1) TDP-43 is a splicing factor that plays roles in various aspects of RNA metabolism and whose dysfunction is central to ALS and FTLD pathogenesis. 2) Deletion of the Drosophila homologue of TDP-43 leads to locomotive defects in flies, and expression of human TDP-43 can rescue this. 3) The C-terminal tail of TDP-43 contains a Q/N domain that mediates its interaction with other hnRNP proteins and is essential for TDP-43 aggregation. Tandem repeats of this domain induce TDP-43 aggregation.

1. International Centre for Genetic Engineering and Biotechnology. Trieste (Italy)
The role of the nuclear factor TDP 43 in neurodegeneration.

2. The cellular RNA world:

3. Pre-mRNA splicing
• Essential step in gene expression
• >15% of human genetic diseases involve splicing errors
• Important regulatory step in gene expression
Alternative splicing
AUG
AUG UGA
UGA

4. Basic elements involved in the RNA splicing process
5’ splice site: recognized by U1snRNP
3’ splice site:
a) 3’ junction (AG): recognized by U2AF35
b) Poly-pyrimidine tract (PPT): recognized by U2AF65
c) Branch Point (BP): recognized by the Branch-point Binding Protein
AGguragu
U1snRNP
mBBP
(SF1)
ynyurac ( y)nnyag
5’ss BP PPT 3’ss
35
65
U2AF
subunits
U2snRNP

5. Exon 1
5’
pGU (Py)nA
Branch site
AGp
3’
5’-splice site 3’-splice site
SF1
U2AF65 U2AF35
Regulatory Elements in pre-mRNA splicing
Exon 2
Regulatory
sequence
Regulatory
sequence
U1
Enhancer Silencer

6. Exon 1
5’
pGU (Py)nA
Branch site
AGp
3’
5’-splice site 3’-splice site
SF1
U2AF65 U2AF35
Regulatory Elements in pre-mRNA splicing
Exon 2
Regulatory
sequence
Regulatory
sequence
U1
Enhancer Silencer

7. Over the years, a great number of Enhancer and Silencer factors have been identified:
Enhancers Silencers
SR protein family hnRNPs (such as A/B family, PTB,
CELF protein family hnRNP H…etc).
hnRNP L TDP-43
Tra2
YB-1
NOVA
In many cases exclusion/inclusion may thus determined by the resulting balance of power

8. Splicing mutations can be found in virtually any intron-containing gene. The frequency depends
on overall length and individual susceptibilities

9. Classical and non classical CF

10. Five “neutral” substitutions out of 19 induce exon skipping
The probability of inducing exon skipping is about 26%
In CFTR exon 12 synonymous sites are under selective pressure to maintain
the exon inclusion
The same sense mutations A49G, C40T and C52T are found in patients and
cause CFTR exon 12 skipping.
CERES 2

11. The coding sequences of CFTR exon 9 overlap with splicing
regulatory sequences.WTexon9
G424S
D443Y
I444S
A455E
V456F
9 +
9 -
Q414X
N418S
Q452P
percentofexon9inclusion
exon 9
100
0
10
20
30
40
50
60
70
80
90
WT Q414X N418S G242S D443Y I444S Q452P A455E V456F

12. 241 2 3 4 5 6a 6b 7 8 10 1112 14a14b 15 16 191817b17a 222120 23
TAGATG
13
mRNA
CFTR
Exons 9
Normal splicing
Aberrant splicing
CFTR
protein
MSD 1 MSD 2
NBD1
NBD2
Dominio
R
Plasmatic membrane
NH2 COOH
8 9 10
8 10
8 9 10Pre-mRNA
The degree of skipping of normal exon 9 is associated with the presence of
polymorphic variants of the polypirimidine tract at the 3’ end of intron 8 (TG
repeats from 9 to 13 and T9, T7, T5, T3)
TTTTGATGTGTGTGTGTGTGTGTGTTTTTTTTTAACAG
exon9
3’ss
(TG)m
Skipping of exon 9 is associated to monosymptomatic forms of CF (Congenital Bilateral
Aplasia Vas Deferens, bronchiectasia, pancreatitis) and produce a non functional protein.
(T)n

13. Clinical
phenotype
Normal
Non-classical CF
Cystic Fibrosis
CFTR
genotype
T9
T7
T5
T5
T3
(TG)m (T)n
98 10
Inclusion
Skipping
This is one of the best clinical associations between a non coding
genotype and a disease outcome:
TG9
TG10
TG11
TG12
TG13
TTTTGATGTGTGTGTGTGTGTGTGTTTTTTTTTAACAG
3’ss
TG T9 9
TTG
13 3
3’ss
TTTTGATGTGTGTGTGTGTGTGTGTGTGTGTGTTTAACAG
high inclusion
low inclusion

14. Minigene system applied to CFTR exon 9 splicing allowed us to
replicate the effects of the UGmUn polymorphisms observed in
patients.

