Lecture 7. Post-Transcriptional Regulation of Gene Expression 2

Question 1

Where does RNA editing occur?

Answer 1

In eukaryotes: tRNA, rRNA, mRNA and miRNA (micro RNA)
Also in archaea and bacteria (mitochondria and plastids)

Question 2

Where does extensive RNA editing of some kinetoplast genes take place?

Answer 2

Trypanosomes

Question 3

Where does RNA editing occur in vertebrates?

Answer 3

In the nucleus

Question 4

What is the structure of Trypansoma brucei?

Answer 4

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

Question 5

What is a kinetoplast and kinetoplast DNA (kDNA)?

Answer 5

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

Question 6

What does maxicircle kDNA encode?

Answer 6

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

Question 7

What are some maxicircle genes?

Answer 7

Encrypted, and their transcripts must be decrypted by RNA editing prior to translation

Question 8

How does base addition and deletion occur in the maxicircle kRNA?

Answer 8

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

Question 9

Is RNA editing (base addition and deletion) unique to trypanosomes?

Answer 9

No mitochondria of trypanosomes and plants utilise it
Some plastid genes are also edited this way

Question 10

What is RNA editing by APOBEC?

Answer 10

Cytosine conversion into uracil through deamination (NH₂ on cytosine replaced by double bonded O with neighbouring nitrogen gaining a hydrogen)

Question 11

What can RNA editing by APOBEC result in?

Answer 11

Editing can alter protein sequences and may therefore alter protein function

Question 12

What is APOBEC?

Answer 12

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

Question 13

What area does APOBEC not work in and why is this significant?

Answer 13

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)

Question 14

What does APOBEC do in the intestine?

Answer 14

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

Question 15

What is RNA editing by ADAR?

Answer 15

Adenosine conversion into inosine through deamination (NH₂ on adenosine replaced by double bonded O)

Question 16

What is inosine a mimic of?

Answer 16

Guanosine

Question 17

What is RNA editing by ADAR?

Answer 17

Editing can alter protein sequences and may therefore alter protein function

Question 18

How does ADAR-mediated RNA editing take place?

Answer 18

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

Question 19

Why would there be inverted repeats in mRNA?

Answer 19

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

Question 20

How many of our genes are prone to ADAR gene editing?

Answer 20

Over 1000 ADAR-edited genes (1 in 20)

Question 21

What is the value of ADAR-mediated RNA editing?

Answer 21

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

Question 22

In humans, ADAR-mediated mRNA editing is nuclear, but where does it occur in the long-finned squid and how do we know this?

Answer 22

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

Question 23

As the introns are removed in the exon hypothesis, what does the spliceosome deposit?

Answer 23

Deposits the exon junction complex (EJC) at sites just upstream of the exon EJC fusion site

Question 24

Why are the EJCs important?

Answer 24

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)

Question 25

How is the messenger RNA particle (mRNP) exported via nuclear export?

Answer 25

1. The NER targets the nuclear pore, and the mRNP is and the mRNP is threaded through. SR proteins are removed. Threaded through 5’ end first. SR proteins removed and recycled
2. The CBC is removed and replaced by eukaryotic Initiation Factor 4E (eIF4E) and circularised with eIF4G (interacts with PABP). NER is returned to the nucleus. mRNA become pseudo-circle and effectively ready for translation

Question 26

How many eukaryotic genes do we think are regulated by miRNA?

Answer 26

At least 1/3 of export eukaryotic genes
The role of miRNAs in regulating gene expression might be as important as that of transcription factors

Question 27

What is microRNA (miRNA)?

Answer 27

Small non-coding regulatory RNAs processed from dsRNA precursor
Transcribed by RNA Pol II

Question 28

What is the structure of miRNA?

Answer 28

Stem-loop double-stranded structure
2-base overhang at the 3’ end
miRNA guide strand typically 20-26 bases long
GC rich

Question 29

What is the canonical pathway in the biogenesis of miRNA?

Answer 29

Found downstream of RNA Pol II promotor
miRNA precursors can be found intergenically and sometimes as clusters transcribed together where there are made as a primary miRNAs (pri-miRNAs)

Question 30

What is the non-canonical pathway in the biogenesis of miRNA?

Answer 30

miRNA precursors can sometimes be encoded in an intron (known as a mirtron)
Exons either side

Question 31

How does nuclear processing occur in the non-canonical pathway?

Answer 31

Spliceosome cuts out mirtron lariat containing the miRNA (as well as mRNA)
The lariat is debranched and base-paired to get the miRNA
Now follows the canonical pathway

Question 32

How does nuclear processing occur in the canonical pathway?

Answer 32

pri-miRNAs interact with an RNAse III family member Drosha (found in the nucleus)
Drosha makes specific cuts at cleavage sites generating pre-miRNA
Doesn’t matter what pathway is taken as end product always the same

Question 33

How does processed miRNA leave the nucleus if it does not have a 5’ cap of a poly-(A) tail? How does nuclear export of pre-miRNA occur and what does it require?

Answer 33

The 2-base overhang at the 3’ end of the pre-miRNA is recognised by exportin 5
Association with Ran-GTP allows export through the nucleus where the complex falls apart cytosolically
Releases the pre-miRNA into the cytosol (Ran-GDP and exportin 5 recycled)

Question 34

Once the pre-miRNA has ended up in the cytoplasm, what happens to it?

Answer 34

Cytosolic pre-miRNA is now further processed by Dicer (cytosolic RNAse III family member)
Dicer binds to and removes the terminal loop resulting in the mature duplex which contains a guide strand and a passenger strand
The mature duplex is loaded onto a remodelled AGO (argonaute) in the open conformation (opened by cytosolic chaperones) and passenger strand is removed
AGO + guide strand = RISC (RNA-induced silencing complex) which can regulate the expression of other genome

Question 35

What does RISC (RNA-induced silencing complex) do?

Answer 35

If the guide strand in RISC has a strong homology for a gene, it will bind very strongly to the target mRNA
Slices and deadenylated target mRNA resulting in rapid mRNA degradation via enzymes
If guide strand has weak homology, then there is no splicing but the efficiency of translation is reduced until mRNA eventually degraded

Question 36

What are examples of miRNAs involved with neuron and muscle development?

Answer 36

miRNA specific to stem cell (miR-184) promotes neural stem cell identity and self-renewal, inhibits differentiation (targets Numbl RNA)
miRNA specific to differentiated cell (miR-1 or miR-206): antagonises stem cell identity, inhibits stem cell renewal, promotes muscle cell differentiation (targets Pax7 RNA)

Question 37

How do alcoholics alter their gene expression?

Answer 37

Alcoholics have altered expression of miRNAs, which means that alcoholics have mis–regulation of the cell cycle, apoptosis, cell adhesion, nervous system development and cell signalling

Question 38

What happens when Dicer is inactivated?

Answer 38

Dysregulation of protein expression
Severe developmental defects e.g. in limb formation, heart defects
Cancer
Neurological defects, behavioural effects Infertility
Dicer is inactivated in fertilised eggs, blocking the generation of all miRNAs at this developmental stage