Lecture 7: Post-translational control Flashcards
1
Q
What is RNA editing?
A
- Occurs in eukaryotic genes (e.g. tRNA, rRNA, mRNA n miRNA)
- Also found in archaeal n bacterial systems
- In trypanosomes there is extensive RNA editing of some kinetoplast genes.
- In vertebrates RNA editing occurs in the nucleus
- In cephalopods, RNA editing also occurs in
2
Q
Describe the trypansoma brucei’s mitochondrial DNA
A
- Mixture of maxicircles (~23kb: copy number, ~50)
- Heterogenous set of minicircles (~1kb: copy number 5k-10k)
Cateanated/interlocked to form aplanar network that resembles chain mail
- Heterogenous set of minicircles (~1kb: copy number 5k-10k)
3
Q
What is the structure of the maxicircle gene?
A
- 23kb
- Variable region
- Conserved region
○ 2 of them encode ribosomal RNAs
○ Extensively edited genes - Genes w limited editing (cytochrome B, cytochrome oxidase II)
- Remainder of the genes aren’t edited, just expressed
- 12 ORF are edited to be translatable
4
Q
Why do mitochondria of trypanosomes and plants utilize extensive RNA editing?
A
- Mitochondria of trypanosomes and plants use a primitive genetic system, derfvied from the RNA world -> offers few opportunities for other forms of transcriptional control.
- Extensive editing is regulated to produce different mRNAs under different conditions, using different minicircles to provide different gRNAs.
- Is RNA editing a primitive way to change the expression of genes?
- Is it a relic, perhaps, of mechanisms that operated in the early RNA world?.
- Different gRNA can be expressed, depending on the environment
5
Q
How do trypanosomes modify their RNA transcripts?
A
- Each circle encodes guide RNA (gRNA)
- gRNA binds to 3’ of primary transcription
○ Every loop is a site where on the gRNA, there is an A - Information from gRNA is copied to mRNA
○ Requires specialized proteins that can recognize addition/deletion site
○ Break either strand, add in U/take U out of mRNA
○ RESULT: mRNA w Us inserted n deleted - Editing provides anchor site for gRNA 2
- gRNA 2 encoded on a different minicircle
- gRNA 2 acts as a template for the addition/deletion of Us in the primary transcript
RESULT: fully edited mRNA
- gRNA binds to 3’ of primary transcription
6
Q
How are some maxicircles extensively edited?
A
- More bases added than there were in the original transcript
- Addition leads to formation of start codon AUG n stop codon uAG
- In between, continuous ORF of triplets
- Maxicircle genes are encrypted n hv to be decrypted before message can be read
7
Q
What is mammalian nuclear APOBEC-mediated mRNA editing?
A
- Cytosine deaminated to generate uracil
○ Change of amine group to oxygen
Editing can alter protein sequences -> alter protein function
8
Q
What does an editosome consist of?
A
○ A cytidine deaminase activity is involved – APOBEC (ApoB mRNA editing enzyme catalytic subunit)
○ Another protein, ACF (APOBEC Complementation Factor) is also required
○ Both recognize sequences flanking the C to be edited
9
Q
What is the function of the APOBEC editosome?
A
- ApoB is required for uptake n cholesterol transport
○ Editing site: Exon 26 (CAA)- LIVER
○ No editing in the liver [differential control of ApoBEC, if not expressed in the liver, no editing]
○ Expression of ApoB- 100 (protein 100kd in size)
§ Lipid associated
§ Binds to LDL receptors - INTESTINE
○ Strong expression of APOBEC -
○ APOBEC recognizes editing site in exon 26 -> converts to stop codon (UAA)
○ RESULT: truncated version of ApoB (ApoB-48)
○ Lipid associated
○ Does not bind to LDL receptors
TLDR: one gene two functions separated by cell specific expression
- LIVER
10
Q
What is mammalian nuclear ADAR-mediated mRNA editing?
A
- Deaminate adenosine to inosine
- Protein required: adenosine deaminase acting on RNA (ADAR)
- Inosine is a guanosine mimic
- Native sequence
○ AAG UCA
○ Lys Ser - Edited sequence
○ IAG UCI
○ Glu Ser - ADAR recognizes dsRNA
- Requires a fold back in mRNA (i.e. stem loop structure of complementary bases in the RNA)
- Convert specific adenines to inosines
- I is translated as G.
○ If the editing site is in an exon, this can change the protein sequence. - I mimics G.
○ If the editing site is in an intron, it can create new splice sites. (Lecture 6: the 5’ splice site is exon|GU: the 3’ splice site is AG|exon).
Thus, ADAR can regulate both protein function and alternative splicing.
11
Q
What are Alu elements?
A
- Class of primate-specific retrotransposons ~300 bases long
- Sub-group of SNEs
- More than 1m Alu dispersed in our genomes (~11% of our genome) -> opportunity to derive dsRNA.
12
Q
Why would there be inverted repeats in mRNA?
A
If 1 Alu element inserts into gene n the other inserts in the opposite direction -> complementarity in mRNA -> allows ADAR to bind
13
Q
What is the value of ADAR-mediated RNA editing?
A
- It may have evolved as a defence system to inactivate retroviruses/ retrotransposons.
- Enhances genome plasticity (new proteins, alternative splicing).
- ADAR can also recognise DNA:RNA hybrids and has a role in DNA repair.
- There are three ADAR gene isoforms in us, and in mice, with different targets.
- Neurobiology link?
○ Mice lacking ADAR1 have frequent epileptic seizures, and die soon after weaning
○ Flies lacking ADAR display neurodegeneration
○ Glutamate receptor expression depends on ADAR1 activity
Serotonin receptors are expressed from edited transcripts
14
Q
How is mRNA edited for nuclear transport?
A
- As introns are removed, spliceosome deposits exon junction complex (EJC) at sites upstream of exon fusion site
○ Upstream of the exon/intron are 2 exon borders
○ ECJs are important bc they interact w SR proteins- At a late stage in splicing, a nuclear export receptor complex is transferred from the CTD of Pol II.
- This provides a nuclear export signal (NES).
- mRNA is not naked
Clothed with proteins and is a complex compacted particle of RNA and proteins – it is a messenger RNA particle (mRNP).
15
Q
How does nuclear export of mRNA occur?
A
- The NER targets the nuclear pore, and the mRNP is threaded through.
- SR proteins are removed
- The CBC is removed and replaced by eukaryotic Initiation Factor 4E (eIF4E, LF104 lecture 15) and circularised with eIF4G.
○ eIF4G interacts w PABP -> pseudo circularization of mRNP
NER is returned to the nucleus.