15. The first well documented role of TDP-43 was the inhibition of
CFTR exon 9 splicing and interactions with other hnRNPs.

16. TDP-43 cellular functions are mediated by its binding to GU rich
RNA sequences and its interactions with other hnRNPs.
Its structure and biological properties are evolutionary conserved from
Drosophila to Man

18. RNA binding is essential for TDP-43 function in splicing

19. Transgenic TDP 43 expression in HEK 293 cell lines down regulates the
endogenous gene:

20. Transgene TDP-43 expression in stably transfected HEK293 cell lines cause
reduction in endogenous TDP 43 gene expression:
GAPDH
Induced TG TDP-43
WT F147/149L
72h Induction
2 3 4 51 6 7 8
2.8 kb
2 3 4 51 6 7
coding sequence
untranslated sequence
TDPBR
TDP-43 mRNA species in stably transfected HEK293 cell lines are downregulated following transgene
TDP 43 cDNA induction:
2 3 4 5 6
pA4
pA1
TG-TDP43
TDP-43 RNA binding function is essential for self regulation

21. coding sequence
untranslated sequence
TDPBR
x7
Tet
7
7
- +
endog.
2 3 4 51 6 7 8
int6 int7
Tet - +
7
7
a
anchor
TTTT
1
2
a-anchor
endog.
GFPfw-anchor
tag
2.8 kb
GFP
reporter
– +Tet – +
DiGFP
X7 7 8GFP
X7-TDPBR 7 8GFP
gt ag
gt ag
tag
tag
The TDPBR in the 3-UTR is needed for the regulatory feedback loop
GFPfw-anchor
7
7

22. valuesnormalizedtoU6snRNA
- Tet
+ Tet
BrUTP nuclear
Run-on
X
1
X4i
nt4
X6c p
A
1
h2p
A4
3’p
A4
10000
0
20000
30000
40000
50000
X1 X4int4
2 3 4 51 6 7
h2pA4
3’pA4
X6c pA1
8
endog.
coding sequence
non-coding sequence
TDPBR
RNA polymerase II stalls in correspondence to the
TDPBR region:
Low TDP-43 concentrations
(-Tet)
High TDP-43 concentrations
(+Tet)
What events are associated with stalling?
Tet - + - +
N C
1
2
1
g
2
71
72
6
TDPBR AAAAAAA
+
Two events: intron 7 skipping and a pA switch from pA1 to pA2
TDPBR
6

23. X7-sup5’-3’
GFP
GFP
TDP-43 TDP-43
int7
Cytoplasm
Nucleus
Improved 3’ss-5ss
Rapid nuclear
degradation
Reduced amounts of
mRNA and protein
produced
GFP
Cytoplasm
Nucleus
No degradation
High amounts of
mRNA and protein
produced
X7-in7 cDNA
Intronless construct
GFP
int7
* *
Int7
removal
Nuclear
retention
GFP * *
Unproductive
spliceosomal
complex
Model of TDP-43 autoregulation:
The splicing process in a 3’UTR may be recognized by the cell as anomalous
and marked as an intron incompletely processed that must be degraded

24. Protein aggregation in neurodegenerative diseases
A wide variety of neurodegenerative diseases are characterized by the
accumulation of intracellular or extracellular protein aggregates.
Ross C.& Poirier M., 2005
Huntington Parkinson Alzheimer

25. Progressive neurodegenerative disease, that
affects motor neurons that provide voluntary
movements and muscle power.
When muscles no longer receive the messages
from the motor neurons that they require to
function, the muscles begin to atrophy
(become smaller).
Some numbers:
The disease prevalence is ~5 people each
100,000.
Most people develop ALS between 40 and 70
years old (average 55).
Life expectancy of an ALS patient averages
from 2 to 5 years from the time of the
diagnosis.
Amyotrophic Lateral Sclerosis

