BIOCHEMISTRY DISCUSSION 9
Leahh
38RNAProcessinginEukaryotes.pptxBiochemistry: A Short Course Fourth Edition CHAPTER 38 RNA Processing in Eukaryotes
Tymoczko • Berg • Gatto • Stryer
© 2019 W. H. Freeman and Company.
Created by Brett Barbaro
So we’ve talked a lot about the processes which regulate the transcription of genes in Eukaryotes, but that’s not where the regulation ends. Once the genes have been transcribed, the RNA can actually undergo a large number of processing steps which are extremely important for their function. Most of these processes occur in the nucleus which means that they are not available for translation until they get out. And that’s why these processes occur in eukaryotes but not in prokaryotes. When the prokaryotes transcribe a gene, the RNA is immediately available for protein synthesis. But these extra steps in eukaryotes add a lot of layers of complexity to how these genes can affect the cell.
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Chapter 38: Outline
38.1 Mature Ribosomal RNA Is Generated by the Cleavage of a Precursor Molecule
38.2 Transfer RNA Is Extensively Processed
38.3 Messenger RNA Is Modified and Spliced
38.4 RNA Can Function as a Catalyst
Created by Brett Barbaro
Now, remember there are three different types of RNA. There’s rRNA, which is Ribosomal RNA, tRNA which is transfer RNA, and then messenger RNA, mRNA. Now, the ribosomal RNA is generated first as a long molecule of RNA and then cut into smaller pieces that are then functional. Transfer RNA, and there are lots of different kinds of transfer RNA, undergo a great deal of modifications after they are transcribed. And messenger RNA is also modified. One of the main modifications that takes place is splicing. That’s the cutting out of certain parts of the RNA and the leaving in of others. The mRNA can actually also be edited at the base level and we’ll talk about that a little bit and a lot of these things are actually catalyzed by RNA - leading many people to the hypothesis that RNA was actually the first structure that was very important for the development of life.
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Section 38.1 Mature Ribosomal RNA Is Generated by the Cleavage of a Precursor Molecule
RNA polymerase I synthesizes a large precursor RNA (45S) that is subsequently processed to yield 18S, 28S, and 5.8S rRNAs, which are components of the ribosome.
Prior to cleavage, the precursor is altered by modifications of some bases and riboses that are catalyzed by RNA-protein complexes called small nucleolar ribonucleoproteins (snoRNPs).
Created by Brett Barbaro
Well, we’ll start by talking about RNA Polymerase I and it synthesizes a large precursor RNA which then gets cut into smaller pieces to make the ribosomal RNAs. And you’ll see the numbers that’ll correspond to these pieces - this is how they’re described. The 45S is the whole piece, and then it gets cut into 28S, 18S, and 5.8S rRNA pieces. Now, if you’ll note, those don’t add up to 45. That’s because this is not a weight or anything - this has to do with where these RNAs settle down in a centrifuge, which is kind of a difficult way of categorizing them, but that’s how it was done traditionally so people still use that language. Bases and riboses on the RNA are also modified by small complexes called nucleolar ribonucleoproteins. That’s a combination of RNAs and proteins that are in the nucleus that catalyze these reactions.
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Diagram of the Processing of Eukaryotic Pre-rRNA
Created by Brett Barbaro
Figure 38.1 The processing of eukaryotic pre-rRNA. The mammalian pre-rRNA transcript contains the RNA sequences destined to become the 18S, 5.8S, and 28S rRNAs of the small and large ribosomal subunits. First, nucleotides are modified (indicated by red lines). Next, the pre-rRNA is cleaved and packaged to form mature ribosomes in a highly regulated process in which more than 200 proteins take part.
So at the top of this diagram we see the pre-rRNA, which is quite a bit longer than the individual pieces, and the three blue chunks that get cut out of it are the ones that are the final products. Before the cutting occurs, the nucleotides are modified. And a lot of these modifications are methyl groups added to the nucleotides. But some of them are the alteration of the uridine into pseudouridine. And you can see in the lower right hand corner what the pseudouridine looks like. It’s almost the same as a uridine, except for normally the base is attached to the ribose at the blue nitrogen but it’s now switched its position.
