Exam 3- L14 Flashcards
Where does all of this RNA processing happen on the mRNA?
- Capping -> 5’ End
- Splicing -> Internal Area
- Poly-Adenylation -> 3’ End
Describe the brief overview of RNA processing.
In euk, RNA processing will occur DURING transcription. Introns will have to be spliced out and capped as soon as transcription has taken place. To become a mature RNA, you need to have all of the introns spliced out and a poly-A-tail at the end of you pre-mRNA strand.
What induces capping?
Phosphorylation of Serine-5.
What is capping?
Capping of an RNA molecule means you are going to add a NT to the 5’ end of the RNA, which will serve as protection.
Describe a unique feature of capping.
The addition of the NT (guanine) is in the wrong orientation. It is connected to the strand of the RNA at the 5’ end through its 5’ phosphate. Which means you will have a 3’ OH on the end of the RNA on the 5’ end of the RNA.
What is also going to happen to the newly added NT (guanine)?
It is going to be methylated. This allows it to bind to proteins (CBC- cap binding protein).
The CBC is connected to the CTD tail, protecting it from exonuclease activity and the cap is important in translat
What NT is typically used for capping?
Guanine.
So the 5’ end of Guanine will be added to the 5’ end of RNA.
What are the four reactions of capping?
- Phosphohydrolase- loss of 1 of the phosphates on the 5’ end of RNA (remember that they have three)
Preps for a nucleophilic attack
- Guanylyltransferase -> nucleophilic attack of the beta phosphate on the 5’ end of RNA onto the alpha-phosphate of G (guanine). Lost of PPI.
Attaches on guanine.
- Guanine 7- methyltrasnferase -> methylation of the guanine w/ adoMET (SAM)
Note: methylation can also occur randomly along other NucleOtides.
At the end of transcription, what do you need to add?
When transcription stops, you need to add a poly-A-tail.
What is a poly-A-tail? Why do you add it on?
A poly-A-tail is a repeating sequence of adenine that is added onto the 3’ end of mRNA.
In order to get the end of transcription, you have to have cleavage of the mRNA. Once it is cleaved from RNAP, a polymerase will hook up to the mRNA and it will add adenosine 80-250 times.
The only RNA’s that have poly-A-tails are those that are produced by RNAP II
A Poly-A-Tail is needed for translation and it serves as protection.
What are the three steps for polyadenylation?
- CPSF =(cleavage/polyadenylation specificity factor) Recognition of the AAUAAA sequence in the RNA’s 3’ UTR and binds to it.
- Recruits CstF = cleavage factors
- Recruits PAP = Poly-A-polymerase
Describe the process of polyadenylation.
As RNAP is synthesizing mRNA, it is going to reach a Pol-A signal sequence on DNA that will synthesize the sequence AAUAAA on 3’ RNA.
This sequence is recognized by proteins factors (CPSF and CstF) associated with RNAP and they will jump off and bind to that sequence. They are basically cleavage factors.
CPSF is the one that recognizes the AAUAAA sequence and binds to the mRNA, while recruiting CSTF to the site.
CSTF recruits endonucleases which will cleave the mRNA. After cleavage, CSTF dissociates, but CPSF will stay associated with the sequence, recruiting PAP (poly-A-polymerase).
As PAP is extending the end of the 3’ RNA strand with a ton of adenosines, poly-A-binding proteins will add onto the repeating adenosine sequence until the polymerase has dissociated from mRNA.
What are the two different models of termination?
- Torpedo Termination
2. Allosteric Termination
What is termination?
So RNAP will keep on functioning even after the mRNA strand has been cleaved and polyadenylated.
But after reading the poly-A sequence, it will induce termination.
Explain the Torpedo Termination mechanism.
The torpedo model is the preferred hypothesis.
On the RNAP there is an exonuclease that tracts along with RNAP. As it is ending RNA synthesis and the RNA is cleaved, that nuclease (Rat1/hXrn2) is going to hop off of the polymerase and hook up with the RNA and start cleaving it, until it pulls the RNA out of the polymerase. F
inally, you have no RNA associated with the polymerase, signaling termination and the dissociation of RNAP.
Explain the Allosteric Termination mechanism.
This one is based on the fact that as soon as you have that cleavage from the polyadenylation reaction, there are other protein factors that may associate with the RNAP.
They alter the conformation of the RNAP in such a way it falls off the DNA and no more RNA synthesis happens.
Larger animals will have more _____ than smaller ones.
Introns.
What is having more introns advantageous?
Alternative splicing can take place, expressing different genes even though they ultimately originated from the same type of information.
What are the four classes of introns?
