Transcript S1 mapping of the 3’ end of a RNA

RNA Editing
Definition: any process, other than splicing, that results
in a change in the sequence of a RNA transcript such
that it differs from the sequence of the DNA template
• Discovered in trypanosome mitochondria
K. Stuart
L. Simpson
• Also common in plant mitochondria
• Also occurs in a few chloroplast genes of higher
plants, and at least a few nuclear genes in mammals
Discovery of RNA Editing in
Trypanosome Mitochondria
• Unusual Mitos. called Kinetoplasts
• DNA:
– Maxicircles (22 kb in T. brucei), contains most of
the genes
– Minicircles (1-3 kb), heterogenous
• Sequencing of genomic Mt DNA (Maxicircles)
revealed apparent pseudogenes:
– Full of Stop codons
– Deletions of important amino acids
Kinetoplast DNA from a trypanosome visualized by EM
Fig. 16.13
Where were the real functional genes?
• Investigators generated cDNA clones to some of
the kinetoplast mRNAs and sequenced them
• Sequences were partially complementary to
pseudogenes on maxicircle DNA
cytochrome oxidase
subunit II
– the COXII DNA sequence above is missing 4
Us found in the mRNA
• Called this “Editing” because it produced functional
mRNAs and proteins from pseudogenes
Some genes are very heavily edited!
COXIII
Cytochrome
oxidase III
From Trypanosoma
brucei
Lower case Us
were inserted by
editing.
The deleted Ts
(found in the
DNA) are
indicated in
upper case.
Fig. 16.15
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’
Guide RNAs Direct Editing
in Trypanosomes.
Editing is from 3’ to 5’ along an unedited RNA.
16.17,18
Editing
Mechanism
with the
enzymes.
TUTase, or terminal uridylyl
transferase, adds U(s) to the 3’
end created by cleavage of the
pre-mRNA
from Fig. 16.20
Other Systems with RNA Editing
1. Land plant (C  U) and Physarum (slime
mold) mitochondria (nt insertions)
2. Chloroplasts of angiosperms (C  U)
3. Some nuclear genes in mammals
– Apolipoprotein B, C  U
– Glutamate receptor B, A  I (inosine)
4. Hepatitus delta virus (A  I)
5. Paramyxovirus (G insertions)
Editing of Oenothera
mitochondrial RNAs
Determined by comparing
sequences of cDNA copies of mt
RNAs with the corresponding
genomic gene.
Editing of Angiosperm Mt RNAs
1. Most RNAs are edited
2. Most events are C  U, but also U  C
3. Preferential editing of coding regions,
but introns and untranslated regions
are also edited.
4. Editing produces translatable RNAs, and
restores conserved amino acids (i.e,
functional proteins).
Possible mechanism for plant Mt editing:
Deamination of cytosine (to uracil) by a
cytidine deaminase
NH2
O
N
N
H20
=O
N
Cytosine
=O
N
Uracil
Plant mt RNA Editing Mechanism
(cont.)
• Cytidine deaminases are known, and in
fact one is involved in ApoB editing in
mammals. Plant enzyme not identified
yet.
• How are editing sites recognized?
– No guide RNAs have yet been found in
angiosperm mitochondria.
Editing of Apolipoprotein B in
Mammals
1. Large nuclear gene
2. Editing is C6666  U6666 in exon 26 of the
14 Kb mRNA
3. This creates a Stop codon, producing a
truncated form of the protein
- both forms circulate in blood but have
different functions
- the long form is endocytosed via the
LDL receptor; the short form is not
Molecular Consequences of Editing ApoB pre-mRNA
(Splicing precedes editing)
Produced by Unedited mRNA
Produced by Edited mRNA
Editing of Apolipoprotein B –
The Editosome
1. A cytidine deaminase activity is involved
– apobec (apoB mRNA editing enzyme
catalytic subunit)
2. Another protein, ACF (apobec complementation
factor) is also required
3. Both recognize sequences flanking the C to be
edited
RNA editing in brain tissue:
Adenosine to inosine
ADA
Adenosine
Inosine
Inosine has long been known from
purine metabolism
Inosine also
acts as a
signaling
molecule.
ADA
deficiency is
a metabolic
disease.
A to I Editing in RNA
• 1st case:
Glutamate
Receptor B
• I read as G
during
translation, R
instead of Q
• Affects Ca2+
permeability,
intracellular
trafficking of
receptor
Important Examples of A to I Editing in Mammals
Mechanism of A to I Editing
•
dsRNA-dependent adenosine deaminase (ADAR)
1. converts A  I in 2 Glut Receptor B exons
(changes the amino acids; I read as G
during translation)
2. recognizes secondary structure around site to
be edited
3. requires intron and exon sequences - acts on
unspliced receptor pre-mRNA
4. has dsRNA binding domains as well as a
catalytic center similar to the cytosine
deaminase
ADARs – adenosine deaminases that work on RNA
<- key for editing of GlutR
Other possible functions:
- RNA modification (other
types)
- RNAi
- Chromatin remodeling
dsRBD- dsRNA binding
Z – Zn, DNA binding
R-rich – Arg rich