These modifications give the RNA some extra catalytic ability because they increase the diversity of the bases on the RNAs and therefore make them more able to interact with specific elements. This is important because when you’re talking about ribosomal RNAs, the RNA components of the ribosome are very important for the catalytic functions of the ribosome. So these modifications give the RNAs a greater diversity of tools to work with, much like the use of prosthetic groups in proteins.
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Section 38.2 Transfer RNA Is Extensively Processed
RNA polymerase III catalyzes the synthesis of precursors to tRNA.
RNase P and RNase Z remove nucleotides from the 5’ and 3’ ends of the precursor, respectively, whereas tRNA nucleotidyltransferase adds nucleotides to the 3’ end.
Bases and riboses are also modified.
Many eukaryotic tRNA precursors contain an intron that is removed by an endonuclease, and the resulting products are joined by a ligase.
Created by Brett Barbaro
tRNA is synthesized by RNA polymerase III. There are four primary modifications that occur to tRNAs after they’re transcribed. The first one is the removal of nucleotides from the 5’ end by RNase P. RNase, by the way is a general class of proteins that destroy RNA or cut RNA. As it turns out, there are a ton of proteins that cut up RNA. RNA is sort of fragile in that way. And that’s a issue in the laboratory when we’re working with RNA is that there’s natural RNases that occur everywhere. So if we’re not very careful those RNases can get into our mixes and destroy our RNA. So we have to clean the benches very carefully and use special equipment for that.
The second modification that occurs is the addition of the nucleotides CCA to the 3’ end of the transfer RNA. {Third} Many bases and riboses are modified in tRNA, much like they are in ribosomal RNA to give the bases a greater diversity. Remember you have 20 different amino acids, and there’s a different tRNA for each amino acid, so it’s very important to be able to tell them apart. They’re all very similar in structure so these modifications introduce important points for recognition of the different tRNAs. And finally {fourth} many eukaryotic tRNAs have a few bases that need to be removed in order for them to reach their mature form.
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Diagram of Transfer RNA Precursor Processing
Created by Brett Barbaro
Figure 38.2 Transfer RNA precursor processing. The conversion of a yeast tRNA precursor into a mature tRNA requires the removal of a 14-nucleotide intron (yellow), the cleavage of a 5 leader (green), the removal of the 3 trailer (blue), and the attachment of CCA at the 3 end (red). In addition, several bases are modified.
So here we can see on the left an immature tRNA, and on the right we see a mature form. And on the left hand side we have an extra 8 bases at the 5’ end that need to be cut off {Leader}. There are 4 bases on the 3’ end that need to be cut off. There needs to be a CCA sequence attached there at the 3’ end. The yellow bit there at the bottom of the immature tRNA needs to be removed {Intron}. And a number of bases need to be modified. We can see the GUA in the immature tRNA gets transformed into a G-psi-A, which is a pseudouridine instead of a regular uridine. There are also several other modifications that occur - methylations and various other things that you can see in this diagram.
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Section 38.3 Messenger RNA Is Modified and Spliced
Learning objective 4: Explain how RNA is processed after its transcription in eukaryotes.
Messenger RNA precursors (pre-mRNA) are synthesized by RNA polymerase II and then subsequently processed in a number of ways.
The 5’ end is modified by the addition of the 5’ cap in which a GTP is added to the precursor in an unusual 5’–5’ linkage. The cap may also be methylated.
The 3’ end is cleaved by a specific endonuclease, and a stretch of polyadenylate is added by poly(A) polymerase to form the poly(A) tail about 250 nucleotides long.
Noncoding stretches of RNA called introns are removed and the products ligated to form mature mRNA.
Created by Brett Barbaro
But now we get to messenger RNA - our favorite RNA, because this is the one that encodes the proteins. And mRNA is synthesized by RNA polymerase II. And after it’s synthesized it’s modified in a number of ways. #1, the 5’ end is modified with a “cap” in which a GTP is added to it in a strange linkage, and there are some methylations that occur. Second, #2, is the 3’ end is cleaved and then polyadenylated. A bunch of ATP is used to create a long string of adenine bases at the end of the mRNA. And finally an extraordinarily important and complicated mechanism removes parts of the RNA, making it into a mature RNA. And this is called splicing and we’ll get into that in a minute.
