Post transcription processing in eukaryotes Flashcards
Whats the main eukaryotic transcription promotor and what does it do?
The core promoter is the TATA box
- upstream sequence elements
- enhancers
- general transcription factors for RNA Pol I (TF)
Whats the first TF that TFIID binds to?
The TATA box, then other TFs join it
Whats happens when RNA Pol II binds?
It transcribes mRNA proteins (larger than bacterial)
Once some TF have attached to the TATA box
More TF bind
Now the pre-initiation complex (PIC) is assembled and its the initiation of transcription
What is the PIC composed of in terms of subunits?
What is a key step in the start of elongation?
9 subunits: ATPase, Helicase, Protein Kinase
TFIIH is a key step in the start if elongation
What happens in the elongation stage of eukaryotic transcription?
- C-terminal domain (CTD) is phosphorylated
- There is a conformational change which tightens drip
- General TFs dissociate
- This aquires new proteins- including elongation factors
- phosphorylation allows loading of RNA processing machinery
What does the C-terminal domain (CTD) contain?
- 52 tandem repeats of 7 AA
- each contains 2 series (Ser2 and Ser5)
- Phosphorylated
Name the stages to post transcriptional processing
Give a general overview of what each one is
- Addition of the 5’ cap- protection and marking a mRNA
- 3’ processing and polyadenylation- Marking as the end of the mRNA and protection
- Splicing- To remove non-coding regions
- Editing (rare but important)- Similar to mutating the mRNA to produce alternative final product
- Transport- To cytoplasm for translation
What is RNA splicing?
- This is the third thing to happen to RNA
- There are coding and non-coding sections of DNA known as Exons and introns respectively
- When the RNA is transcribed everything is converted into RNA
- The RNA is transcribed then capped at thr 5’ end and the PolyA tail is added
- Then the introns between the coding sequences are removed by the process of splicing
When were introns first detected?
In 1977
Then, little was known about their function but likely to allow generation of multiple products from the same gene
How are introns removed?
- conserved sequence of motifs indicate exon/intron boundaries
- introns are removed by 2 transesterification reactions
What is the steps for RNA splicing?
Dont go into detail just state them
- Cleavage at the 5’ splice site
- Cleavage at the 3’ splice site and joining of the exons
In step 1 of RNA splicing (Cleavage at the 5’ site), what are the steps to this part?
- The hydroxyl group (OH) of the A within the branch acts as a nucleophile and attacks the 5’ splice site
- introns folds back on itself
- phosphodiester bond between A (BP) and G (5’ SS)
Whats happens in stage 2 of RNA splicing (clevage at the 3’ splice site and joining of the exons)?
- The newly liberated 3’OH group of the 5’ exon becomes a nucleophile and attacks the phosphoryl group at the 3’ splice site
- This is mediated by the OH group of the last base of the exon
- This performs a nucleophilic attack on the 3’ splice site
- This results in cleavage at the 3’ splice site
- And this looped intron is released
- This intron with the looped structure is also describes as a lariet and this is then degraded in the cell
- The two exons are then simply ligated together
How is the splice site selected?
Co-transcriptional loading
- RNA Pol carries with it many of the proteins involved in splicing
- Much of the machinery of splicing assembles as the gene is being transcribed
Whats a spliceosome?
A spliceosome is a large and complex molecular RNA identity found primarily within the nucleus of eukaryotic cells
Whats are spliceosomes comprised of?
Both RNA (5) and protein (150)
- RNA component: small nuclear RNAs (snRNAs)- there are 5 of these U1-6
- Protein component: snRNPs- there are 6-10 proteins which form this
- different complexes come in at different stages
- uses ATP
Tell me about the binding of spiceosomes to the pre-mRNA?
- First U1 snRNP binds to the 5’ splice site (SS)
- U1 snRNP consists of U1 snRNA + Proteins
Tell me about the U1 small nuclear ribonuclear protein
- This is the U1 RNA and you can see it has extensive secondary structure of stem loops
- What it also has is a sequence at the 5’ end which is complementary to the 5 SS of the gene
- This is what targets the U1 snRNP to the 5 SS
Commitment/ early complex
Pre-spliceosome/ A complex
How is a spliceosome formed?