26. Pol II
Transcriptional
regulation
miRNA processing
pre-mRNA
splicing
Stress granule
formation
AAAAAA
m7G
AAAAAA
m7G
Translation
and protein
homeostasis
m7G Autoregulation
AAAAAA
m7G
mRNA stability
Nuclear/cytoplasmic
shuttling
mRNA transport
TDP Binding
region
(TDPBR)
Stress, oxidizing
agents, ageing
autophagy,
ubiquitin-proteasome
system (UPS)
Normal degradation
pathways
Chromatin
hnRNPs
FUS/TLS
P
P
P
P
P
Ub
Ub
Ub
P
P
Ub
Ub
P
Aggregation
TAR DNA Binding Protein (TDP 43) is a splicing factor that belongs to the
hnRNP family and plays a role in many aspects of RNA metabolism.
Its aggregation/dysfunction is central to ALS and FTLD pathogenesis
Buratti and Baralle, 2012, TiBS 37: 237.

27. Ubiquitinated, misfolded and hyper phosphorylated TDP 43 was identified as the major
component of the pathological inclusions found in the brain of FTLD and ALS patients
Neumann et al., 2006 Science 314: 130-136

29. Deletion of the Drosophila homologue of human TDP-43 )TBPH) leads to a paralytic phenotype:
But cannot get out of the pupal
cage without external help, are
deficient in locomotion and have a
very short life span
Flies apparently
develop normally

30. W1118; TBPH/TBPHW1118; +/+ control
Deletion of TBPH does not affect the external
morphology of mutant flies
But flies develop locomotive effects
W1118;TBPH+/+

31. Genetic Expression of Human TDP-43 in
Motoneurons can rescue fly motility
TBPH23/-; D42-G4/ UAS-hTDP-43
TBPH23/TBPH23
TBPH23/-; Elav-G4/ UAS-hTDP-43

32. TBPH LOF affects Synaptic Growth and Bouton Shape
W1118 TBPHΔ23 TBPHΔ142
W1118 TBPHΔ23 TBPHΔ142 siRNA Tg-hTDP43

33. Microtubule Organization
TBPHΔ23/D23TBPHΔ142/Δ142
W1118Rescue
anti-HRP anti-MAP-1B merge

34. Ubiquitinated, misfolded and hyper phosphorylated TDP 43 was identified as the major
component of the pathological inclusions found in the brain of FTLD and ALS patients
Neumann et al., 2006 Science 314: 130-136

35. Transcriptional
regulation
pre-mRNA
splicing
miRNA
processing
Autoregulation
mRNA
transport and
stability
Aggregation
TAR DNA Binding Protein (TDP 43) is a splicing factor that belongs to the
hnRNP family and plays a role in many aspects of RNA metabolism.
Its dysfunction is central to ALS and FTLD pathogenesis
RRM1 RRM2
C-terminal tail
NLS
N C
NES
RRM domainsN-terminal tail
Q/N
1 414342 366
TDP Binding
region (TDPBR)
AAAAAA
m7G
Translation

36. A peptide containing the 321-366 region of TDP-43
can efficiently compete with hnRNP A2 for the
binding to TDP-43
321AMMAAAQAALQSSWGMMGMLASQQNQSGPSGNNQNQGNMQREPNQA366
C-terminal tail of TDP-43 contains a
Q/N domain that mediates its
interaction with hnRNPs.

37. 
p342-366
p342AAAA
p363AAAA
p352AAAAA
(UG)6
(UG)6
(UG)6
peptide
THE Q/N RICH REGI0N IS ESSENTIAL A2 INTERACTION AND AGGREGATION

38. --
+ p321-366 (2,5M)
5 10 20 GST-A2 288-341 (M)5 10-
TDP-43 (1M)+ + + + + + + +
-
%ofsignalremained
inthewells
4 5 62 31 7 8
0
20
40
60
80
100
120
1
+ + + + + + + +
GST-A2 288-341 (M)
TDP-43
5 10 20 405 10
+ p321-366 (2,5M)
GST
288 341
GST- A2-(288-341)
A2
Aggregate formation induced by the p321-366 peptide can be competed by addition
of an excess amount of hnRNP A2 C-terminal sequence

39. 0
10
20
30
40
50
60
70
1FLAG-TDP-43
+ EGFP
FLAG-TDP-43
+ EGFP-12xQ/N
IN
S
P
%ofFLAG-TDP-43
-C
-C
331-369
-C
-C
Tandem repeats of the Q/N rich sequence induce in vitro and in vivo aggregation
of TDP 43
IN S P IN S P
FLAG-TDP-43
+ EGFP-12xQ/N
WB:anti-FLAGWB:anti-GFP
FLAG-TDP-43
+ EGFP
30
46
58
46
1 2 3 4 5 6
SDS
0,5%
0,25 uM
+ + + + +
WB:anti-TDP-43
GST-TDP-43
EGFP-constructs
0,125 uM

