messenger RNA (mRNA) – a topic about which there is much to say! DNA tends to get the most attention because it’s what our cells use to store our genetic blueprint (genome). But SARS-CoV-2, the novel coronavirus that causes the disease Covid-19, keeps it’s blueprint in RNA. So we’ve been hearing a lot about RNA. But thing is, in my (admittedly biased) opinion, we should have been hearing about RNA for a long time. Because, even though humans and all organisms that we know of (yeah, viruses technically aren’t “living” even though they are quite ingenious), RNA plays a crucial role in every cell in every living thing. It can serve different roles, but one of the main roles is in the form of messenger RNA (mRNA), which acts as an intermediary between proteins and their DNA “recipes” (genes).
How it works is that the genome is kinda like a set of cookbook volumes (chromosomes), where the recipes are genes, many of which contain instructions for making proteins. These “original copies” of the recipes are really important to protect because they’re permanent & get passed onto all the cells made from that cell. So, when a cell wants to make a protein, it makes RNA copies of the DNA genes, edits them to cut out regulatory regions (introns) and add a cap and a tail, and passes these copies off to the protein-making complexes (ribosomes). Not only does this protect the original DNA, but it also allows you to make lots of copies of the encoded protein at once. And, when you’ve made enough, you can degrade the recipe copies – and RNA is less stable than DNA, so this is easier.
Yesterday, I told you about how we can deliver mRNA directly to cells to get them to make proteins, which is the strategy being taken with mRNA vaccines – introduce mRNA for viral proteins, get the cells to make those proteins -> immune system learns to recognize them without person ever getting sick. https://bit.ly/modernamrnavaccine
Our cells don’t normally have viral mRNA in them, but they do have a lot of mRNA of their own, so here’s a review of mRNA in general – note that since coronaviruses are positive-stranded RNA viruses, they can make proteins directly from that original genome they stick into our cells. And then they can make more mRNA from a copy of that. But since our original genome is in DNA, we always have to make mRNA before protein, no going straight from the original.
And we can do this because RNA is DNA’s molecular “sibling” – they both have nucleotide “letters” consisting of a sugar-phosphate base that allows them to link into strands and 4 unique nitrogenous “bases” that stick off and allow for base-specific pairing between strands. RNA & DNA differ in that RNA has an “extra” oxygen in its sugar and it has the letter “U” instead of “T” – but U & T can both bind the letter A; and C binds G, so you can use DNA as a template for making RNA, or vice versa.
If you think of proteins as “baked goods,” genes are the “recipes” for making them. All the recipes for all the proteins you should ever need throughout your life are collected together into “cookbook volumes” called CHROMOSOMES that are held in a membrane-bound compartment of your cells called the NUCLEUS. The nucleus is like a “reference section” of a library – you can’t check out these books, but you can make copies. And, if you want to bake a recipe, you *have* to make a copy because the bakers (ribosomes) are in the general interior of the cell (cytoplasm).
Not only can you not check out the original genes, but security in the reference section is very tight. So, after a (RNA)letter-for-(DNA)letter copy’s made in a process called TRANSCRIPTION, some of the information in the genes gets “redacted” (regulatory “margin notes” called INTRONS that are important for transcription but not translation) get removed and the remaining information (EXONS) get pieced back together in a process called SPLICING.
And even these “redacted” versions can’t leave the nucleus without undergoing a security check – certain proteins bind to specific features on the mRNA and enable it to pass through nuclear pores into the cytoplasm. 2 of the features nuclear security looks for are a cap and a tail. Most of the text in the mRNA is “templated” meaning that it comes word-for-word from the DNA version. But mRNA also has extra “generic” words put on in the form of a polyadenylate (poly-A) tail and a 7mG at the beginning.
These are kinda like the “front matter” and “back matter” of the recipe. You know how some books have blank pages at the end? And (if it’s not a library book and you’re really desperate) you might tear out one of those pages to use for scratch paper? This might anger some book-lovers, but it doesn’t change the integrity of the text. The poly-A tail’s kinda like that. It protects the “true text” – deadenylases (A erasers) can modify the length of the tail without touching the unique templated part.
This is important because the cytoplasm has “security agents” of its own in the form of RNA nucleases (RNAses) – proteins that cleave like scissors (endonuclease) or chew from the ends (exonucleases) unauthorized visitors.RNA letters (nucleotides) usually link up “left arm” (5’ phosphate) to “left leg” (3’ OH). So the “first” letter in the chain has a free left arm (5’ phosphate) and the last letter has a free left leg (3’ OH). Different exonucleases are specialized to recognize & chew these different ends.