- U1 snRNP bind to the 5’ splice site
- BBP binds to the branch point
- U2AF35/65 bind to the 3’ splice site
- SR provides a framework
- U2 snRNP can now bind to the branch site which is aded by U2AF and displaces BBP
- Branch point A residue is excluded creating a single nucleotide bridge
- U4,5 and 6 snRNPs can now bind
- The U2AF proteins are displaced
- This brings the 5’ splice site close to the branch point and the 3’ splice site
What are the three classes of RNA splicing?
- Nuclear Pre-mRNA splicing
- Group II self-splicing
- Group I self-splicing
Tell me about Group II self-splicing?
- Some eukaryotic genes (Rare)
- Two transesterification reactions (branch site A)
- RNA enzyme encoded by intron (ribozyme)
- e.g. rRNA, tRNA, mRNA or organelles in fungi, plants and bacteria
- self-splicing introns- folds itself into a specific conformation within the mRNA and catalyses the chemistry of its release
Tell me about group I self-splicing
- Some eukaryotic genes (rare)
- Two transesterification reactions (branch site G)
- RNA enzyme encoded by intron (ribozyme)
- e.g. Introns in eukaryotic viruses
- Self-splicing introns - folds itself into a specific conformation within the mRNA and catalyses the chemistry of its release
What are the two things that can occur which is when the selection of the splice site isn’t correct/ common?
- Exon skipping- there may be a 5’ on 1 and 3’ on 3 which means its cleaves there and skips 2
- Cryptic splice site selection- splices in a site that isn’t a splice site but only resembles one
What are SR proteins?
What do they bind to?
What do they recruit?
SR proteins are rich in serine (S) and arginine (R)
SR proteins bind to sequences called exonic splicing enhancers (ESEs)- they are located within the exons
SR proteins recruit the splicing machinery to the nearby splice sites
Whats alternative splicing?
A process by which the RNA can be spliced in more than one way
This can result in the formation of 2 or more proteins from the same gene
e.g. Theres a 6 exon gene
the thyroid can splice at 4, produce a protein then via proteolysis make calcitonin
Whereas;
The brain can splice all 6 but not 4, produce a protein and then make CGRP
This is an example of how two different proteins can be made by the same gene
Whats RNA editing?
A process in which information changes at the level of mRNA
This is not a mistake- this is a specific controlled reaction
Codons
What type of reaction is RNA editing?
What occurs on this reaction?
Editing is a deaminase reaction
In which the amino group on the nucleotide ring is removed
This editing reaction is catalyzed by cytosine deaminase with a rna binding domain
26 bp sequence around the CAA editing site which allows recognition of this site then converts c to u
On what receptors does RNA editing also occur?
What happens here?
Glutamate receptors
- transmitter gated ion channel (in nervous system)
- here there is an A to I (inosine) change
- Catalysed by adenosine deaminase acting on RNA (ADAR) – there are 3 in humans
- acts on double stranded RNA
- alters the Ca2+ permeability of the channel
Why is RNA editing vital?
ADAR knock out mice: epilepsy and die
All stages are essential for full functional proteins
Mutations in the promoters or any sequence involved processing results in no protein and can lead to genetic disorders.
One disorder is Thalassemia what is this?
Thalassemia is a genetic disorder in which there is decreased production of one of the globin chains found in haemoglobin.
Adult haemoglobin
consists of 2 alpha globin and
2 beta globin chains
What are the mutation hotspots of the beta globin gene?
How does this effect globin production?
What type of mutation is it?
