mRNA Processing - Online Tutor, Practice Problems & Exam Prep

RNA processing is essential for converting pre-mRNA into mature mRNA, which involves three key steps: 5' capping, polyadenylation, and RNA splicing. The 5' cap, a methylated guanine, protects mRNA from degradation. Polyadenylation adds a poly(A) tail to the 3' end, enhancing stability. RNA splicing removes introns and joins exons, facilitated by the spliceosome, which consists of small nuclear RNAs (snRNAs) and proteins. Alternative splicing allows for diverse protein products, influencing gene expression. RNA editing can also occur, making small nucleotide changes to the transcript.

mRNA Processing Overview

Hello, everyone. In this lesson, we are going to talk about RNA processing. Okay. So whenever an mRNA is made or it is transcribed, it's not entirely ready to go. It's not just an mRNA. It's actually called a pre-mRNA or an mRNA transcript. And pre-mRNAs don't have the same characteristics as mature mRNAs. In fact, we actually have to do different things to the pre-mRNA to make it into a mature mRNA. And this processing occurs in the nucleus of the cell, and it is required before the pre-mRNA or the mature mRNA can even leave the nucleus. So we know that transcription happens in the nucleus because that's where the DNA is, but processing also occurs in the nucleus and once the whole event all the steps of processing have been finished then the mature mRNA is allowed to leave the nucleus because it has all of the characteristics that it needs.

So why do we do this? What is the point? Well, the processing does distinguish mRNA from other RNAs. So mRNA processing is a unique thing. Also, there is tRNA processing which is also unique. There's ribosomal RNA processing which is also unique and this is going to give characteristics to these RNAs so that they have their own distinguishing characteristics. They are recognizable. So mRNA processing allows mRNA to be unique and recognized in comparison to other types of RNAs and this process happens then translation happens. But actually, as RNA processing happens then translation happens. But actually, as the mRNA is being transcribed into pre-mRNA, it is at the same time being processed to turn into mature mRNA, and then it will go on to translation.

So what are the different processes that you are going to need to know? Well, there are going to be 3 major things that happen in RNA processing. The 5 prime cap is going to be added to the 5 prime end. The poly-A tail is going to be added to the 3 prime end, and RNA splicing is going to occur. That is going to be the removal of exons or the removal of introns, excuse me. The removal of introns and the keeping of exons. Now remember, mRNA does not have introns, but pre-mRNA does and it has to be removed. And that is going to be a process called RNA splicing, which is going to be one of the steps of mRNA processing.

So what is this generally going to look like? Well, generally, you're going to have your mRNA. So this is your mRNA here. And it is going to have no introns. It is going to be composed of only exons. Okay? And then it is going to have a 5 prime end and a 3 prime end. And on the 3 prime ends you're going to have a poly-A tail. And on the 5 prime end you're going to have this cap. And, this is going to be your 5 prime cap. And, these things are utilized to distinguish mRNAs from other types of RNAs and also protect the mRNA from degradation because once it enters into the cytoplasm, it can be destroyed by other proteins if it doesn't have all of these mechanisms and these characteristics.

So let's talk about how this processing actually occurs. So we're going to talk about the C-terminal domain of the RNA polymerase number 2. Remember RNA polymerase number 2 is going to be the specific RNA polymerase that actually transcribes mRNA from DNA. And it is going to have this special domain called the C-terminal domain, which carries proteins that are responsible for RNA processing and they're going to be utilized in mRNA processing. So as the RNA polymerase builds the pre-mRNA transcript to the C-terminal domain is interacting with that transcript and processing that transcript. So actually this C-terminal domain which is also abbreviated CTD. The C-terminal domain is involved in a lot of things. It's actually involved in the initiation of transcription. It helps that process begin. It's involved in the capping of mRNA and it's involved in the attachment of the spliceosome to the pre-mRNA transcript.