Group I -> self splicer
Group 2 -> self splicer
Group 3* (eukaryotes) -> spliceosomal introns
Group IV (some tRNA) -> requires ATP and an endonuclease.
Explain the intron class: Group I and Group II.
Group I and II are ‘self splicers’. They don’t require any specific factors or ATP.
Explain the intron class: Group III.
They are only found in eukaryotes and are specific to mRNA. They are called spliceosomal introns that require a spliceosome.
A spliceosome is a huge protein/machine that about the size of a ribosome.
Explain the intron class: Group IV.
Specialized intron splicers that are associated with tRNA.
What two reactions have to take place in order to have splicing?
You have to have two transesterfication reactions. Whether you are a self-splicer or an intron that requires a spliceosome. These two reactions must take place.
What does Group I and Group III use to initiate the first transesterifcation reaction?
They both use the 2’ OH group on adenine as their nucleophile, which will attack the 5’ end intron-exon junction.
What does Group II use to initiate the first transesterifcation reaction?
It uses the 3’ OH of a free guanine as a nucleophile, which will attack the 5’ end intron-exon junction.
What is the end result of a Group I and III splicing?
You will end up with a lariat structure and your combined exons in a linear form.
What is the end result of Group II splicing?
You will end up with a linear intron and a linear exon.
Explain the mechanism of splicing for Groups I.
- You will have an external guanine that will act as a nucleophile. The 3’ OH end of the guanine will attack the 5’ intron-exon junction, creating a free 3’ OH end on the 5’ end (no phosphodiester bond is formed here).
- The free 3’ OH end on the 5’ end will now perform a nucleophilic attack on the 3’ intron-exon junction to create a phosphodiester bond, linking the two exons together.
Net result: Linear Exon + Linear Intron.
Explain the mechanism for splicing for Groups II and Group III.
- You will have an internal adenosine that will act as a nucleophile. The 2’ OH of the adenine will attack the 5’ intron-exon junction, creating a free 3’ OH end on the 5’ end AND a new phosphodiester bond is made between the adenine and the end of the exon.
- Then the free 3’ OH end on the 5’ end will now perform a nucleophilic attack on the 4’ intron-exon junction to create a phosphodiester bond, linking the two exons together.
Net result: Linear exon + Lariat Structure
Does splicing require energy?
It requires a lot of energy.
Again, what does Group III utilize?
It utilizes a spliceosomal machinery.
How does the spliceosomal machinery know where the intron-exon junctions are located?
There are specific set of nucleotide sequences that the spliceosomal machinery can recognize. There are the bases GU on the 5’ end (right after the first exon) and the bases AG on the 3’ end (right before the second exon).
These nucleotides serve as a recognition point for the machinery, and once it finds those nucleotides, it will attach to the mRNA and base-pair with it, continuing the specificity of the location and accuracy.
In other words, U1 and U2 will bind to the mRNA via base-pairing to ensure accuracy on location.
What dictates whether or not an intron will get spliced out? (3)
There are three features that will dictate whether or not an intron will be sliced out.
First you have this junction on the exon-intron on the 5’ end that is associated with this G-U sequence. This sequence is conserved about 100% of the time, it is highly conserved, it is the first two bases of the intron.
Then, you have a sequence that is composed of A-G, which is the final NT of the 5’ exon that is going to be conversed, but less highly conserved. Your U1 will have to make base-pair between these NT.
Secondly, you will see highly conserved sequences between the intron and the 3’ exon junction, this A-G site, again is highly conserved.
Third, you will always see this adenine that is relatively close to the 3’ end of the intron. It is part of what we call a pyrimidine tract (a set of sequence that is high in pyrimidines) this is the adenine that is going to use its 2’ OH to attack the phosphodiester bond between this guanine of the intron and the guanine of the exon at the exon-intron junction to produce a lariat byproduct and a linear exon.
What are snRNPs? What are they involved in?
Small nuclear ribonucleoprotein particles.
They are involved in splicing. (i.e. U1, U2, U4, U5, and U6)`
What does a snRNP contain?
It contains one of five small nuclear RNAs (snRNAs) and about 10 different proteins.
What forms a spliceosome?
snRNP + pre-mRNA
What is the relative size of a spliceosome?
It is the size of a ribosome and its assembly require ATP.
Describe a snRNP.
Your spliceosomal activity is dictated by these small particles called snRNP that are associated with snRNA.
They are going to have this 3-D structure where they have sequences that can actually interact with the mRNA.
To make a snRNP, you are going to have proteins that are going to interact with these RNA molecules.
In addition to the snRNPS, you are going to have accessory proteins that are going to help the snrNPS.
What constitutes the during early splicing?
Base pairing of U1 and U2 to pre-mRNA.
