nucleus. These original copies can’t leave the nucleus (like a reference section of a library), so there’s a lag time between when a cell realizes it needs a protein and when that protein’s ready to use; it has to make a copy of the genetic “recipe” for that protein (transcribe the DNA sequence into messenger RNA (mRNA)) and send that mRNA copy to the “chefs” (ribosomes) that translate it into protein, linking together the protein letters (amino acids) the RNA letters (nucleotides) say to.
One way to help cells respond more quickly is to stockpile the mRNA, but this introduces new complications; you have to tightly regulate its translation to prevent problems like cancer from occurring and you need a way to get rid of mRNAs for proteins you know you won’t need again for a while so that, instead of them taking up space and resources, you can recycle their pieces to make mRNA for proteins you will need.
mRNA is regulated in large part by a fundamental, evolutionarily-conserved process called microRNA (miRNA)-mediated regulation, or RNA interference (RNAi) and it involves types of RNA you don’t usually think of regulating the type of RNA you usually think of (hopefully you think of RNA 🙂 ).
See, RNA can have other functions than as a DNA-protein “go-between” “recipe” role. That’s just one type of RNA – messenger RNA (mRNA). It tends to get most of the attention but there are lots of other types of RNA that are the “end of the line” (i.e. they’re not instructions for some other product, they *are* the product). These are sometimes referred to as “noncoding RNAs” or “functional RNAs”
The type I study most is microRNA (miRNA) – they may be “micro” in size but they’re “macro” in importance! – they work to regulate over half of all our genes. miRNAs have their own recipes in DNA and get written (transcribed) just like the like protein-coding genes do, but they get processed differently. They’re written (transcribed) as long hairpins but they get chopped a couple times and, after processing, they’re ~20 nucleotide long & single-stranded. On their own they can’t do much but when bound to a protein called Argonaute (Ago) it’s like an address typed into the GPS of a self-driving car.
Different miRNAs contain different “addresses” for a specific mRNA or set of mRNA “targets” to be silenced (recipes to find & destroy). This address gets entered into a self-driving car (binds a protein called Argonaute (Ago)), and the car takes it to that address (we call this address-loaded car the RNAi-induced-silencing complex (RISC)) and does its thing.
The car knows where to “park” because the miRNA’s sequence is complementary to a sequence in the mRNA target. RNA letters can base pair with each other – like matching puzzles pieces – “A” binds “U” and “C” binds “G.” So you can get 2 complementary sequences to bind to each other and a sequence can act as a guide to direct silencing machinery to a complementary sequence
Once parked, the car can either directly silence it or recruit other helper proteins to repress translation of the mRNA (temporarily keep it from being made into protein) and/or degrade the mRNA completely so they can’t be used to make more protein (don’t worry – these are just copies of the “original” DNA instructions that are still held safely in the nucleus).
Which way it goes depends in part on how well the sequences match. There’s a critical “seed sequence” of ~6-8 letters in the beginning that serves as the “code word” – this “has” to match, but the whole thing doesn’t have to match in order for Ago to bind and recruit mRNA degradation machinery. But if the whole sequence *does* match, and it’s in Ago2 (the only one of the 4 human versions of Ago that can slice) Ago can cut the target sequence. This type of fully-complementary guide often comes from a different type of small RNA (sRNA), which comes from double-stranded RNA (dsRNA) that usually comes from exogenous (outside sources).
Plants and some other critters use siRNA for antiviral defense and stuff but we have a more complex immune system to take care of those, so our RNAi system has shifted to miRNA. But our cells still have the capacity to deal with siRNA, so we can hijack the system to direct Ago to specific genes. Often if cell biologists want to see what a specific protein does in cells, they’ll use RNAi – they put double-stranded RNA that gets chopped into siRNA containing the protein’s mRNA address into those cells to selectively take that item off the menu. This is often called “knockdown”
But most of the time, our cells are dealing with miRNA & its imperfect matches, so Ago calls for backup, recruiting (with the help of a scaffolding protein called GW182) deadenylation (poly(A) tail removing) complexes & decapping complexes which remove the mRNA’s protective ends so they can be chewed up.
Instead of managing a restaurant, your body uses miRNA-mediated mRNA regulation as a sort of thermostat to regulate levels of proteins being made inside each of your billions of cellular homes. It’s always working (or it better be!) and It works on tons of targets using miRNA it makes.
But what miRNA to make? Depends on what recipes you want to take off the menu. And there are LOTS of options! If you had to have a totally unique one for each recipe that’d be way too complicated. So how to make things easier? Combine and conquer!
The “code words” that match the miRNA are usually in the 3’ untranslated region (3’UTR) of mRNA – this 3’UTR is like the “backmatter” of a recipe book (the index & glossary & stuff that’s there to help but doesn’t contain actual info about what to add when. mRNA have many target sites in their 3’ UTRs, so they can be targeted by multiple miRNAs. And the same miRNA address can take Ago to many different recipes because different mRNAs can have some of the same sites. This allows many proteins to be targeted by the same miRNA (or copies of it). And this is great for regulating functionally related proteins that are needed (and not needed) at the same time. Even if you want them at the same time, you want to be able to regulate them individually in case you overestimate demand for one of them, etc. So in addition to having some shared sites, you also have some different sites. So you can target different groups of proteins at the same time (think Venn-diagrammy)
If there’s an avocado shortage, it doesn’t just affect avocado toast – it also affects guacamole. So a restaurant might have to stop making both. If both of their recipes have the same “code word” the same miRNA can “erase” them both -> Often cells want to regulate the levels of multiple related proteins at the same time, so they often contain sites for the same miRNA.
But it’s not just an avocado shortage that might make you want to take guacamole off the menu. What if the restaurant finds that no one’s ordering it at breakfast time – they can take it off the breakfast menu – but they don’t want to take off the avacado toast at that time because people want it – this is why recipes have different combos of miRNA sites, allowing you to control related genes separately. And that’s where combos come in.
So, let’s all “tip” RNAi for keeping each of our cells running like a 5-star restaurant! And let’s thank the @IUBMB for giving me the chance to talk about my favorite topic in this week’s Bri*fing from the Bench, so I can “Do My Happy Dance” with Snoopy (I LOVE SNOOPY!)! And I also love all the awesome stuff the IUBMB does, so be sure to follow them for great biochemistry from around the world!
Since RNAi is my favorite topic, I’ve done several more in-depth posts on various aspects of all this stuff, and you can find links to them (and tons of other topics) here 👉 http://bit.ly/2OllAB0