Remember I said those introns have to be spliced out and those exons have to be glued back together? The spliceosome complex of proteins is going to do that job. So C-terminal domain very, very, very important on the RNA polymerase because it helps with transcription and RNA processing. Okay. So now we're gonna talk about some other proteins which their name is Heterogeneous Nuclear Ribonuclear Proteins which are simply HNRNPs and they are going to be our protein and RNA complexes. So they're gonna be proteins and specialized RNAs that are bound together and they are going to bind to RNA inside of the nucleus. And generally, they're going to bind to the RNA in the nucleus while it is being transcribed and while it is being processed. And why would it and which means it can complementarily

5' RNA Capping

Okay, so the first mRNA pre-mRNA processing event that we're going to talk about is five prime capping. This RNA capping occurs at the five prime end of the transcript, which is why it's named that. So, how does this work? Well, there are three enzymes responsible for adding this cap, and what this cap is, it's a special methylated guanine. Remember, a methyl is a chemical group, right? So, that gets added to a guanine and then that methylated guanine gets added to the five prime end of the transcript.

So, here's how this works: you have this phosphatase, and it removes a phosphate from the five prime, leaving this binding site open. Then another enzyme comes in, the guanylyl transferase, and they add a GMP, which is kind of very similar to an AMP. But it uses guanine instead. So, guanylyl transferase is actually a unique protein that adds guanine in this unique way. You don't necessarily need to know anything about it, but generally, things don't bind five prime to five prime. But in this case, it does; it binds five prime to five prime, and it's just sort of unique.

And then, once the guanine has been added in this really unique way, you then have a methyltransferase that adds a methyl group to the guanine at a certain position on its sugar on the ribose ring. So, I've highlighted a lot of things, what do you need to know? You need to know that the five prime cap adds a methylated guanine onto the five prime end. And if you really want to get special, get some extra credit or whatever, sort of know that the guanine occurs in three steps: with a phosphate being removed, a guanine being added in this unique 5-5 way and then it becoming methylated.

So, once the five prime capping, oh so when does the five prime capping occur? It occurs pretty immediately after transcription. Once there's been about 25 nucleotides of RNA transcribed, the five prime cap is added, and the purpose of the five prime cap is for processing, and you need it. But it also has a really important function in preventing RNA bases, which are enzymes that can break apart and cut apart RNA, from destroying the transcript as it's being transcribed because you kind of you don't want RNA scissors coming along, chopping up your transcript as RNA polymerase is chugging along; you want to be able to maintain it and prevent it from being degraded. And so, the five prime cap does that.

So, if we're to look at what the five prime cap actually looks like, here you have your mRNA, and here's the five prime end. You can see that here. And here you have your methylated guanine. Here's the structure of it, and you'll notice here that it binds uniquely five prime to five prime. This cap gets added on every pre-mRNA transcript that's going to eventually become a protein. So now let's move on.

3' Polyadenylation

Okay, so now we're going to talk about 3' polyadenylation, which is a second form of pre-mRNA processing that must occur. So what does this mean? What is RNA polyadenylation? Well, it is the addition of repeating adenine nucleotides at the 3' end of the transcript. So, this is at the end of the transcript. This edition of A's over and over again can actually get fairly long. But so, this is at the end and so RNA. So how does it start? Well, the first thing that happens is RNA is cleaved at a specific sequence. There are two cleavage signals that exist. One is this, and one is a GU-rich region. Just, you don't necessarily need to memorize these sequences. Just know that RNA is cleaved at really specific cleaving sequences. And there are a couple of factors called cleavage stimulatory factors that come in, bind to these sequences, and say, okay, this is where RNA is going to be cleaved so that the A's can be added. And if you are interested, you may read about them in your textbook, but CPSF and CSTF are acronyms for the two main cleavage stimulatory factors.

Now, once RNA is cleaved, what happens? Well, this polymerase comes in called the poly A polymerase, and it just starts adding A's, you know, that's what it does. So it begins at an AU-rich site located right near where the cleavage site was. Generally, the first 12 A's are added fairly slowly. But then, once it gets past that hump of 12, it goes much faster. And there are around 150-200 A's that begin adding. Now, this is just because it's just repeating As. It doesn't need a template, like other things like transcription and DNA replication need a template. But this doesn't need a template because it's just adding As.

