mRNA Processing - Video Tutorials & Practice Problems
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1
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mRNA Processing Overview
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9m
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Hello everyone in this lesson. We are going to talk about RNA processing. Okay so whenever an M. RNA is made or it is transcribed it's not entirely ready to go. It's not just an M R N. A. It's actually called a pre M R N. A. Or an M RNA transcript. And pre M R N. A. S don't have the same characteristics as mature M. RNA. In fact we actually have to do different things to the pre M. R. N. A. To make it into a mature M. R. N. A. And this processing occurs in the nucleus of the cell and it is required before the pre M. R. N. A. Or the mature M. RNA can even leave the nucleus. So we know the transcription happens in the nucleus because that's where the D. N. A. Is. But processing also occurs in the nucleus. And once the whole event all the steps of processing have been finished then the mature M. RNA 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 M. RNA from other RNA. So M RNA processing is a unique thing. Also there is T RNA processing which is also unique. There's ribs OMA RNA processing which is also unique. And this is going to give characteristics to these RNA. S. So that they have their own distinguishing characteristics they are recognize. So M RNA processing allows M RNA to be unique and recognized in comparison to other types of RNA. And this process is actually going to occur at the same time as transcription. Usually when we're taught this we're taught that transcription happens. Then RNA processing happens, then translation happens. But actually as the M R N A is being transcribed into pri M RNA, it is at the same time being pro processed to turn into mature M RNA and then it will go on to translation. So what are the different processes that you are going to need to know where they're going to be? Three major things that happen in RNA processing? The five prime cap is going to be added to the five prime end. The poly a tail is going to be added to the three prime end and RNA splicing is going to occur. That is going to be the removal of exons or the removal of in theron's, excuse me, the removal of n tron and the keeping of exons. Now remember M RNA does not have introns but pre M RNA does and it has to be removed. And that is going to be a a process called RNA splicing, which is going to be one of the steps of M RNA processing. So what is this generally going to look like? Well generally you're going to have your M R N. A. So this is your M RNA here and it is going to have no introns is going to be composed of only exons. Okay, and then it is going to have a five prime end and a three prime end. And on the three prime ends you're going to have a poly a tail. And on the five prime end you're going to have this cap And this is going to be your five prime cap. And these things are utilized to distinguish M. R. N. A. S from other types of RNA S. And also protect the M. RNA 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 two. Remember RNA polymerase number two is going to be the specific RNA polymerase that actually transcribes M RNA 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 M. RNA processing. So as the RNA preliminary build the pre M RNA 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 C T. D. 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 M. R. N. A. And it's involved in the attachment of the splice zone to the pre M. RNA transcript, remember I said those introns have to be spliced out and those exons have to be glued back together the splices own 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 going to talk about some other proteins which their name is gigantic. So usually we just use the abbreviation we have the heterogeneous nuclear rebo nuclear proteins which are simply H. N. R. N. P. S. And they are going to be our protein and RNA complexes. So they're going to be proteins and specialized RNA. 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 need to do this? Well remember M RNA is going to be single stranded and which means it can complimentary lei bind with other nucleic acids including itself. So H N. R N. P. S are going to prevent RNA from forming secondary structures and keep it straight because a lot of the time RNA can form structures that kind of look like this where the R. N. A binds to itself. This structure is actually called a hairpin structure which you will learn more about in different biology classes. But if the M. RNA binds like this, it's really hard to translate. You can't really get to it. So we don't generally want M. RNA to bind to itself and form these secondary structures like this hairpin structure because it makes it much more difficult to work with. So the H. N. R. N. P. S. Are going to bind to RNA, make sure it doesn't do that. Also H. N. R. N. P. S are going to be a signal that tells the cell that the M. R. N. A. Is not finished processing. So H N. R N. P. S. Are bound to pre M. R. N. A. To do demonstrate that it has not finished RNA processing yet. This is a signal to tell the cell that this M. RNA transcript should not leave the nucleus yet because the whole process has not finished. So R N. A H. R N. P. S are going to be kind of like red flags. Like hey we're not done yet. Don't ship this out of the nucleus just quite yet. Also remember I told you that introns are going to be removed. They're going to be spliced out which we will talk more about in later lessons. But HN our mps are going to bind to those introns that have been removed from the M. R. N. A. And they're going to do something very, very important. They're going to target this entrance for degradation. This entrance are removed. What happens to them? Do we get rid of them? Yeah, we do. And the way that we know that these particular segments of M R N. A need to be destroyed is because they're bound to H N R N P. S. So these proteins are very important for the whole RNA processing event. Now this drawing down here is going to represent RNA polymerase number two, which is going to be the RNA polymerase that transcribes M. R. N. A. You can actually see the M. RNA right here and this is the pre M. R N A. Because it is being actively built at this time and this is going to be the three prime end that is actively being added to because remember nucleic acids can only be added to on the three prime end and we have the C terminal domain or the card box all tail domain, same thing, see TD, car box tail domain, C terminal domain, car box terminal domain, same thing. Just a whole bunch of different names for the same thing. So you'll see a lot of people just simply abbreviated to C. T. D. Basically, it's one of the ends of the RNA polymerase number two and it's going to be this kind of projection end and it's going to be bound to proteins and it's all also very commonly phosphor related to change its function depending on what the RNA polymerase needs it to do. So it will commonly be phosphor elated and it will also have a lot of proteins attached to it. So that's what these different peas are going to be representing. Phosphate groups and different proteins that are utilized for RNA processing. And remember that this car box cell domain or the CT deep domain are going to be actively processing the pre M R N A. As it is being made. So these things are happening at the same time and the way it's able to happen at the same time is it's attached to the same protein that does the RNA transcription. So these two things are going to happen at the same time. We're going to talk more about these different processes of RNA processing in depth. In our later lessons. Okay, everyone, let's go on to our next topic.