A complex called the exosome (not to be confused with the membrane-bound vesicles that can bud off from cells) can chew in the 3’-5’ direction. And 5’-3’ can be handled by Xrn1 and friends. But it will only chew “naked ends” (it needs a 5’ phosphate) and the 5’ cap “hides” the 5’ phosphate -> like putting a glove on it. Biochemically, it’s a “backwards letter” -> a G put on 5’ to 5’ instead of 5’ to 3’. And it has a methyl (-CH3) group added onto the 7 position to make it extra special.
This cap serves as a sort of “copyright page” It tells the cells – this is a legit recipe. If there’s no cap, it could be a “forgery” – foreign RNA such as that from viruses doesn’t have a cap (but some viruses have evolved clever tricks to “steal caps” from legit ones or make their own – SARS-CoV-2 makes its own for example)
This cap has to be removed before the 5’-3’ chewing can begin, and usually this is “egged on” by chewers at the opposite end. It may seem strange, but the 5’-3’ mRNA degradation pathway, which is the predominant pathway our cells use, starts with de-tailing and that’s at the 3’ end.
Those chewers (deadenylases) are more picky about what they chew – they only want that A. And they often don’t “eat it all” – instead they “trim” the tail to affect how efficiently the recipe gets translated and how long it lasts. There are a couple main chewing complexes: PAN2/PAN3, where PAN2 does the chew – and PAN3 helps keep it in the right place and help it hang out with the right friends – it binds poly(A) RNA, PABP, and – in the case of miRNA-mediated mRNA decay, GW182.
That RNA interference (RNAi) form that uses small pieces of RNA called microRNA, guided by a protein called Argonaute (Ago) to target specific mRNAs with complementary sequences but instead of cutting it, Ago “needs help” – and GW182 helps scaffold it to various things – including PAN2/PAN3, and the second de-tailing complex, CCR4/NOT (where CCR4 is the chewer) – this is how you can connect Ago binding to mRNA decay. More on miRNA-mediated decay here: https://bit.ly/rnainterference
But you don’t need miRNA, Ago, or GW182 to do decay the “normal way” When the tail gets short enough, deadenylases recruit decapping factors, which provides a direct link between deadenylation and decapping.
The protein can still get made with a shorter tail, but, once you take the cap off, party’s over. for a couple of reasons. In addition to freeing the end to get chewed up by 5’-3’ exonuclease like XRN1, without a cap you “don’t have a kitchen” – you lose the proteins that help get the ribosomes going. The cap also serves as a sort of “kitchen.” All recipes basically need the same prep work, so it’s ok that the cap’s generic. Actually, it’s more than just “okay” – it’s a major time/resource saver!
At the biochemical level, what’s going on is that different protein “workers” can bind to the cap – and the proteins that bind to the cap – or the proteins that bind to the proteins that bind to the cap -> you can get a whole interconnected network of players that come and go at different points.
In the nucleus, the cap is bound by a “nuclear cap binding complex” (CBC) that includes NCBP2 & NCBP1, which recruit proteins involved in splicing, 3’-end processing, export & the first (pioneer) round of translation.
Translation is where a ribosome travels along the mRNA and links together the amino acids (protein letters) dictated by the recipe and brought by transfer RNA (tRNA) to make a long polypeptide chain that folds up into a functional 3D protein. This happens in the cytoplasm.
So, once the mRNA is in the cytoplasm, it swaps out the nuclear components for the eIF4F protein complex that promotes translation initiation: eIF4E binds the cap; eIF4G acts as a scaffold to hold things together; and eIF4A (or other isoforms) acts as a helicase (unwinder) to help with RNA remodeling. eIF4F recruits initiation factor eIF3 -> that recruits initiator tRNA & 40S ribosome subunit.
So how does the mRNA get decapped? Depends on the pathway. In addition to the 5’-3’ pathway, there’s also a 3’-5’ pathway where the deadenylases hand off the detailed RNA to the exosome which is much less picky and much more hungry. It chews most of the way, but it can’t handle that special linkage. So you need a decapper. The different pathways call in different ones which leave different marks – You have to break a triphosphate linkage and 3 isn’t an even number… like splitting a wishbone different ways -> generate slightly different (but still chewable) products.
In the 5’-3’ decay pathway, the Dcp2-Dcp1 complex does it. Dcp2 does the actual cutting, but Dcp1 helps. Dcp2 is in the “Nudix” family (which refers to its ability to cleave NUcleotide DIphosphates linked to some thing “X” not the fact that it makes RNA “nude”). When it cuts, the G gets 2 phosphates & the chain only gets 1: m7GpppRNA -> m7Gpp + pRNA.
In the 3’-5’ pathway, where the main chewer’s the exosomal complex, a “scavenger,” DcpS, cuts it off. When it cuts, the G only gets 1 phosphate & the chain gets 2: m7GpppRNA -> m7Gp + ppRNA.
DcpS doesn’t serve as the decapper in the other pathway in part cuz it’s only good at this if the RNA is short, like once it’s already been chewed most of the way from the other end
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