So let's look at this. Let me back out of the way. So here we have RNA polymerase, and it's working its way this way, transcribing. So here's the start of the transcript, and we know it's the start of the transcript because right here, it has its 5' cap, and it's also from the 5' end. So those are two reasons why we know. So it's been transcribing, transcribing, transcribing, and it gets to a polyadenylation signal, a cleavage signal, and this G-rich sequence. Now remember, these two are sequences that are both cleavage signals. So what happens is something comes in and cuts this out. So now you're left with just this. And once you're here, what happens is the poly A polymerase (PAP) comes in, binds this AU region, and starts adding As. So, what you get left with at the end is you have a 5' transcript with a 5' cap, your transcript, and it ends with a bunch of A's, approximately 200 or more. And so that is two processing steps that lead to the formation of an mRNA. So now, let's turn the page.

RNA Splicing Overview

Okay, so now we're going to talk about a third mechanism of mRNA processing, and this is RNA splicing. Now, I'm going to talk a lot about RNA splicing, so much so that I've actually split it up into two videos. It's a big topic and is going to include a lot of information. This first video is going to focus on just an overview of RNA splicing. So let's get started.

What is RNA splicing? RNA splicing occurs when introns, which are the non-coding segments of the transcript, need to be spliced or removed from the pre-mRNA. RNA splicing removes introns from pre-mRNA. This can be kind of difficult for enzymes. Okay, first, how do enzymes recognize where the introns are? This can actually be really difficult because introns vary greatly in size. So when scientists go in and try to find all the introns, they really have difficulty finding them because they're just all over the place and they don't code for anything, which makes it hard to find them. What scientists do is they actually use exons to find introns, and the proteins in the cell do the same thing. Exons, which are the coding sequences if you remember, average about 150 nucleotides long. So when you get to an exon and you travel 150 nucleotides, you say, "Okay, well an intron is beginning somewhere in here," and that's exactly what the proteins do. We'll talk more about that in future videos.

Introns contain specific sequences between 30 and 40 nucleotides long that signal for splicing. Generally, these are really close to exons. There are three that we are going to talk about: the 5' splice site, which begins with a GU sequence or an AU sequence (GU is more common than AU); the 3' splice site, which begins with an AG sequence or an AC sequence (AG is more common); and the branch point, which are special sequences located several dozen nucleotides upstream of the 3' end. These sequences here, which have consensus sequences or more common sequences, are GU or AG depending on 5' or 3', and then there's also the branch point.

These are sequences that say, "Hey, I'm here, come splice me out." Because it's confusing when I talk about it, I realize that it's also kind of confusing for the proteins themselves, but they get it right most of the time, which is good because we are alive. However, improper splicing actually accounts for 15% of genetic disorders, which is a pretty high amount. So they're not really that great at it, but this is the system we have. So this is what we've got to learn.

If we're looking at an overview of RNA splicing, here you have your pre-mRNA, you have your exon and your intron is here. You can see these nice sequences, GU at the 5' end, and AG at the 3' end, and the signal they say, "Okay, come in here and splice me out." And that's what happens; the intron gets spliced out and you are left with the joining of the exons together. So, what's the protein that comes in and actually splices the introns out? Well, this is called the spliceosome and this is responsible for splicing most RNA. The spliceosome consists of small nuclear RNA, or snRNA, and small nuclear ribonucleoproteins, or snRNPs. The snRNA comes in five groups and we're going to talk about them a lot more in future videos. The snRNPs are formed by about 6 to 10 proteins that bind to the RNA. The spliceosome isn't just a protein, it's a complex composed of RNA and proteins. The snRNPs are responsible because they form this sort of core of the spliceosome and they are actually what recognize and bind to the splice sequences. They bind to the 5' splice sequence, 3' end, recognize the sequences that say, "Hey, cut me out," and then the spliceosome can act. So that's the overview of RNA splicing. Bear with me, it's going to get more complicated. But let's now turn the page.

