2!!! - Control of gene expression: RNA Flashcards
What are isoforms?
Multiple proteins made from the same gene
What are 3 ways that isoforms made?
From:
- Alternative splice sites
- Alternative start sites
- Alternative polyadenylation (the end of the DNA)
What do some mRNAs have that can be regulated independently?
A second ORF
What 3 ways gene expression be regulated?
- Transcription
- Splicing
- Translation
- Regulated nuclear transport
- RNA stability
How many genes in the human are alternatively spliced?
75%
How many genes in the drosophila are alternatively spliced?
40%
What are 4 ways that alternative splicing can occur?
1) Optional exon
2) Optional intron
3) Mutually exclusive exons
4) Internal splice sites
What are mutually exclusive exons?
Exons that don’t occur at the same time
When do mutually exclusive exons usually occur?
When the exons code for similar proteins
What is an internal splice site?
Where the 3’ OR 5’ end splice site is in the middle of the intron instead of the end
How does the cell alternatively splice sites?
- Splice donor and acceptor sequences are very small (only 2 base pairs), meaning they are VERY FREQUENT
- Splice sites are relatively simple
- Mechanism isnt very precise - taken advantage of to produce different splice sites
How do RNA binding proteins assist alternative splicing?
They bind around the donor/acceptor splice sites and influence where splicing occurs
What affects the choice of splice site?
- RNA binding proteins
- Other sequences and secondary structure in the RNA
Why is the splicing mechanism not very precise?
To take advantage of it
What is the most common spliced gene?
Dscam in drosophila
What is Dscam involved in?
Specifying neurons in the brain of the drosophila
In what way is the Dscam gene alternatively spliced?
Has mutually exclusive exons
How many different possible isoforms are there in the Dscam gene?
38,000
What process is used to help to understand alternative splicing in humans?
Sex determination in drosophila
How is sex determined in drosophila?
By the amount of X chromosomes that the fly has:
- Male = X
- Female = XX
- This is then translated into sex characteristics
What are the three genes that regulate male or female differentiation?
1) sxl (sex lethal)
2) tra (transfomer)
3) dsx (double sex)
How are MALE characteristics determined in the drosophila?
- Transcripts sxl and tra are spliced to give INACTIVE protein isoforms
- dsx is ACTIVE
What does dsx do in the active form?
- Gives rise to a male specific repressor protein, which represses transcription from genes required for female development
How are FEMALE characteristics determined in the drosophila?
- MORE transcription of sxl due to two X chromosomes
- SMALL AMOUNT of functional sxl protein made using an alternative promoter
- sxl binds to its own transcript in a positive feedback loop to make MORE of the active protein by REPRESSING SPLICING at this site
How does sxl block splicing of the sxl transcript?
sxl is a binding protein - binds to transcript:
- Blocks the splice acceptor site that is used in males
- Causes splice over and removal of the central exon
- Activation of an alternative acceptor - joining the outside exons together (alternative combination that in the male)
- The joining of these exons together form the active form of tra
What does the active form of tra do in females?
What does this result in?
Binds to dsx mRNA with tra-2 to activate a splice acceptor in dsx mRNA:
- This results in a change to the carboxy terminus at the end of the protein
- Producing the female specific isoform of dsx transcription factor
- This isoform represses male differentiation genes
How do RNA binding proteins work to specify sex?
- Can repress splicing by HIDING the splice acceptor, forcing an alternative splice site to be chosen by the splicing complex
- Can enhance splicing at a new site by binding to the splice site and active it
How can the site of polyadenylation of the mRNA be regulated to produce different protein isoforms?
- B lymphocytes produce the
How do B lymphocytes produce 2 different antibody isoforms?
Why is this important in the immune response?
By altering where the RNA transcript is cleaved and polyadenylated
Important because B lymphocytes originally want to make antibodies that are NOT secreted and are tethered to the membrane
As these antibodies are perfected - need to be secreted, no longer have a transmembrane domain
How many possible sites in antibodies are there for cleavage and polyadenylation?
2 possible sites
What tells where to cut the preRNA to make mRNA?
Sequences inside the preRNA
What is the mechanism of altering the site of cleavage and polyadenylation in antibodies?
- When the cell produces the preRNA, the first stop codon is spliced out, resulting in the translation of a transmembrane domain
- Switch to cleavage and polyadenylation earlier within the intron
- Loss of the splice acceptor (and second stop site)
- Intron no longer recognised, can’t be removed
- First stop codon not removed
- Results in the antibody being secreted
How are alternative starts sites regulated?
- Many different start sites (AUG sequence)
- Not all created equal - ribosome can skip AUG sites
What is ‘leaky scanning’?
Where the ribosome skips AUG sequences
What is the optimal sequence for the small subunit to stop and transcription to happen?
KOZAK sequence:
ACCAUGG
How do proteins differ when they have alternative start sites and why?
Only in their N-terminus
As all the alternative starts are all in the SAME reading frame
How do cells modify the use of their alternative start sites?
