Lecture 7. Post-Transcriptional Regulation of Gene Expression 2 Flashcards
Where does RNA editing occur?
In eukaryotes: tRNA, rRNA, mRNA and miRNA (micro RNA)
Also in archaea and bacteria (mitochondria and plastids)
Where does extensive RNA editing of some kinetoplast genes take place?
Trypanosomes
Where does RNA editing occur in vertebrates?
In the nucleus
What is the structure of Trypansoma brucei?
Basal body with long flagellum intermediately associated with an undulating membrane allowing movement via swimming
Contains long, singular mitochondrion with a kinetoplast at the front end of it
What is a kinetoplast and kinetoplast DNA (kDNA)?
Stacks and stacks of mitochondrial DNA with an unusual arrangement
A mixture of maxicircles (~23 kb: copy number, ~50) and a heterogeneous set of minicircles (~1 kb: copy number 5,000 - 10,000) catenated/interlocked to form a planar network that resembles chain mail
What does maxicircle kDNA encode?
Encodes two rRNAs (9S, 12S0, one ribosomal protein (RPS12) and seventeen protein-coding sequences
12 of the open reading frames require RNA editing in order to be converted into transferable mRNAs
What are some maxicircle genes?
Encrypted, and their transcripts must be decrypted by RNA editing prior to translation
How does base addition and deletion occur in the maxicircle kRNA?
A guide RNAs (gRNAs) encoded on a minicircle binds the target maxicircle RNA transcript by base pairing at its 5’ ‘anchor’ and 3’ ‘tail’ sequences
It provides a template that can be copied into the final edited 5’ mRNA
As opposite of Us present on gRNA1 determining whether or not Us will be inserted or deleted
gRNA2 is encoded on a different minicircle and so on until fully edited mRNA achieved
Is RNA editing (base addition and deletion) unique to trypanosomes?
No mitochondria of trypanosomes and plants utilise it
Some plastid genes are also edited this way
What is RNA editing by APOBEC?
Cytosine conversion into uracil through deamination (NH₂ on cytosine replaced by double bonded O with neighbouring nitrogen gaining a hydrogen)
What can RNA editing by APOBEC result in?
Editing can alter protein sequences and may therefore alter protein function
What is APOBEC?
A cytidine deaminase activity is involved – APOBEC (ApoB mRNA ACF
editing enzyme catalytic subunit)
Two domains (catalytic and pseudo-catalytic) and turned off until associated with ACF (APOBEC Complementation Factor)
Both recognise sequences flanking the C (cytosine) to be edited
Might be called the editosome
What area does APOBEC not work in and why is this significant?
Liver
Allows for the formation of ApoB-100 which is lipid-associated and binds to LDL receptors (ApoB is required for uptake and transport of cholesterol)
What does APOBEC do in the intestine?
APOBEC recognises binding site in exon 26
Converts CAA into UAA
ApoB-48 produced instead of ApoB-100
ApoB-48 is lipid-associated but does not bind to LDL receptors
What is RNA editing by ADAR?
Adenosine conversion into inosine through deamination (NH₂ on adenosine replaced by double bonded O)
What is inosine a mimic of?
Guanosine
What is RNA editing by ADAR?
Editing can alter protein sequences and may therefore alter protein function
How does ADAR-mediated RNA editing take place?
ADAR recognises dsRNA.
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 (the 5’ splice site is exon| GU: the 3’ splice site is AG|exon)
Thus, ADAR can regulate both protein function and alternative splicing
ADAR can generate new splice sites
Why would there be inverted repeats in mRNA?
Alu elements (class of primate-specific retrotransposons ~300 bases long, sub group of SINES)
There are more than one million Alu elements dispersed in the our genomes (~11% of our genome): this provides plenty of opportunity to derive dsRNA
If one Alu is inserted into the gene, and another one inserted in the opposite direction, there will be complementarity in the mRNA which will allow ADAR to bind
How many of our genes are prone to ADAR gene editing?
Over 1000 ADAR-edited genes (1 in 20)
What is the value of ADAR-mediated RNA editing?
It may have evolved as a defence system to inactivate retroviruses/retrotransposons
It 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 target
In humans, ADAR-mediated mRNA editing is nuclear, but where does it occur in the long-finned squid and how do we know this?
In the cytoplasm
An antibody specific to squid ADAR (not rat ADAR) shows cytosolic location of ADAR in transfected cells
Cytosolic fraction of squid tissue have no nuclear contamination but can convert pA to pI
Squid responds to environment without relying on nuclei
As the introns are removed in the exon hypothesis, what does the spliceosome deposit?
Deposits the exon junction complex (EJC) at sites just upstream of the exon EJC fusion site
Why are the EJCs important?
EJC interacts with SR proteins and they compact mRNA
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: it is clothed with proteins and is a complex compacted particle of RNA and proteins – it is a messenger RNA particle (mRNP)
mRNP is mature enough to be exported (double nuclear membrane)