2
concept
5' RNA Capping
Video duration:
3m
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Okay so the first um M. RNA pre M. RNA processing event that we're going to talk about is five prime capping. So um 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 is it's a special methylated guanine. Remember metal is chemical group. Right? So that gets added to a guarani and then that methylated guanine gets added to the five prime end of the transcript. So they're kind of how this works is you have this phosphate eight and it removes a phosphate from the five prime and leaving this binding side open. Then another enzyme comes in the guanciale transfer ASIS and they add a GMP which is kind of very similar to a Mp. But instead of but it uses guanine instead. And so it actually guanciale transfer is is actually a unique protein um That adds wanting in this unique way. Um you don't necessarily need to know anything about it. But generally things don't bind five prime to five prime. But in this space it does it binds five prime to five prime. And it's just sort of unique. And then once the guangming has been added in this really unique way, you then have a methyl transfer race that adds a methyl group to guanine at this certain position. You don't need to know that position um on its sugar on the Revo sugaring. So I've highlighted a lot of things, what do you need to know? You need to know that if I prime cap adds a methylated guangming onto the five prime end. Um 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 away and then it becoming methylated. So um once the five time capping, Oh so when does the five prime capping occur um occurs pretty immediately after transcription. Once there's been about 25 nucleotides of RNA transcribed, um 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 R. N. A. From destroying the transcript as it's being transcribed because you kind of you don't want you know pretty much 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 prep cap actually looks like here, you have your M. R. N. A. And here's the five prime end. You can see that here and um here you have your methylated guanine. Here's the structure of it and you'll notice here um that it binds uniquely five prime to five prime. Um And this cap gets added on every pre M. RNA transcript that's going to eventually become into a protein. So now let's move on.
3
concept
3' Polyadenylation
Video duration:
3m
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Okay, so now we're gonna talk about three prime poly ventilation which is a second form of pre m. RNA processing that must occur. So what does this mean? What is RNA polyu ventilation? Well it is the addition of repeating a nucleotides at the three prime end of the transcript. So this is at the end of the transcript. Um And it's just this edition of A's over and over and over again can actually get fairly long. But um so this is at the end and so R. N. A. So how does it start? Well, the first thing that happens is RNA is cleaved at a specific sequence. So there are two cleavage signals that exist. One is this and one is a G. U 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 buying to these sequences. And um you know, say, okay this is where RNA is going to be cleaved so that the A. S. Can be added. And so if you are interested, you may read about them in your textbook, but cps F and C T C S T F are acronyms for the two main cleavage stimulatory factors. Now, once RNA is cleaved, what happens while this polymerase comes in called the poly a polymerase and it just starts adding a, you know, that's what it does. So it begins at an au rich site located right near where the cleavage site it was. And generally the 1st 12 A's. That are added are added fairly slowly. But then once it gets kind of past that hump of 12, it goes much faster. Um And they're around you know 202 150 that begins adding. Now this is just because it's just repeating. Aces doesn't need a template. Like other things like transcription needs a template. DNA replication needs a template. But this doesn't need a template because it's just adding a space. So let's look at this. Let me back out of the way. So here we have RNA polymer race and it's you know, 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 five prime cap um and it's also for five prime. So those are two reasons why we know. So it's been transcribing transcribing transcribing and it gets to a Polident elation signal, a cleavage signal. And this G rich sequence. Now remember these two are sequences that are both kind of 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 the poly polymer raise the pap comes in, binds this au region and starts adding eight. So what you get left with at the end is you have a five prime transcript with a five prime cap, your transcript. And it ends with A Bunch of A's Times 200 ISH More. And so that is, you know, two processing steps that lead to the formation of um or at least the first couple of things that lead to the formation of an M r n A. So now let's turn the page.