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Diagram of Capping the 5’ End of Eukaryotic mRNA
Created by Brett Barbaro
Figure 38.3 Capping the 5 end. Caps at the 5 end of eukaryotic mRNA include 7-methylguanylate (red) attached by a triphosphate linkage to the ribose at the 5 end. None of the riboses are methylated in cap 0, one is methylated in cap 1, and both are methylated in cap 2.
So here’s a diagram of a cap that would occur at the 5’ end of an mRNA. The triphosphate group at the 5’ end gets modified by a methyl guanylate residue, which is in red at the top. And this gives the mRNA a unique structure that can be recognized by other proteins in the cell. Methylation can also occur on the first couple of bases - on the ribose elements of the bases actually, and this creates a variety of recognition sites for different binding partners in the cell. One interesting feature of the cap is that the 5’ carbon of the cap is bonded, but the 3’ carbon is not bonded - this makes it chemically similar to the 3’ end of the RNA molecule, and therefore resistant to 5’ exonucleases.
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Diagram of Polyadenylation of a Primary Transcript
Created by Brett Barbaro
Figure 38.4 Polyadenylation of a primary transcript. A specific endonuclease cleaves the RNA downstream of AAUAAA. Poly(A) polymerase then adds about 250 adenylate residues.
Now we’ll talk about the polyadenylation of the mRNA. And that occurs after the RNA has been transcribed. A specific endonuclease - and remember an endonuclease is something that cuts in between nucleotides – this endonuclease recognizes the signal AAUAAA and then cleaves the RNA a little bit downstream of that point. Now at that point, there’s another protein, called poly(A) polymerase, which takes over and adds a string of adenines {adenosines} to the end of this mRNA. This is thought to enhance the stability of the RNA, but its actual role is still a little bit unclear.
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Sequences at the Ends of Introns Specify Splice Sites in mRNA Precursors
Most genes in eukaryotes consist of exons (coding regions) and introns (noncoding regions). Introns vary in length from 50 to 10,000 nucleotides.
The exons are joined by splicing.
Intron–exon junctions have several common features:
The 5’ end of the junction has the consensus sequence 5’ AGGUAAGU 3’ with the first GU from the 5’ end demarcating the beginning of the intron.
The 3’ end of the intron is marked by a stretch of pyrimidines (polypyrimidine tract) followed by any base, a C, and then the intron ends with AG.
The branch site is located 20–50 nucleotides from the 3’ end of the intron.
Splicing video: https://www.youtube.com/watch?v=aVgwr0QpYNE
Created by Brett Barbaro
Now we’re going to talk about splicing, which is an extremely important and complicated part of RNA processing. There’s a video that does a very good job of representing mRNA splicing, and I encourage you to take a look at it before going on - and probably take a look at it again afterwards so you can review what we just talked about. I’ve provided a link to the website at the top of this slide.
So most genes in eukaryotes have parts that are expressed, and those are called exons, and then there are parts that are not expressed - those are called introns. Maybe to help you remember it, you can say that those are just stuck “in” to the gene. But these introns can be very long. And there can be a whole lot of them. So they need to be removed before the translation of the mRNA starts taking place. The splicing machinery or spliceosome, as it’s called, is a very large complex of proteins - another one of these huge protein complexes, just like your polymerases - which is very complicated and has a lot of regulatory elements. This spliceosome recognizes the beginning of an intron by a specific sequence. And it’s listed here that the sequence is AGGUAAGU, but I think that might be a little bit too specific. The sequence varies a great deal. But that first GU is usually the most important thing that marks the beginning of an intron. When introns are cut out they begin with that GU.
The 3’ end of the intron has usually got a stretch of pyrimidines - and remember the pyrimidines are cytosine and uracil - and then this stretch can be followed by a cytosine at the end and then the intron ends with an A and a G. So this particular consensus sequence is what the spliceosome looks for when it’s trying to find the end of an intron.
The third part that is necessary for an intron to be spliced out is called a “branch site”, and that can be 20-50 nucleotides from the 3’ end, and that is an adenine residue.
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Diagram of the Transcription and Processing of the β-globin Gene
Created by Brett Barbaro
Figure 38.5 The transcription and processing of the -globin gene. The gene is transcribed to yield the primary transcript, which is modified by cap and poly(A) addition. The introns in the primary RNA transcript are removed to form the mRNA.
