I may wear the lab coat cape, but I’m not a real superhero – today I want to introduce you to a real (really tiny) one: meet Miranda the miRNA – one small RNA on a mission to make a big difference (in cellular protein production). In a sort of cellular David vs. Goliath story, micro-RNAs (miRNAs) direct protein-recipe-destroying machinery at specific “target” recipes (mRNAs) to prevent production of specific proteins. What’s this tiny RISC-taking superhero’s origin story? The birds and the bees of microRNA BIOGENESIS
Not all genes hold instructions for proteins. You might have heard of the “central dogma” of molecular biology – that sequences of DNA called GENES get copied into RNA (TRANSCRIPTION) which gets used as instructions for making a PROTEIN by protein-making complexes called ribosomes (TRANSLATION)
But not all genes have this fate, only “protein coding” ones. And not all RNA serves that DNA-protein “go-between” “recipe” role. That’s just one type of RNA – messenger RNA (mRNA). 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”
One of my favorite types is microRNA (miRNA). These are small (only ~22 nucleotides (nt) (RNA letters) long) but mighty! Their sequences match sequences in specific mRNAs (protein recipes), so they’re able to bind those mRNAs. And they bring mRNA-destruction machinery with them (before they bind the targets they’re loaded into a protein called Argonaute (Ago) which can shut down some targets on its own and call for backup as needed.
Just like storks don’t really bring babies, miRNAs don’t just magically appear. They have to be made. They’re born similarly to their enemies (they are both transcribed from DNA genes) but then their paths diverge early on and they mature differently. mRNAs don’t leave their birthplace (the nucleus) until they’re fully mature, but miRNAs leave home before they’re fully mature, finishing their maturation in their “final destination” the cytoplasm.
mRNAs are transcribed into pre-mRNAs, which get edited through a process called splicing that removes regulatory introns and stitches back together the protein-coding exons. They also get a 5’ (starting end) 7mG cap (a modified backwards nucleotide) and a 3’ (ending end) poly(A) tail (lots of the RNA letter A) added “generically.” The cap & tail help tell the cell that those are mRNAs. It helps them get trafficked out of the nucleus and into the cytoplasm, where protein-making machinery recognizes and binds them and gets to work.
Since miRNAs are NOT mRNAs, they don’t need (or want) those mRNA identifiers. So, even though they do get a cap when they’re born (cuz that’s coupled with transcription) and some get a tail, those are removed during processing, where the miRNA gets cut out of the middle of the original transcript.
The cap & tail also provide protection for mRNA’s ends because there are RNA exonucleases (end chewers) that degrade “raw ends.” Since miRNAs don’t have this protection, they wouldn’t last long without protection from proteins, so they’re passed off from one protein to the next.
miRNAs often have their own genes (though multiple miRNAs can be transcribed from the same gene and then separated) and their sequences are such that they fold back upon themselves into long hairpins (aka stem-loops)(you might be familiar with “base pairing” between strands of DNA or RNA? well, base pairing can also occur within a strand, and RNA often tends to do this).
These long hairpins are called primary miRNA (pri-miRNA) are bound in the nucleus by a complex called Microprocessor that contains a pair of RNA scissors (RNA endonuclease) that chops of the non-loop end of the hairpin to form a shorter hairpin (~60nt) called pre-miRNA.
Microprocessor is heterotrimeric – it as 3 (tri) parts and the parts aren’t all the same (hetero). The scissors-part (endonuclease) is a protein called Drosha and the other 2 units are copies of its helper, DGCR8 (aka Pasha in flies and worms) which helps recognize it as a pri-miRNA in need of processing and holds it in place for Drosha to cut. Drosha has 2 pairs of scissors (2 RNase III) domains. One pair cuts on one side of the hairpin & the other pair cuts across from it (with a 2nt offset so it leaves a little overhang)
This (still premature) miRNA then gets transported into the cytoplasm. Since it’s not an mRNA and it doesn’t have a cap complete with binding partners to help shuttle it out the mRNA way, it uses a different helper, Exportin 5 (Xpo-5). Xpo-5 clamps onto the base of the hairpin, protecting its ends. And it also binds another protein called Ran, which binds GTP to provide energy to power the process. Xpo-5 helps shuttle the pre-miRNA out, then Ran GTPase-activating proteins in the cytoplasm convince Ran to “spend” the GTP (hydrolyze it to GDP), causing it to change shape and release the pre-miRNA and the exporters get recycled.
In the cytoplasm, instead of being bound by protein-making workers, pre-miRNA is bound by another pair of scissors called Dicer. Dicer lops of the loop, turning the pre-mRNA hairpin into an miRNA duplex. Like Drosha, Dicer cuts both strands at an offset, so the duplex has ~2nt 3’ overhangs on each side (each side being ~22nt long)
This duplex gets loaded into another protein called Argonaute (Ago), to form a pre-RISC (RNA Induced Silencing Complex). Pre??? Yup – you’re still not done!
The targeting power of miRNA comes from it containing a sequence that’s complementary to (can base pair with) a sequence in the target mRNA (usually in the target’s 3’ UTR (untranslated region) (the sequence past the end of the protein instructions but before the generic tail).
But in the duplex form, the guide strand miRNA’s sequence is being hidden by the “passenger strand” (the other half of the duplex that was across from it in the hairpin). So Ago ejects the passenger strand to form a mature RISC and positions the guide strand to go searching for targets. When it finds one, it shuts down protein production. more on how 👉 http://bit.ly/2BuEcpr
I like to think of RNAi working a bit like GPS, where miRNAs contain an address for a specific mRNA or set of mRNA “targets” to be silenced, which gets entered into a self-driving car (binds Ago), and the car takes it to that address (we call this address-loaded car the RNAi-induced-silencing complex (RISC)). The car knows where to “park” because the sRNA’s sequence is complementary to a sequence in the mRNA target. 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).
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.
Ago can also give rides to miRNA’s “cousins” – small interfering RNA (siRNA) which come from chopping double-stranded RNA that using dsRNA that comes from outside sources – plants and bacteria use it to protect against foreign viral invaders. Mammals like us rely on more complex immune systems for this, so our RNAi system mostly uses miRNA. But it can also process siRNA which scientists often use to their advantage.
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 crank down that protein’s thermostat knobs. This is often called RNAi knockdown.
more on topics mentioned (and others) #365DaysOfScience All (with topics listed) 👉 http://bit.ly/2OllAB0