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concept
RNA Splicing Overview
Video duration:
5m
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Okay, so now we're gonna talk about a third mechanism of em are pre m RNA processing and this is RNA splicing. Now, I'm gonna talk a lot about RNA splicing, I'm talking about it so much that I've actually split it up into two videos. Um So it's a big thing, it's gonna have a lot of information. Um So this first video is going to focus on just kind of an overview of RNA splicing. So let's get started. Now, what does RNA splicing? RNA splicing occurs when n tron, which are kind of the non coding segments of the transcript need to be spliced or removed from the pre M. R. N. A. Can also say splice here, it's kind of another verb. And so RNA splicing removes and trans from pre M. R N. A. Now this can be kind of difficult for enzymes you say. Okay, well first how do enzymes recognize where the introns are? Well, this can actually be really difficult because introns very greatly in size. So when scientists go in and say, okay, well where all the N tron we really have difficulty finding them because they're just all over the place and we don't know they're not coding for anything, it's kind of hard to find them. So what scientists do is they actually use Exxon's to find n tron and the proteins in the cell do the same thing. So Exxon's which are the coding sequences if you remember average about 100 and 50 nucleotides long. So when you get to an Exxon and you travel 150 Nucleotides, you say. Okay, well an entrance beginning somewhere in here and that's exactly what the proteins do. We'll talk more about that in future videos. So entrance contain specific sequences between 30 and 40 nucleotides long, that signal for splicing. And generally these are really close to Exxon's. So there are kind of three that we're going to talk about. 123. And so the first one is the five prime splice site and this begins with a G. U sequence or an au sequence. Now G. U. Is more common than you. And um the second one is the three splice site and this begins with an A. G sequence or an A. C sequence. This is more common. And then the third one is the branch point and these are special sequences located several dozen nucleotides upstream for the three prime in. So this is kind of just vocab. Let's write this down, memorize this. Um But in just thinking about it conceptually, what what are we talking about here? We're talking about Exxon's located by n tron and somewhere near that site, they're all they're going to be these sequences. So there's one at the five prime and one of the three prime end and then there's this branch point which happens also at the three prime end. So these sequences here um You know, have these sort of consensus sequence or more common sequences are G. URAG depending on five or 3. Um And then there's also the branch point. And so these are sequences that say, hey, I'm here, right, here's an N tron come splice me out. And so um Because I mean it's confusing when I talk about it, I realize that it's also kind of confusing for the for the proteins themselves, they get it right most of the time, which is good because we are alive, we're alive. But 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. So if we're looking at kind of an overview of RNA splicing, um what we get here is you have your pre M R N. A. You have your X on your n tron is here. And you can see that there are these nice sequences G. U at the five prime and a G at the three prime end. Um and the signal they say, okay, come in here and splice me out. And that's what happens the entry and get spliced out and you're left with joining of the exxons together. So what's the protein that comes in and actually splices the entrance out? Well, this is called the splices own and this is responsible for splicing most RNA. So what is it consists of? Well, it consists of these things called small nuclear RNA or snow RNA, which you'll hear it referred to as the acronym more than the full name. And also the small nuclear rival proteins, or SNR and PS. Now the small nuclear RNA comes in five groups and we're going to talk about them a lot more in future videos. And the small nuclear rival proteins are formed by about 6 to 10 proteins that bind to the RNA. So the splices home isn't just a protein, it's a complex composed of RNA and proteins. And so the SNR and PSR responsible because they form this sort of core of the splice zone and they are actually what recognize and bind to the splice sequences. And they buy into the five prime splice sequence of three prime and recognize the sequences that say, hey, cut me out. And then the splices own can act. So that's the overview of RNA splicing. Bear with me, you're gonna get more complicated. But let's now turn the page
5
concept
Splicing Steps
Video duration:
4m
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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 style 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 um and sort of understand that, you know, these steps are complicated, you're in cell biology. Um 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 um you know how the splices own works and how splicing actually occurs, so I'm gonna disappear so that you guys can see these pictures that are off to the side. So the very first step of splicing occurs when the S. N. R. And P. Um And also the U. One S. N. S. Snow RNA binds to M. RNA at the five prime. So what does this mean? You have your five prime sequence here and you have your you one snow RNA binding here. Now, step two occurs when the youtube, Snow RNA binds to the branch point sequence. So what do we have? We have this branch point sequence. I didn't even put up here, five prime splice site and you have your youtube Snow RNA. Now the binding of the U. One and the U. Two 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 Snow RNA that get recruited to the N. Tron. Okay step one, step two, step three walkthrough, fairly simple. You one is recruited you two is recruited that recruits more snow RNA. Now this is when we start cleaving. So once we get everything recruited to this area the pre M. RNA is cleaved at the five prime splice site