So here’s an overall view of what happens with splicing. You start out with a primary transcript (up at the top) and it contains three blue exons and two orange introns. First, the mRNA is capped on the 5’ end and the poly(A) tail is added on the 3’ end. And then the splicing apparatus comes in and removes the introns, leaving you with a capped, polyadenylated, exon-only, mature beta-globin mRNA.
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Diagram of Splice Sites
Created by Brett Barbaro
Figure 38.6 Splice sites. Consensus sequences for the 5 splice site and the 3 splice site are shown. Py stands for pyrimidine.
So here’s an anatomy of an intron. We have at the 5’ splice site the GU and its surrounding nucleotides that get recognized by the spliceosome. In the center you see an adenine which is a branch site. And then at the end there’s a string of pyrimidines, followed by an N (for any base), and then a cytosine, and then an AG. And that marks the 3’ splice site of the intron.
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Small Nuclear RNAs in Spliceosomes Catalyze the Splicing of mRNA Precursors
Splicing is facilitated by small nuclear ribonucleoprotein complexes (snRNPs). These complexes associate with the pre-mRNA to form the splicing apparatus called the spliceosome.
U1 snRNP binds at the 5’ splice site, followed by U2 snRNP binding at the branch site.
Binding of U4-U5-U6 tri-snRNP completes spliceosome formation.
The U2 snRNA and the U6 snRNA, components of their respective snRNPs, are the actual catalysts of splicing.
The catalytic snRNAs facilitate transesterification reactions that remove the introns and join the exons.
Created by Brett Barbaro
Now, the splicing of mRNA is actually catalyzed by other RNAs. These RNAs are combined with proteins to create small nuclear ribonucleoprotein complexes, or snRNPs, or “snurps” (it’s always nice to have a handy word to describe these things). And all of these parts come together to form the spliceosome. So the U1 snurp binds at the 5’ splice site, and the U2 binds at the branch site – it says here that they’re in sequence. I don’t know if that’s always the case. Probably not. You know, in biology there’s almost an exception to every single rule you can come up with. But generally the 5’ splice site will be synthesized before the branch site, so that would make it easier to bind the 5’ splice site first. Then there is a complex called the U4-U5-U6 tri-snRNP, and the snRNA parts of the U2 and U6 snurps are the actual parts that do the catalysis of the splicing. The exons on either side of the intron are joined by a transesterification reaction and the intron is removed and gets degraded.
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Diagram of Spliceosome Assembly and Action
Created by Brett Barbaro
Figure 38.7 Spliceosome assembly and action. U1 binds the 5 splice site, and U2 binds to the branch point. A preformed U4-U5-U6 complex then joins the assembly to form the complete spliceosome. Extensive interactions between U6 and U2 displace U1 and U4. Then, in the first transesterification step, the branch-site adenosine attacks the 5 splice site, making a lariat intermediate. U5 holds the two exons in close proximity, and the second transesterification takes place, with the 5 splice-site hydroxyl group attacking the 3 splice site. These reactions result in the mature spliced mRNA and a lariat form of the intron bound by U2, U5, and U6. [After T. Villa, J. A. Pleiss, and C. Guthrie, Cell 109:149–152, 2002.]
Here’s a diagram of that whole process. And you’ll notice there is a “pG” at the beginning of the intron and a “p” at the end. That’s nothing special but that’s just to show you that there’s a phosphate there - and there’s always a phosphate there, so there’s no real need to actually include that, but they chose to include that in this diagram. Don’t get confused. So the branch site, which is an adenine residue, is probably marked by other sequences in the intron but you know we’re still trying to figure this stuff out, to be honest with you. The U1 and U2 snRNPs come in and bind to those two sites, and then the U4-U5-U6 complex comes in, puts everything in the right place, the U1 and U4 snRNPs get released, and then the transesterification reactions occur, catalyzed by the U6 and U2 elements.
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Diagram of the Splicing Catalytic Center
Created by Brett Barbaro
Figure 38.8 The splicing catalytic center. The catalytic center of the spliceosome is formed by U2 snRNA (red) and U6 snRNA (green), which are base-paired. U2 is also base-paired to the branch site of the mRNA precursor. [After H. D. Madhani and C. Guthrie, Cell 71:803–817, 1992.]
