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RNA editing
- process by which nucleotides not coded by a gene are introduced at
specific positions in an RNA molecule after (or during) transcription
– discovered 1986 (Rob Benne) some mRNAs in African trypanosomes
differed from their genes – insertions changed coding properties
- occurs in wide range of organisms – only in eukaryotes, diverse
mechanisms
1. Subsitution editing - chemical modification of individual nucleotides
catalyzed by enzymes that recognize a specific target sequence of nucleotides
occurs in both pre-mRNAs and tRNA
- cytidine deaminases that convert a C in the RNA to uracil (U);
-adenosine deaminases that convert an A to inosine (I), which the ribosome
translates as a G.
2. Insertion/Deletion editing: Insertion or deletion of nucleotides in the RNA
- alterations are mediated by guide RNA molecules that
- base-pair as best they can with the RNA to be edited (pre-mRNA) and
- serve as a template for the addition (or removal) of nucleotides in the target
Possible Consequences:
Insertion/Deletion editing
Substitution editing
changes in splice site selection
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Subsitution editing - chemical modification of individual nucleotides catalyzed
by enzymes that recognize a specific target sequence of nucleotides
cytidine deaminases convert a C in the RNA to uracil (U)
Factors required: APOBEC protein family
human: APOBEC-1, hAID, APOBEC-2, APOBEC3A-3H,APOBEC-4
// C //
C // AAA-3’
Cis-element
C // AAA-3’
U // AAA-3’
Specificity
factor
transaminase
Transcription
Mechanism of RNA editing in mitochondria and plants – in organelles
Pre-edited RNA
Gene
Assembly of
multicomponent
complex
Editing
Edited mRNA
Specificity factor binds to cis-element
and recruits transaminase
Dissociation of editing complex
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Lipoprotein metabolism
Mammalian apolipoprotein B
Two forms of apolipoprotein B in mammalian serum - both transcribed from same gene
In vivo - need spliced and polyadenylated transcript, occurs in nucleus
introns suppress editing
Apo-B100 - transport of cholesterol
in blood
Apo-B48 - absorption of lipids
from intestines
4563 aa 2351 aa
Cytosine deaminase
with RNA-binding
specificity
Glu stop
editosome
APOBEC – apolipoprotein B mRNA-editing catalytic polypeptide
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The mooring sequence and the 3′ efficiency element form a double-
stranded (ds) stem that is predicted to position the edited cytosine in a
favorable configuration for deamination.
Model for the apolipoprotein B editing site
Original Transcript > 14,000 residues
minimal sequence within ~30 nts flanking edited base*
secondary structure important
Mooring sequence - 11 nts located
4-6 nt downstream of edited base
Editosome
apobec-1 (apob editing catalytic complex)-
catalytic deaminase (CDAR-cytidine deaminase
acting on RNA)
ZDD- zinc-dependent deaminase domain binds
to AU-rich sequences
binds to AU-rich elements in 3’ UTR of RNAs and
regulate mRNA stability
additional proteins
ACF apobec-1 complementation factor -
adaptor protein binds deaminase and RNA
substrate,
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Subsitution editing - chemical modification of individual nucleotides catalyzed by
enzymes that recognize a specific target sequence of nucleotides
- adenosine deaminases that convert an A to inosine (I)
Can lead to codon change
• Most widespread in higher eukaryotes
•Complexity of machinery and number of targets
increases from lower to higher organisms
• occurs in ds RNAs – mostly in 3’UTRs or in
non-coding regions
Adenosine deaminases that act on
RNA (ADARs) - recognize the adenosine
to be edited not by a surrounding
consensus sequence but by the structure
of the duplex that is formed between the
editing site and an editing site
complementary sequence (ECS) that is
usually located in a downstream intron.
Adenosine deaminases that act on RNA (ADARs)
dsRAD - double stranded
RNA adenine deaminase
Target recognition
basepaired region
with mispairing
A to I editing
All ADARS contain 1-3 double stranded binding motifs (dsRBDs) mediate the binding to the duplex.