first. And this forms a kind of loop and this loop is called Illyria. So what you can see down here you can actually see this occur. So here we have a larry it and it just forms this loop. And then what happens is the three prime slice by three prime splice site is cleaved by another reaction. And so this joins the two Exxon's together, forming the processed M. R. N. A. And then all the other snow RNA. S. And slices on they leave. And so um 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 exxons form together the exon junction complex is recruited which is just more proteins recruited to the newly joined exxons. And this helps the transcript to actually get out of the nucleus and into the side it's all 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 hand out 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 exxons could be joined together. Now these movements are really facilitated by RNA RNA interactions. Remember the exxons and the N. Tron R. R. N. A. And then these things coming in the U. One, the youtube. These are all RNA as well. The RNA RNA arrangements are just kind of interactions between these two different RNA S that are responsible for most of these steps but also um 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. I feel free to go back over, you know, take it slow, watch this video again um and really get these steps down because it is important that you understand these steps. So let's move on
6
concept
Splicing Forms
Video duration:
4m
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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 um RNA splice using the seven steps that I talked to you about before. Don't worry, I'm not gonna walk you through another seven steps. Um But just know that some entr ons are called self splicing, N tron and they do exactly what they say, like what they sound like they do, they self splice and they actually splice without a splices own. So how does it do this get? Not another seven step process, But these entrance fold into complex secondary structures. And because there are N. A. That allows them to either recruit more proteins or you know, splice themselves. Um 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's this process called alternative splicing, which we've mentioned before and you may remember from your inter class, but alternative splicing allows for different combinations of Exxon's 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 um so that's one form of how splicing can regulate gene expression. But another way that it can do this is to actually um chroma TIN structure. So very condensed chroma tin can actually slow the rate of transcription and therefore slow the rate of processing of RNA processing. And so how well the chroma 10 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 X. Or an N. Tron that's supposed to be cut out or an Exxon that's supposed to be joined in with a different one is accidentally just skipped because the chroma 10 is so loose that the process is moving so quickly that it just gets skipped. Um Another way that chrome A tin can really affect splicing is through histone modifications. Remember protein chroma tin is made up of histone proteins. And um so these histone modifications can recruit proteins that are in a. And they can actually control transcription or processing speeds and regulate it. So spicing is much more than just cutting entrance out. It can be a way of regulating gene expression. So we're gonna look at alternative splicing. Let me back out here. So here we have our D. N. A. And it has all of the exxons and N. Tron on the double helix, the D. N. A. Then we have the R. N. A. Now this is remember single stranded and again we have all the exxons and the enTR ons. Now, alternative slicing occurs and you can get these three forms of M. R. N. A. 12 and three and you can see that these have different combinations of Exxon's four or five that has all of them, this is missing three, this one's missing four. 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.
7
concept
RNA Editing
Video duration:
2m
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Okay so now we're going to talk about another way that RNA can be processed. And this is the RNA editing now. All the other ways the five prime Kapali ventilation splicing. These are things that have to occur for an M. R. N. A. To be processed. Now RNA editing isn't something that has to occur. Doesn't happen that often actually it's kind of a ray 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 R. M. RNA. 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. So one of the more common ways that this has done is so. Delamination. So what is the domination? 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 to kind of forms of nucleotides that can happen after domination is the formation of a Euro Dean. Um And that's from that is created from A. D. Emanated side of scene. And in a sign which is A. D. Emanated out. I mean Mhm. And so how is it? I mean this of course has to be controlled. You can't just willy nilly kind of change any nucleotide you want. So what controls this our guide RNA. So if we're gonna look at this, let me back out of the way again. Here we have our D. N. A circle it here. So this is our D. N. A. Sequence. Then we get our RNA. So this is our pre RNA sequence which is complementary of course has to be. And so um so from here we have our Guide RNA is come in and they say here here are some nucleotides we're gonna change. And so these get removed or the D. Animation comes in and changes this or whatever is gonna happen here happens to these nucleotides. And in this case what you get is the additions of your cells. Um and so €3 sales get added in place of Adam means. And so this is the process of RNA editing. Now. You might think this is kind of crazy because you know these aren't necessarily these sequences that are conserved in the D. N. A. 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
8
Problem
Problem
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
9
Problem
Problem
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
10
Problem
Problem
Guide RNAs are responsible for what?
A
Guiding the spliceosome to the correct splicing sequence