And here’s a diagram of the catalytic center where all of this splicing takes place. The U6 and U2 snRNAs are attached and form a structure which is presumably important for the catalysis of this reaction (but you can’t really see what that structure is in this diagram). And then the U2 snRNA combines (or at least pairs) with elements around the branch point. This results with that critical adenine sticking out - and that is then used to attack at the 5’ splice site and break that bond. That’s the essential catalytic reaction that’s occurring in this case.
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Clinical Insight: Mutations that Affect Pre-mRNA Splicing Cause Disease
CLINICAL INSIGHT
Mutations that Affect Pre-mRNA Splicing Cause Disease
Mutations in either pre-mRNA or splicing factors can result in pathological conditions.
Defects in splicing may cause up to 15% of all genetic diseases.
Some thalassemias, diseases resulting from defective hemoglobin synthesis, are caused by mutations at splice sites in the pre-mRNA for the β chain of hemoglobin.
Retinitis pigmentosa, a disease of acquired blindness, is due to a mutation in the U4-U5-U6 tri-snRNP.
Created by Brett Barbaro
Now, splicing is actually a really important part of gene expression. And it turns out that if there’s a problem with your splicing factors, or with the pre-mRNA, that can lead to serious diseases. And this can happen - they estimate maybe up to 15% of all genetic diseases are problems with splicing. One example is the thalassemias, which are defective hemoglobin molecules. And this is caused by mutations at the splice sites in the pre-mRNA for hemoglobin. Retinitis pigmentosa, a kind of blindness, is the result of a problem with the U4-U5-U6 tri-snRNP.
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Diagram of a Splicing Mutation that Causes Thalassemia
Created by Brett Barbaro
Figure 38.9 A splicing mutation that causes thalassemia. An A-to-G mutation within the first intron of the gene for the human hemoglobin chain creates a new 5 splice site (GU). The abnormal mature mRNA now has a premature Stop codon and is degraded.
So here’s an example of how a mutation in an intron can actually cause a problem. We have here (at the top) a gene for the beta chain of human hemoglobin. And you can see in the middle of the first intron there, there’s a stop codon. This means that when the protein is translated it would stop translating at that point. But normally that’s not a problem, because that whole intron is removed, and so the translation occurs from one exon to the next without any break. However, if there is a mutation that changes an A to a G in that intron, after that stop codon, then it can change this AU to a GU - and then that GU can be recognized as a 5’ splice site. It doesn’t mean it would always be recognized as a 5’ splice site, but a good percentage of the time, a significant percentage of the time, it is. So you end up producing, on the one hand, a normal mature RNA - but on the other hand you’ve got an abnormal mature RNA which has this stop codon in it. And it says here that that becomes degraded -well, yeah, that’s probably true, but it probably also gets translated into proteins. And those proteins will be non-functional - and not only non-functional, but they will probably interfere with the normal function of other proteins and that is what causes the disease.
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Table 38.1 Selected human diseases attributed to defects in alternative splicing
Created by Brett Barbaro
Well, just an interesting overview of some of the diseases that are caused by splicing problems. And some of them I’m sure you are familiar with. Breast cancer, cystic fibrosis, frontotemporal dementia, which is related to Alzheimer's disease. So yeah, splicing is a really important part of gene expression.
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Clinical Insight: Most Human Pre-mRNAs Can Be Spliced in Alternative Ways to Yield Different Proteins
CLINICAL INSIGHT
Most Human Pre-mRNAs Can Be Spliced in Alternative Ways to Yield Different Proteins
Alternative splicing is a powerful mechanism for expanding protein diversity.
In alternative splicing, a pre-mRNA can be spliced in different patterns, generating proteins with different functions. For example, in one splicing pattern, an antibody may be membrane-bound, while in another splicing pattern, the antibody is secreted.