When theres a mutation in intron 2, what can it lead to?
beta0 major- no globin synthesis
creation of a cryptic splice site
Explain what happens in the first stage of addition of a 5’ cap (addition of guanosine at the 5’ end of RNA)
- Removes the gamma and beta phosphates of GTP
- Added in a GMP (guanosine monophosphate) form
- The GMP (guanine) is added to the 5’ terminal base of the transcript
- GMP is added to the RNA in reverse orientation from all other nucleotides forming a 5’-5’ linkage
- We now have a G linked to phosphate which comes from the GTP linked to the remaining 2 phosphates of the rna
- Now the way in which this G is added to the end of the rna is actually in the reverse orientation compared to every other nucleotide in the chain
- Because what the enzyme is doing is forming a 5’ to 5’ link
- So, if you look at the rna chain they are all lined 5’ to 3’ then with this 5-5 link on the end which is a very unusual link for rna and DNA
Explain what happens in the second stage of addition of a 5’ cap (adding a methyl group to guanine at the 7th position)
- Adds methyl group to guanine (7th position of the purine)
- What then happens is the second step of this reaction
- In this second step the guanosine that we have just added to the rna is now converted to 7 methyl guanosines
- This is done by the enzyme guanine methyltransferase
- What this methyltransferase enzyme does is simply add a methyl group onto this 7th position on the purine ring
- This then provides what is called a cap which protects the 5’ end of the rna from degradation in the cell
Why is there an addition of a 5’ cap?
- Addition of 7 methyl-guanosine to the 5’ end of RNA
- The cap helps to distinguish mRNA from other RNA
- Helps mRNA be properly processed and exported from the nucleus
- Protects it from degradation in the cell
What does 3’ processing and polyadenylation involve?
Where is the signal for this stage encoded?
What happens?
- The 3’ end of the mRNA is polyadenylated
- This involves the addition of a strong of Adenosines (roughly 200) to the end of the transcript
The signal for polyadenylation is encoded in the DNA (conserved 3’ control region)
- Proteins bind to the poly A tail and help stabilise the RNA
- What you find is the sequence AATAAA followed by 10-30 bases by a CA then 10 – 20 bases by a GT rich region
- The whole of this gets transcribed into rna and becomes the rna sequence AAAUAAA then ca then GU
- These sequences are recognized by a set of proteins
- So, what happens is firstly we get binding of the protein cpsf which stands for…
- So, this binds to the AAAUAAA sequence
- You then get binding of PolyA pol to the CA sequence
- This is followed by binding of the cleavage stimulation factor to the GU rich region
What are the 3’ processing and polyadenylation sequences recognised by?
- These sequences are recognized by a set of proteins
- So, what happens is firstly we get binding of the protein cpsf which stands for…
- So, this binds to the AAUAAA sequence
- You then get binding of PolyA pol to the CA sequence
- This is followed by binding of the cleavage stimulation factor to the gu rich region
What the the components of the 3’ processing and polyadenylation brought together by?
RNAP II
What do the 3’ processing and polyadenylation proteins work together to do?
How do they do this?
Cleave to RNA
- They cut the rna just after this CA
- What then happens is you get addition of what’s known as a poly A tail
- This is essentially about 200 ribonucleotides all of which contain the adenine base
- The addition of the poly a tail is catalyzed by the poly a pol
- What this poly a tail does is essentially protect the 3’ end of the rna from degradation in the cell
- Occurs in the nucleus alongside transcription
Alternative polyadenylation
dysregulated alternative polyadenylation
overview of eukaryotic gene expression
How is RNA transported?
- Only way to leave the nucleus is through the nuclear pore complex
- Small molecules can move through but larger molecules like RNA and proteins have to be transported across through an energy dependant process
- REF proteins bind to the splicing junction by associated with spliceosome
- After splicing they remain attached to the RNA at the exon/exon boundary
- Transport relies on a set of proteins called ref proteins
- Ref proteins targeted by transport proteins
- bound by 2 transport proteins TAP and MEX – these bring the rna to the nuclear pore (they are transport molecules)
- Dock with proteins at the pore
- And the rna is transported through into cytoplasm of cell
- This ensures that only fully spliced rna is transported
- REF proteins are targeted by transport proteins
- bring RNA to nuclear pore & dock
Transport of RNA