Found in wide range of organisms from yeast to mammals
mRNAs, tRNAs, viral RNAs, non-coding RNAs are substrates for ADAR
Subsitution editing - adenosine deaminases that convert an A to inosine (I), which the
ribosome translates as a G
Substrate - partially ds RNA structure
involving exonic and intronic sequences
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translated
exon
untranslated
exon
Experimentally validated structure
Re-coding editing
Single amino acid changes
Types of RNA subject to editing by ADARS
glutamate receptor subunit GRIA2 exon 11 Q/R site
More than 98% of all pre-mRNAs subject to Alu mediated RNA editing
Most Alu repeats in introns and non-translated exons - editing will not directly influence
protein function but can indirectly alter protein expression or function
• can create cryptic splice donor or acceptor site to induce alternative pre-mRNA splicing
• can modify splicing enhancer or inhibitor sequences to modulate alternative splicing efficiency
• may lead to the nuclear binding, storage, degradation or release of these I-containing RNA s.
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Can modulate miRNA structure or miRNA binding sites
Can modulate rate of processing by RNase Drosha or can prevent the further
maturation and expression of the miRNA
miRNA – small regulatory RNA molecules having diverse roles in development,
differentiation and cell cycle regulation
16% of all human miRNA genes are subject to A-to-I modification.
miRNA excised from longer, hairpin-
structured precursors through the
sequential action of the RNases Drosha
and Dicer
A to I conversions in tRNAs
I present at positions 34 and 37 in
all eukaryotic tRNA Ala of higher
eukaryotes
Conclusion- A34 and A37 modification may influence translation at different levels
Functional signifcance
--can influence the stability and structure of tRNAs
and improve the fidelity and efficiency of tRNAs in
decoding the genetic message
-- can improve reading frame maintenance
-- can alter cellular distribution and/or function in
protein synthesis
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ADAR - adenine deaminase that acts on RNA
Undergoes self-editing
In mice - ADAR null mutation is lethal
Highly conserved in vertebrates, insects, worms, absent in protozoa, yeasts, plants
ADAT - adenine deaminase that acts on tRNA
Z-DNA binding motif
In A to I editing - splicing occurs AFTER editing because the double-
stranded RNA must be formed between the upstream Exon and the
downstream Intron
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RNA editing can regulate splicing by targeting the
adenosines involved in splicing
Rat ADAR2 edits its own pre-mRNA such that a 3′ splice site is generated,
the use of which adds 47 nucleotides to rat ADAR2 and changes the predicted
open reading frame.
Internal translation initiation then leads to the production of an active enzyme, but
lower protein levels are expressed, because the internal translation initiation is
relatively inefficient.
Self-editing results in a lower ADAR2 concentration, so this process can be
thought of as a negative autoregulatory mechanism whereby rat ADAR2 can
regulate protein expression by changing a downstream splice site.
AUG
Factors affecting A to I pre-mRNA editing
Likely that splicing and editing components interact with each other
Relative localization of methylation and editing machineries important in
determining kind and rate of modification made
Stability of dsRNA structure sensitive to temperature, availability of RNA
helicases, splicing rate regulate editing levels
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Examples of mammalian A to I editing
Most of genes in mammals, Drosophila, Caenorhabditis found to undergo
AI editing are expressed in nervous system
Cytoplasmic RNase activity specifically cleaves I-dsRNA - may be part of
cellular mechanism to process hypermodified dsRNA (viral or cellular)
molecules
Disruption of the RNA editing balance
Direct causal relationship
Correlations
Possible cross-connections
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APOBEC and HIV infection
APOBEC3G is a
cellular restriction
factor which inhibits
infection with HIV, but
to HIV has evolved a
protein Vif – viral
infection factor which
targets APOBEC3G for
ubiquitination and
degradation.
Retrovirus production in a nonpermissive
cell expressing APOBEC3G results in the
incorporation of APOBEC3G into viral
progeny
which are able to invade host
cells and undergo the early
steps of reverse transcription
the APOBEC3G cytidine deaminase induces rapid
accumulation of deoxyuridine residues
HIV has evolved a protein Vif – viral infection factor which
targets APOBEC3G for ubiquitination and degradation
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1. Subsitution editing - chemical modification of individual nucleotides
catalyzed by enzymes that recognize a specific target sequence of nucleotides
- cytidine deaminases that convert a C in the RNA to uracil (U);
- adenosine deaminases that convert an A to inosine (I), which the ribosome translates as a G.
