RNA editing
Network of kinetoplast minicircles and maxicircles from Leishmania
tarentolae; circular DNAs; catenanes
Comparison of COII gene of a trypanosome with its mRNA product
R. Benne et al., 1986
cryptogene
inserted to mRNA
Deleted from DNA
Pan-edited
Sequence of the COIII mRNA of T. brucei
Us added to the mRNA, (Us or Ts) deleted from mRNA
RNA editing happens after transcription.
1. Unedited found with edited
2. It happens also in Poly(A) tail
The mechanism of editing:3’ to 5’ end
clue: partial edit only at but not 5’ end
Mechanism of editing
Wild-type
K. Stuart et al.
1988
Mutant w/o mt.DNA ( negative control)
3’edited cDNA (Positive control)
PCR analysis of direction of editing
What determines where to edit?
L. Simpson et al. 1990
Computer search
Model for editing using gRNAs
Simpson et al
Evidence for gRNAs, Northern blot probed with labeled
oligonucleotide
Mechanism of RNA editing
Pre-mRNA
S. Seiwert & Stuart
Side reaction, not biologically significant
Evolution of RNA Editing
Model for evolution of editing
D. Maslove & Simpson
Post-Transcriptional Control of Gene Expression
J. Hartford: “ cellular mRNA levels often correlated more
closely with transcript stability than with transcription rate “
P. Chambon et al
Casein mRNA stability
Effect of prolaction on half-life of casein mRNA
RNA half-life
(hr)
Species of
RNA
-prolactin
+prolactin
rRNA
>790
>790
Poly(A)+RNA
(short-lived)
3.3
12.8
Poly(A)+RNA
(long-lived)
29
39
Casein mRNA
1.1
28.5
Transferrin Receptor (TfR) mRNA stability
Transferrin, iron transporter,
TfR when cell requires iron,
while ferritin 
if too much iron,  ferritin concentration,  TfR
J Harford et al (1986)
D. Owen &
Lukas Kuhn
(1985)
• Iron Response Element
– Effect of the 3’UTR on the iron- responsiveness of cell surface
concentration of TfR
Harford et al
Hemin, Desferioxamine
Effect of deletion within the 3’UTR on iron-responsiveness of TfR mRNA
concentration.
ferritin
Comparison: 5 stem-loops in 3’UTR of TfR mRNA and IRE in 5’UTR
ferritin mRNA
+competitor
Summary- 3’UTR of
TfR mRNA contains 5
stem-loops (IREs ). 
2 or more IRE in
tandem  response of
iron
2. TfR mRNA; 3. Ferritin mRNA; 4. b-globin
• Presence of IRE binding
proteins -Gel mobility shift
assay
Rapid turn over determinant
Human and chicken IREs of 3‘UTR of TfR mRNAs
 IREs A and E, large central loop
TfR 3’UTR  response of iron O.K..
Assay of deletion mutants in the 3’UTR
for iron-responsiveness
 all of the IREs renders TfR mRNa
stable; i.e. desptroys the rapid turnover
determinant.
Effects on iron-responsiveness
of deletions in the IRE region
on the TfR 3’UTR
 one of two non-IRE stem-loops
renders TfR mRNa stable; I.e. desptroys
the rapid turnover determinant.
Effects on iron-responsiveness of deletions
non-IRE stem-loops from the TfR 3’UTR
Protein binding assay
Summary
• IREs A and E, large central loop of the TfR
3’UTR can be deleted without altering the
response to iron.
• Each of the non-IRE stem-loops and one of
IREs B-D are part of rapid turnover
determinant
TfR mRNA stability
Mullner & Kuhn
Effect of iron on stability of TfR mRNA
Effect of iron on stability of wild-type and mutatn TfR mRNAs
Harford and colleagues
Summary - [iron], TfR mRNA decay , TRS-3 w/o rapid
turn over determinant, constitutively stable, TRS-4, unable to
bind IRE-binding proteins, constitutively unstable
TfR mRNA degradation pathway
Appearance of a shortended TfR mRNA upon hemin treatment
Short exposure
longer exposure
The 4.9 kb transcript had
been cut by an
endonuclease within
3’UTR including the
poly(A)
TRS-1
3‘ fragment released from the TfR mRNA
The 3’endonucleolytic product of the
TfR mRNA retains a long poly(A)