Splicing Steps

Okay, in this video, we're going to be going over the seven steps of splicing. So, you're going to notice a lot moving forward in the cell biology class that we're going to be going over a lot of steps that are going to kind of look like this, where you have these steps, I've numbered them and off to the side, there's an image of what's going on and so you just need to get ready and sort of understand that, you know, these steps are complicated. You're in cell biology. But you know, let's try to do the best that we can to get through them and understand exactly what's going on so that we can understand how the spliceosome works and how splicing actually occurs. I'm going to disappear so that you guys can see these pictures that are off to the side.

The very first step of splicing occurs when the SNRNP and also the U1 snRNA binds to mRNA at the 5' prime. So, what does this mean? You have your 5' prime sequence here and you have your U1 snRNA binding here. Now, step two occurs when the U2 snRNA binds to the branch point sequence. So, what do we have? We have this branch point sequence. I didn't even put up here, 5' prime splice site and you have your U2 snRNA. Now the binding of the U1 and the U2 allows for step three to occur. And step three is the recruitment of other proteins. And so you can see here you have all of these other snRNA that get recruited to the intron.

Okay, step one, step two, step three walkthrough, fairly simple. U1 is recruited U2 is recruited that recruits more snRNA. Now, this is when we start cleaving. So once we get everything recruited to this area the pre-mRNA is cleaved at the 5' prime splice site first. And this forms a kind of loop and this loop is called a lariat. So, what you can see down here, you can actually see this occur. So here we have a lariat and it just forms this loop. And then what happens is the 3' prime splice site is cleaved by another reaction. And so this joins the two exons together, forming the processed mRNA. And then all the other snRNA. and spliceosome, they leave.

And so, step seven is important but I actually don't have an image of it. There's not a very clear image of it but it's another vocab word. And this is called the exon junction complex. So once you actually have the exons form together the exon junction complex is recruited which is just more proteins recruited to the newly joined exons. And this helps the transcript to actually get out of the nucleus and into the cytosol for translation.

Now we've gone through these steps and I don't want to scroll back up. But if you just kind of look at your handout and scroll through the steps, you'll notice there's a lot of movement. RNA and proteins kind of moving all over the place, looping things and cutting things so that the exons could be joined together. Now, these movements are really facilitated by RNA-RNA interactions. Remember the exons and the intron are RNA. And then these things coming in the U1, the U2. These are all RNAs as well. The RNA-RNA arrangements are just kind of interactions between these two different RNAs that are responsible for most of these steps but also and each one of these steps, these interactions are always being disrupted and reformed to promote the next step from the next step of occurring. So, I guess that was complicated. Feel free to go back over, you know, take it slow, watch this video again and really get these steps down because it is important that you understand these steps. Let's move on.

Splicing Forms

Okay, so in this video, we're going to focus on slicing. But instead of focusing on the steps, we're going to talk about various considerations we need to really know about. We're going to really consider splicing as a whole. So the first thing that we need to know is that not all RNA splice using the seven steps that I talked to you about before. Don't worry, I'm not going to walk you through another seven steps. But just know that some introns are called self-splicing intron and they do exactly what they sound like they do, they self-splice and they actually splice without a spliceosome. So, how does it do this? Not another seven-step process, but these introns fold into complex secondary structures. And because they are RNA that allows them to either recruit more proteins or you know, splice themselves. So that's one thing that you need to know that not everything is sort of this very clear seven-step program.