40. FUS-V5
(anti-V5)
FLAG-TIA-1
anti-FLAG
EGFP-12xQ/N Merge
FUS-V5
(anti-V5)
FLAG-TIA-1
anti-FLAG
EGFP-12xQ/N Merge
EGFPEGFP-1xQ/NEGFP-4xQ/NEGFP-12xQ/N
EGFP
Endo TDP-43
(anti-TDP-43) DAPI
Merge
EGFP/anti-TDP-43
Enlarged image

41. FLAG-TDP-43 WT + GFP-
12xQ/N
FLAG-TDP-43 321-366 + GFP-12xQ/N FLAG-TDP-43 F147,149/L + GFP-
12xQ/N
anti-FLAGGFP-12xQ/NMERGE
Enlargedimagefrom
eachcondition
*
*
*
FLAG-TDP-43 321-366 + GFP-12xQ/N
The Q/N rich TDP 43 region is essential for the recruitment of TDP 43 to the
aggregates while the RNA binding function is not needed

42. +Tetracycline
-Tetracycline
TDP-12xQ/N
TDP-43
Aggregate formation:
Red- TDP-12xQ/N
Green – (End. TDP-43)
Yellow (merge)
Blue (nuclei)

43. EGFP-12xQ/N Anti-TDP-43 Merge
Anti-Flag Anti-TDP-43
MERGE
anti-Flag/anti-TDP-43
Aggregation can be enhanced by linking 12xQ/N to TDP 43 itself

44. Tet + + --
variant 1
variant 2 47.5
α-POLDIP3
variant 1
variant 2
SPLICING CHANGES AFTER AGGREGATION ARE SIMILAR TO THOSE OBSERVED
AFTER TDP 43 DEPLETION BY RNAi

45. Climbing
Solubility
IHC
Movement
IHC
Adult fly 3rd instar larvae
Looking for a phenotype…
Driver: Elav, nSyb, GMR, etc.
Temperature: 25 or 29°C
Growth cycle: Adult fly or 3rd instar larvae
TDP-43 aggregation model in flies

46. Climbing during aging
29°C
e
la
v
/W
1
1
1
8
e
la
v
/E
G
F
P
#
3
e
la
v
/G
F
P
1
2
x
#
3
0
2 0
4 0
6 0
8 0
1 0 0
2 9 C D 1 E L A V
G e n o ty p e
%Topclimbingflies
e
la
v
/W
1
1
1
8
e
la
v
/E
G
F
P
#
3
e
la
v
/G
F
P
1
2
x
#
3
0
2 0
4 0
6 0
8 0
1 0 0
2 9 C D 7 E L A V
G e n o ty p e
%Topclimbingflies
**
*
e
la
v
/W
1
1
1
8
e
la
v
/E
G
F
P
#
3
e
la
v
/G
F
P
1
2
x
#
3
0
2 0
4 0
6 0
8 0
1 0 0
2 9 C D 1 4 E L A V
G e n o ty p e
%Topclimbingflies
*
**

47. CLIMBING ASSAY ELAV 12XQ/N

48. Larval brain immunohistochemistry
• Temperature: 25°C
• Growth cycle: 3rd instar larvae
• Antibodies:
Anti ELAV (rat): 1:250
Anti TBPH (rabbit): 1:300
Anti GFP (mouse): 1:200

49. +/Elav; +/EGFP2
29°C
Anti GFP
Anti ELAV
Merge
+/Elav; +/GFP12x
29°C
Merge

50. Genetic interaction TBPH and GFP-12xQ/N

51. Genetic interaction TBPH and GFP-12xQ/N
Larval eye disc IHC
• Temperature: 25°C
• Growth cycle: 3rd instar larvae
• Antibodies:
Anti ELAV (rat): 1:250
Anti Flag-TBPH (mouse): 1:200
Anti GFP (rabbit): 1:250

52. GMR/+; TB1/+
25°C
Anti ELAVAnti Flag-TB1
Merge

53. Merge Merge
Anti ELAVAnti GFPAnti Flag-TB1
GMR/+; TB1;12X/+
25°C

54. Finding drugs to clear TDP-43 aggregates
One possible approach could be to enhance degradation of the aggregated protein.
Endogenous TDP-43
Newly produced TDP-43
Will be less sequestered
= “TDP-43 sink”