Using high levels of eIF-EF (initiation factor)
What start site does eIF-4G favour?
The first AUG site
What happens with HIV enters the cell as an RNA genome?
It is converted into DNA by reverse transcriptase and inserted into the host genome
When HIV DNA is inserted into the host genome, what can happen?
The entire genome is transcribed
Can undergo alternative splicing to give rise to many different protein isoforms
Wha is the full-length un-spliced HIV RNA needed for?
To make new virions to infect the the next stage
What is the full-length un-spliced HIV RNA needed for?
To make new virions to infect the the next cell
What helps full-length, un-spliced RNAs to leave the nucleus?
- Retroviruses produce a REV (a protein)
- REV goes back into the nucleus and binds to the introns of a full-sized RNA transcript, escorts the RNA out of the nucleus
Why don’t un-spliced RNAs leave the nucleus?
This is a checkpoint
What monitors the levels of HIV?
Levels of rev protein (produced by reteroviruses)
What targets mRNA to a specific part of the cell?
Signals in the untranslated region of mRNA (UTR)
Where is the UTR in mRNA?
Between:
- The 5’ end and the start codon
- The stop codon and the 3’ polyadenylation site
What structure do the 3’ UTR in the mRNA form?
What is this recognised by and what does this allows?
Stem loop structures
Recognised by cellular proteins which are localised to one side of the cell
This traps the RNA at a specific point in the cell - when translated, there is a higher concentration of the protein on one side of the cell compared to the other
What do many mRNA have in their 3’ and 5’ UTR?
Translational control elements
What can regulate the transcriptional control elements in the 5’ and 3’ UTR?
Ferrtin - proteins that stores ion in the cell (reducing available Fe)
Transferrin - receptor that imports ion into the cell (increasing available Fe)
What happens where is low Fe in the cytoplasm?
- Aconitase binds to the stem loops of 5’ UTR of ferritin mRNA and BLOCK TRANSLATION
- Fe not stored
- Also binds to stem loops of 3’ UTR of transferrin mRNA and BLOCKS DEGREDATION
- More receptors, more Fe into the cell
What type of protein is Aconitase?
An RNA binding protein
How does Aconitase block the translation of ferritin?
Prevents the small subunit from moving from the cap
How does Aconitase block the degradation of ferritin?
By stabilising the mRNA
What happens when there is high Fe in the cytoplasm?
- Aconitase binds to Fe in the cytoplasm and goes through a conformational change
- Aconitase releases mRNAs on ferritin and transferrin
- Ferritin now translated and transferrin mRNA destabilised
Is aconitase specific or not specific to certain mRNAs?
Specific
How can the cell modify ALL translation? (globally)
By using eIF2 and eIF-2B
What 2 situations is global control of translation needed?
1) When the cell becomes infected with a virus - slows down to prevent further infection
2) When the cell starves - runs out of energy
What is the mechanism of the global control of translation by eIF2 and eIF-2B?
Normally:
1) eIF2/GTP binds to Met tRNA - start scanning and progress from the cap
2) eIF-2b binds to eIF-2 and knocks off GDP - allowing GTP to bind and activate eIF-2 and recycling eIF-2b
When the cell enters rest phase:
1) Phosphorylates eIF-2 - causing it to bind tightly to eIF-2, blocking the recycling
2) Less active eIF-2 in the cytoplasm
What must eIF2 be bound to to be active?
GTP
What is required for the dissociation of GDP from eIF2?
eIF-2b
What are IRES?
What do they allow?
Internal ribosomal entry sites
- Stem loops in RNA, between ORFs that can initiate the formation of the ribosome independant of the cap/polyA initiation complex
Allow more than one gene to be present on an mRNA
(2 exons, 2 ORF)
Where are IRES’s common?
In viruses
How does the IRES work?
Imitates the cap structure, allowing the small subunit to work
What initiation factor binds to the IRES stem loop and is required for IRES based induction?
eIF-4G
What are the levels of eIF-4G in the cell?
Regulated
How do viruses favour the translation of their transcripts?
Cleave the hosts eIF-4G into a form that no longer binds eIF-4E but still binds IRES sequence
What is eIF-4G usually required for in the cell?
Binds to the polyA tail and eIF-4E (which is bound to the cap on the mRNA) - to form the looped structure of mRNA to ensure both ends are present before leaving the nucleus
Apart from in viruses, when else is eIF-4G cleaved and why?
During apoptosis:
- Genes required during cell death are usually in the second ORF (favoured during cell death)
- Utilise this to activate the IRES and continue to be translated
How can RNA stability be regulated in order to regulate translation?
1) Exonuclease - chews down the polyA tail
- At 30nt - the mRNA is decapped and recognised for degredation
2) Some mRNAs are favoured and RE-ADENYLATED in the cytoplasm - extending their half life
3) Proteins target RNAs to degrade them more quickly
What does DAN do?
Competes with eIF-4E to bind to the cap and promotes degredation