Now, regulation of splicing is actually a form of regulating gene expression. Because there is this process called alternative splicing, which we've mentioned before and you may remember from your intro class, but alternative splicing allows for different combinations of exons to form. And so what this forms is the same protein, but kind of a different structure. So you have all of these proteins that are essentially the same protein, but different formations of it. And so, the different formations can have slightly different functions, therefore controlling gene expression. So that's one form of how splicing can regulate gene expression. But another way that it can do this is to actually chromatin structure. So very condensed chromatin can actually slow the rate of transcription and therefore slow the rate of processing of RNA processing. And so how well the chromatin is condensed has a major role in RNA processing. So one of the things that can occur is this thing called exon skipping. And so this is where an exon or an intron that's supposed to be cut out or an exon that's supposed to be joined in with a different one is accidentally just skipped because the chromatin is so loose that the process is moving so quickly that it just gets skipped. Another way that chromatin can really affect splicing is through histone modifications. Remember protein chromatin is made up of histone proteins. And so these histone modifications can recruit proteins that are RNA and they can actually control transcription or processing speeds and regulate it. So slicing is much more than just cutting introns out. It can be a way of regulating gene expression.

So we're going to look at alternative splicing. Let me back out here. So here we have our DNA and it has all of the exons and intron on the double helix, the DNA then we have the RNA. Now this is remember single-stranded, and again we have all the exons and the introns. Now, alternative slicing occurs and you can get these three forms of mRNA 1, 2 and 3 and you can see that these have different combinations of exons 4 or 5 that has all of them, this is missing 3, this one's missing 4. And so these proteins, when they are eventually translated, create all these different structures and they're likely going to have different functions as well because their structures are so different. And so alternative slicing can really affect different mechanisms and protein actions within the sound. So now let's turn the page and move on.

RNA Editing

Okay, so now we're going to talk about another way that RNA can be processed, and this is RNA editing. All the other ways: the five prime capping, polyadenylation, splicing — these are things that have to occur for an mRNA to be processed. Now, RNA editing isn't something that has to occur. It doesn't happen that often; actually, it's kind of a rare event, but it can happen. So we need to talk about it. You need to know what it is. So, what is RNA editing? RNA editing is actually small changes in the nucleotide sequence of the pre-mRNA. So this is actually what it sounds like: it's taking the transcript, taking out some of the nucleotides, and replacing them with something else. And so this can insert or remove multiple nucleotides in a transcript.

One of the more common ways that this is done is deamination. So, what is deamination? You don't necessarily need to know that term; it's much more chemistry, but it's just kind of this removal of an amino group of specific nucleotides. And so two kinds of nucleotides that can happen after deamination is the formation of a uridine, which is created from a deaminated cytosine, and inosine, which is deaminated adenosine.

And so, how is it controlled? Of course, this has to be controlled. You can't just willy-nilly change any nucleotide you want. So what controls this are guide RNAs. So if we're going to look at this, let me back out of the way again. Here we have our DNA; I'll circle it here. So this is our DNA sequence. Then we get our RNA. So this is our pre-RNA sequence, which is complementary, of course; it has to be. And so, from here, our guide RNAs come in and they say here are some nucleotides we're going to change. And so, these get removed, or the deamination comes in and changes this, or whatever is going to happen here, happens to these nucleotides. And in this case, what you get is the additions of uracils. And so this is the process of RNA editing.

Now, you might think this is kind of crazy because these sequences that are conserved in the DNA over time and they don't necessarily evolve. But like I said, they happen rarely, but it can happen, and it is a form of RNA processing, so you need to know about it. So, let's move on.

Which of the following is NOT a processing event pre-mRNA undergoes to become mRNA?

• A. The addition of an extended sequence of repeating A nucleotides at the 3' end of the transcript
• B. The addition of a methylated guanine at the 5' end of the transcript
• C. Splicing out exons to connect the introns
• D. Removal of 1+ nucleotides in the pre-mRNA transcript

Which of the following is not a sequence that is required for splicing?

• A. 5' GU sequence
• B. 3' AG sequence
• C. Branch Point
• D. Splice Sequence

Guide RNAs are responsible for what?

• A. Guiding the spliceosome to the correct splicing sequence
• B. Determining the position of RNA editing
• C. Adding the poly-A tail to the mRNA
• D. Adding the 5' cap to the mRNA