2. Insertion/Deletion editing: Insertion or deletion of nucleotides in the RNA
- alterations are mediated by guide RNA molecules that
- base-pair as best they can with the RNA to be edited and
- serve as a template for the addition (or removal) of nucleotides in the target
First found in Trypanosome kinetoplast
a single large mitochondrion
Trypanosome kinetoplast a single large mitochondrion
extends the full length of cell
mRNAs synthesized in kinetoplast can be extensively edited -
usually through addition of U - to increase their size by as
much as 50%
Insertion/Deletion editing: Insertion or deletion of nucleotides in the RNA
Mitochondrial genome is compartmentalized in kinetoplast – consists of large
network of catenated circular DNA molecules
Consists of about 50 maxicircles (between 15kbp and 80 kbp) and 10,000
minicircles (between 0.9 kbp and 2.5 kbp)
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DNA:
Maxicircles (22 kb in T. brucei), contains most of the genes - encodes
apocytochrome b, subunits 1 and 2 of cytochrome c oxidase (cox), and
unassigned reading frames (URFs).
Minicircles (1-3 kb), heterogenous
Sequencing of genomic Mt DNA (Maxicircles) revealed apparent pseudogenes:
Full of Stop codons
Deletions of important amino acids
Sequences of cDNA clones of some of the kinetoplast mRNAs were partially
complementary to pseudogenes on maxicircle DNA
cytochrome oxidase
subunit II
– the COXII DNA sequence is missing 4 Us found in the mRNA
Sequencing of other mitochondrial cDNAs and their comparison to the genomic
sequence showed not only the addition of U’s but also their deletion
1988 - T. brucei coxIII gene shown to be derived from one of the maxicircle URFs
through extensive editing – insertion of 547 U’s and deletion of 41 U’s, doubling the
size of the RNA.
1990 - shown that the editing information was provided by small guide RNA’s
encoded mainly by minicircles
1996 - in vitro systems to examine the mechanisms of editing were developed
extensive editing – e.g. in T. brucei there is a combined total of 3583 inserted
and 322 deleted U’s to generate the repertoire of mitochondrial mRNAs.
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Editing Mechanism
-Post-transcriptional
-Guide RNAs (gRNAs) direct editing
-gRNAs are small and complementary to portions of the
edited mRNA
-Base-pairing of gRNA with unedited RNA gives
mismatched regions, which are recognized by the editing
machinery
-Machinery includes an Endonuclease, a Terminal
UridylylTransferase (TUTase), and a RNA ligase
-Editing is directional, from 3’ to 5’
Three regions:
1. Anchor - can base-pair with region of message immediately beside (3’ to)
region to be edited
2. Region that directs editing - stretch complementary to message to be edited but
containing additional As
3. 3’ polyU stretch - role unclear
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Formation of duplex looped out single-stranded
regions where Us to be inserted
Endonuclease recruited to target mRNA cuts opposite loops
Tutase (3’ terminal uridylyl transferase) transfers Us into gap
RNA ligase closes gap
Editing of trypanosome coxII gene
Pre-mRNA
gRNA
The RNA editing process - proceeds 3’ to 5’ respect to mRNA
Number of Us added
dictated by guide RNA
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RNA editing proceeds 3’ to 5’
Pre-edited mRNA 3’UTR 5’ - -3’
3’ UUUU
5’
Guide RNA (1)
Pre-edited mRNA 3’UTR 5’ - -3’
3’ UUUU 5’
Guide RNA (2)
Edited mRNA
Pre-edited mRNA 3’UTR 5’ - -3’
3’ UUUU 5’
Guide RNA (3)
Edited mRNA
Pre-edited mRNA 3’UTR 5’ - -3’
3’ UUUU 5’
Guide RNA (4)
Edited mRNA
3’UTR 5’ - -3’ Fully-edited mRNA
Pan-editing of the L. tarentolae A6 mRNA
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The final pan-edited A6 mRNA has a single open reading frame
creation of methionine
for translation initiation
ribosome-binding site?
Editosome contains proteins and RNAs encoded by mitochondrial and
nuclear genomes within the Trypanosome
Kinetoplast DNA –
network of maxicircles
and minicircles
Nuclear encoded,
imported into
mitochondria
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Kinetoplastid RNA editing proteins
Model for editosome - multiprotein complex in mitochondrion 20S-40S
Separate catalytic sectors for
insertion and deletion
ligase
Tutase
exoUase
RNase
RNA recognition
Few enzymes required for catalytic steps in editing but numerous specific
dynamic molecular interactions must occur
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The editing cycle
Binding of premRNA and gRNA to editosome
Factors stabilize complex
Several sites edited as
specified by gRNA
First gRNA replaced by second
Each gRNA cycle entails formation of an anchor duplex with edited sequence formed by previous
gRNA each gRNA remains associated with the editosome until decoded and then replaced by
subsequent gRNA
RNA binding proteins may
participate to stabilize association
of RNAs with editosome