https://commons.wikimedia.org/wiki/File:Alternative_splicing.jpg
Created by Brett Barbaro
Now, there’s another thing about splicing, which is that it can actually be used to generate several different alternative transcripts. Often times these exons will represent different domains in a protein. And sometimes you want all of the domains in a protein, and sometimes you don’t. Well, an easy way to get rid of a domain is to splice it out. Instead of utilizing the 3’ splice site that’s attached to the intron you can pair a 5’ splice site with a 3’ splice site that’s farther down the pre-mRNA. And that can be used to cut out 1 or more exons. These different transcripts will produce different proteins that might have different functions, and sometimes all of these functions are wanted and desired. Intron retention I don’t know too much about - it looks kind of weird to me so I’m going to move on. Mutually exclusive exons is you can sometimes include 1 exon and sometimes include the other exon, but you never have both of them. Or you can get an alternative splice site that is in the middle of an exon, or the middle of an intron. And in a lot of cases this can lead to disease, but sometimes that’s exactly what is meant to happen. So you can imagine the machinery required to regulate all of this - it’s extremely complicated, and is a very active area of research, the spliceosome machinery.
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Diagram of Alternative Splicing
Created by Brett Barbaro
Figure 38.10 Alternative splicing. Alternative splicing generates mRNAs that are templates for different forms of a protein: (A) a membrane-bound antibody on the surface of a lymphocyte and (B) its soluble counterpart, exported from the cell. The membrane-bound antibody is anchored to the plasma membrane by a helical segment (highlighted in yellow) that is encoded by its own exon.
Here’s a simple example of how you might want to have two different versions of the same protein. So here you have an antibody. And sometimes you want to have that antibody attached to the surface of a cell. That’s an important function for lymphocytes. So there’s a domain that exists that is hydrophobic and becomes embedded in the membrane, and tethers the antibody to the membrane. But you also need to have soluble antibody molecules that are not attached to the membrane. Well, it turns out that this membrane anchoring portion of the protein is encoded by an exon. So to turn your membrane bound molecule into a soluble molecule, all you need to do is splice that exon out.
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The Transcription and Processing of mRNA Are Coupled
Transcription and processing of mRNAs are coordinated by the carboxyl-terminal domain (CTD) of RNA polymerase II.
Functions of the CTD include
Recruiting enzymes to synthesize the 5’ cap.
Recruiting components of the splicing complex.
Recruiting an endonuclease that cleaves the pre-mRNA to expose the site for poly(A) addition.
Created by Brett Barbaro
It actually turns out that a lot of these functions are coordinated by the carboxyl-terminal domain of RNA polymerase II. And we talked a little bit about that. It’s kind of a long tail that stretches out behind the RNA polymerase. And it’s responsible for getting enzymes to make the 5’ cap on your mRNA, for recruiting the splicing complex (the spliceosome), and also for recruiting endonuclease to cleave the 3’ prime end of the mRNA and get it ready for polyadenylation.
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Diagram of the CTD: Coupling Transcription to Pre-mRNA Processing
Created by Brett Barbaro
Figure 38.11 The CTD: Coupling transcription to pre-mRNA processing. The transcription factor TFIIH phosphorylates the carboxyl-terminal domain (CTD) of RNA polymerase II, signaling the transition from transcription initiation to elongation. The phosphorylated CTD binds factors required for pre-mRNA capping, splicing, and polyadenylation. These proteins are brought in close proximity to their sites of action on the nascent pre-mRNA as it is transcribed in the course of elongation. [After P. A. Sharp. Trends Biochem. Sci. 30:279–281, 2005.]
So you can see in this diagram, on the top, we’ve got your RNA polymerase II complex, and you’ve got the C-terminal domain kind of trailing out behind it. That C-terminal domain gets phosphorylated by transcription factor 2H, which we discussed a couple chapters ago. And that’s important because you don’t want the C-terminal domain to be recruiting all of these enzymes unless it’s already part of an active transcription complex. So once these are phosphorylated, the capping enzymes, splicing factors, and polyadenylation factors associate with that CTD, and then the pre-mRNA, once it gets extruded, kind of just falls into place alongside of this long tail and the enzymes that are attached to that tail can then access it and do their jobs.
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Biological Insight: RNA Editing Changes the Proteins Encoded by mRNA
The coding information of some mRNAs can be altered by RNA editing.
In the case of apolipoprotein B, a protein important in lipid transport, the unedited transcript encodes a 512 kDa protein called apo B-100, which participates in the transport of intracellular lipids in the liver.
In the small intestine, RNA editing of the transcript deaminates a cytidine in the codon for glutamine, forming uridine, which generates a Stop codon.
