RNA Editing

1
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
• Also common in plant mitochondria
• Also occurs in a few chloroplast genes of higher
  plants, and at least a few nuclear genes in
  mammals

L. Simpson
K. Stuart
2
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

3
Kinetoplast DNA from a trypanosome visualized by
EM
Fig. 16.13
4

• 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

5
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
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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

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Guide RNAs Direct Editing in Trypanosomes.
Editing is from 3 to 5 along an unedited RNA.
16.17,18
8
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
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Other Systems with RNA Editing

• Land plant (C ? U) and Physarum (slime mold)
  mitochondria (nt insertions)
• Chloroplasts of angiosperms (C ? U)
• Some nuclear genes in mammals
• Apolipoprotein B, C ? U
• Glutamate receptor B, A ? I (inosine)
• Hepatitus delta virus (A ? I)
• Paramyxovirus (G insertions)

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Editing of Oenothera mitochondrial RNAs
Determined by comparing sequences of cDNA copies
of mt RNAs with the corresponding genomic gene.
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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).

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Possible mechanism for plant Mt editing
Deamination of cytosine (to uracil) by a cytidine
deaminase
NH2
O
N
N
H20
O
O
N
N
Cytosine
Uracil
13
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.

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Editing of Apolipoprotein B in Mammals

• Large nuclear gene
• Editing is C6666 ? U6666 in exon 26 of the 14 Kb
  mRNA
• 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

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Molecular Consequences of Editing ApoB pre-mRNA
(Splicing precedes editing)
Produced by Unedited mRNA
Produced by Edited mRNA
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Editing of Apolipoprotein B The Editosome

• 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

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RNA editing in brain tissue Adenosine to inosine
ADA
Adenosine Inosine
18
Inosine has long been known from purine metabolism
Inosine also acts as a signaling molecule.
ADA deficiency is a metabolic disease.
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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

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Important Examples of A to I Editing in Mammals
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Mechanism of A to I Editing

• dsRNA-dependent adenosine deaminase (ADAR)
• converts A ? I in 2 Glut Receptor B exons
  (changes the amino acids I read as G during
  translation)
• recognizes secondary structure around site to be
  edited
• requires intron and exon sequences - acts on
  unspliced receptor pre-mRNA
• has dsRNA binding domains as well as a catalytic
  center similar to the cytosine deaminase

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ADARs adenosine deaminases that work on RNA
lt- 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