What happens when U2 binds to pre-mRNA?
It causes the nucleophilic adenine to protrude out of the strand, making the branch site.
What portion other structures are important for the catalysis of splicing?
RNA secondary structures (i.e. Stem loops)
What portion of the snRNP is catalytic?
The RNA portion, not the protein portion.
Where does U1 bind to?
The 5’ splice junction.
Where does U2 bind to?
The intron branch point (facilitates 2’ OH, 5’-Phosphodiester bond formation).
Explain the whole process of splicing, starting with the binding of U1.
- The first thing that is going to happen is that U1 will find the 5’ exon-intron junction and base-pair to RNA.
- Then, there will be another set of other proteins that will define the branch site. It is this U2AF65-35 (U2AF) that going to fit on that pyrimidine tract that is going to associated between the adenine (that is the branch point) and the end of the intron.
- That will now recruit a protein that will sit on the BBP (branch point binding protein).
- Once that has been established, this BBP is going to be replaced by U2 which has sequence identity and homology to the branch point, so that the adenine protrudes out.
- Next, comes in the tri-snRNP-particle that has 3 snRNPS associated with it. U4 is masking U6’s ability to interact with U2, and the reason for this is that ultimately, U6 has the catalytic activity, but it needs to interact with U2, which is sitting at the branch point. It is also going to base-pair with U2. U6 has the catalytic activity so it has to base pair with U2.
- So the tri-part particle comes in and interacts with U1 and U2, however, the thing that has to happen is that U6 needs to sit where U1 is sitting. So when splicing is going occur, U1 has to leave so that U6 can have homology at that junction.
- The final thing that has to happen for catalysis to happen is that U4 has to dissociate, so that U6 and U2 can interact, activating the catalytic site to allow the adenine to attack the junction to make the phosphodiester bond, and U6 will allow the release of the exon
What is the role of U4?
It masks the catalytic activity of U6 in the U4/U5/U6 tri-snRNP prior to the actual transesterifcation reaction.
Some extra information about splicing.
Massive rearrangements of base-pairing interactions amount various snRNAs convert the spliceosomes into a catalytically active form, which releases the U1 and U4 snRNP, bringing together U2 and U6.
RNA molecules play key roles in directing the alignment of splice sites (i.e. U1 and U2 base pairing with the pre-MRNA) and in carrying out the catalysis (U2/U6 catalytic center)
What is the role of U6?
It has the catalytic activity.
What does the E complex during splicing look like? (Early presplicing complex)
- U1 snRNP binds to the 5’ splice site.
- It is recruited by ASK.SF2 (a SR protein; Ser-Arg-Rich Region) - U2AF binds to the pyrimidine tract.
- BBP binds toU1 snRNP, Mud2P and the RNA neat the 3’-end of the intron, upstream from U2AF.
What are SR proteins?
They are Ser-Arg rich regions hat mark the specific areas of the exon-intron junctions for the snRNPs to bind.
What happens to U1 when U6 comes it?
U1 dissociates from the mRNA to allow for U6 to base pair.
How does the spliceosome find the splice sites reliably?
- Coupling of proteins allows the processing factors to present at high local concentrations when splice sites and poly-A signals are transcribed by RNAP II, enhancing the rate and specificity of RNA.
In other words, the spliceosomal machinery is associated with RNAP and as RNA is being produced, it will jump off of the CTD tail and splice off the intron, but the jumping off onto the junction require other signals to tel it where to go.
- The association of the splicing factors with phosphorylated CTD also stimulates Poly II elongation. Thus, a pre-mRNA is not synthesized unless the machinery for processing it is properly positioned.
What two things can help the spliceosome find the splice sites reliably?
- Exonic splicing enhancers.
- SR proteins.
They contribute to exon definition and regulate alternative splicing.
How do the ESE (exonic splicing enchancers) and ST proteins know where to go themselves?
Correct 5’ GU and 3’AG splice sites are recognized by splicing factors based on proximity to exons.
ESE’s are binding sites for SR proteins. The SR proteins interact with each other and recruit the splicing machinery to nearby splice sites.
What is alternative splicing regulated by?
It is regulated by activators and repressors.
How can two different hormones be produced from essentially the same gene?
You can have alternative cleavage, alternative polyadenylation, and alternative splicing.
You can have a gene that has two Pol-A-sites. If you end transcription at those sites you will have two different fragments depending on when transcription ended.
After cleavage of the RNA molecule and polyadenylation, you will splice it and they will both consist of different number of exons since they terminated at different sequences.
After translation and all of that has happen, you essentially have different hormones that originated from the same gene.
What does U5 do?
It interacts with mRNA through protein-protein interactions.