The edited transcripts encode for apo B-48, a 240 kDa protein which carries dietary fat in the form of chylomicrons.
Created by Brett Barbaro
Figure 38.12 RNA editing. Enzyme-catalyzed deamination of a specific cytidine residue in the mRNA for apolipoprotein B-100 changes a codon for glutamine (CAA) to a Stop codon (UAA). Apolipoprotein B-48, a truncated version of the protein lacking the LDL receptor-binding domain, is generated by this posttranscriptional change in the mRNA sequence. [After P. Hodges and J. Scott. Trends Biochem. Sci. 17:77, 1992.]
We’re going to talk a little bit now about RNA editing - and this is a slightly different approach to changing RNA. This is when RNA elements in the mRNA are actually altered. So there’s a lot of alterations that occur on ribosomal RNA and tRNA, but not so many on mRNA. When there ARE changes that occur on mRNA though, it can have a big effect. And one example is apolipoprotein B. Now, apo B has a portion that is very important for transport of lipids. Now in the liver this is coupled to LDL receptor binding, so there’s a domain that is responsible for the association with the lipids, and then there’s another domain that’s responsible for the receptor binding. And in the middle of this mRNA there is a sequence, CAA. Normally this codes for the amino acid glutamine - but an editing operation can remove a nitrogen (an amine group) from the C and turn it into a uridine. UAA doesn’t code for any amino acid - that’s a stop codon. So when the edited mRNA is translated it stops there and does not produce the LDL receptor binding domain. Now, this remaining protein, apo B-48, is very important for the transfer of dietary fat in chylomicrons. So both forms of this protein have very important functions, depending on where they are - and which version of the protein gets finally translated is dependent on the editing process.
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Section 38.4 RNA Can Function as a Catalyst
Some RNAs, called ribozymes, function as catalysts.
Introns from certain organisms can self-splice―that is, excise themselves.
Self-splicing or group 1 introns were initially identified in rRNA from Tetrahymena.
Group 1 introns require guanosine as a cofactor.
Created by Brett Barbaro
I’ve talked about RNA as a catalyst before. There’s times when an RNA can actually catalyze a reaction, and RNAs that do this are called ribozymes. One type of ribozyme is able to splice itself. One of these self-splicing introns was identified in the ribosomal RNA of tetrahymena, which is a small organism, and it required guanosine as a cofactor to catalyze the reaction.
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Diagram of Self-Splicing
Created by Brett Barbaro
Figure 38.14 Self-splicing. A ribosomal RNA precursor from Tetrahymena splices itself in the presence of a guanosine cofactor (G, shown in green). An intron (red) is released in the first splicing reaction. This intron then splices itself twice again to produce a linear RNA. [After T. Cech, RNA as an enzyme. © 1986 by Scientific American, Inc. All rights reserved.]
So what happened is the RNA folded into a particular configuration, as RNA is known to do, and in this configuration it had a binding site for a guanosine. So once that guanosine came into that active site on the RNA, it ended up cutting out one part of the intron - and then the remaining part of the ribosomal RNA attacked what would be considered the 3’ splice site of this intron, and the RNA effectively spliced out part of itself.
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Diagram of the Structure of a Self-Splicing Intron
Created by Brett Barbaro
Figure 38.15 The structure of a self-splicing intron. The structure of a large fragment of the self-splicing intron from Tetrahymena reveals a complex folding pattern of helices and loops. Bases are shown in green, A; yellow, C; purple, G; and orange, U.
And here’s a 3-dimensional picture of what that RNA looks like. And it forms this very complicated, but very specific, 3-dimensional structure, which includes the guanosine binding site which is indicated here. And one would probably guess that the 5’ and 3’ splice sites are located right near there, so that once the guanosine bound then the remaining reactions would take place sequentially. So this ability of RNA to catalyze its own reactions is one of the reasons that people have proposed the “RNA world theory”. And you can look that up on the internet (it’s pretty fascinating) - but the idea is that one of the most important things that happened in the development of life on this planet was the development of RNA. Another one of the most important things of course is the development of membranes. So if you have a soup, just a bunch of membranes (lipids) and RNA that happens to be there, the idea is that somehow, sooner or later, they would form these complex structures, which would begin to self-replicate and eventually lead to life on this planet. DNA and proteins, being more specific developments, occurred later on.
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