55. Aggregates induced by transient transfection of EGFP12Q/N display
autophagosomal markers

56. Hek293 GFP-12xQ/N
Tet induction
for 24 hrs
Wash
Compound
treatment for
48 hrs
Cell lysis
SDS-PAGE with urea and DTT
WB anti GFP
Clearance assay using the cellular model of TDP-43 aggregation

57. EGFP 12Q/N aggregates are partially resolved by poly anions effectors

58. C 40uM 100uM
actin
GFP-12xQ/N
TDP-43 aggregation model in cells
C 0.1uM 1uM 10uM 20uM
actin
GFP-12xQ/N
actin
GFP-12xQ/N
C 20uMC 0.1uM 1uM 10uM
FDA approved
drug 1
FDA approved
drug 2
FDA approved
drug 3

59. Possible disease connections following alterations at the TDP-43 autoregulatory loop level

60. INTERNATIONAL CENTRE FOR GENETIC ENGINEERING AND BIOTECHNOLOGY
Trieste, Italy
Emanuele Buratti
Amit Bhardwaj
Mauricio Budini
Laura De Conti
Valentina Romano
Maureen Okuku
Jeremias Herzog
Fatemeh Mohagheghi
Zainuddin Quadri
Simona Langellotti
Maurizio Romano
Cristiana Stuani
Chiara Chiavelli
Frederick Allain
Peter Lukavsky
Zurich CH
Ashish Dhir
Nick Proudfoot
Oxford UK
Fabian Feiguin
Lucia Cragnaz
Raffaella Klima

61. IP HEK-TDP-12X-Q/N stable cell line
(HSP70)
Is there anything else in the aggregates?

62. Anti-TDP-43
(Flag-TDP-12xQ/N and
endogenous TDP-43)
Anti-HSP70 MERGE
anti-TDP-43/anti-HSP70
Anti-HSP70
83
Tet + + + + + +
83
47.5
Anti-TDP-43
Anti-TDP-43
Hsp 70 is present in the early stage aggregates

63. FLAG-TDP-43 EGFP-12xQ/N MergeHA-Ubiquitin
FLAG-TDP-43 EGFP-12xQ/N Mergep409/p410
AGGREGATES ARE UBIQUITINATED AND PHOSPHORYLATED
AS IN THE PATIENT’S BRAIN INCLUSIONS

64. GFP
reporter
– +Tet
coding sequence
untranslated sequence
TDPBR
endog.
2 3 4 51 6 7 8
int6 int7
DiGFP
tag
2.8 kb
A
X7-Δin7cDNA 7GFP
7GFP
7GFP
B
X7 7 8GFP
8
8
X7-pA2
X7-2pA1
X7-TDPBR 7 8GFP
gt ag
7GFP 8X7-gt-ag
gt ag
gt ag
gt ag
tag
gt ag
tag
tag
tag
tag
tag
gt ag
– + – + – + – + – +
Flag-TDP-43
TDP-43 end.
Tub.
X7
X7-
TDPBR
X7-
gt-ag
X7
-pA2
X7
-Δin7
cDNA
X7-
2pA1
1 2 3 4 5 6 7 8 9 10 11 1
2
C
D
1 2 3 4 5 6 7 8 9 10 11 1
2
GFP/DiGFPnormalizeddata
1.0
0.8
0.6
0.4
0.2
0.0
Analysis of the relative importance for self regulation of pAs quality and splicing

65. - +
X7
- +
X7-pA2 X7-2pA1
- +TetTet Tet
77
7
8´ 8´´7
Cryptic
pASV40
7
7
87
1
2
1
3
4
6
1
X7 7 8GFP
GFPfw
A
B C D E
anchor
TTTT
7
Autoregulation Yes Yes Yes
X7 gt-ag
71
- +Tet
No
F
5
G
- +Tet
No
X7 TDPBR
- +
X7-Δin7cDNA
Tet
72
No
7
7
7
8
Autoregulation
mRNA isoforms in self regulation

66. TDP-43 continuosly shuttling
between the nucleus and the
Cytoplasm.
Physiological conditions
TDP-43 localization
Pathological conditions
Exported to the cytoplasm to
form insoluble aggregates.
GOF LOF

67. Climbing during aging
25°C
e
la
v
/W
1
1
1
8
E
la
v
/e
g
fp
2
E
la
v
/1
2
X
3
0
2 0
4 0
6 0
8 0
1 0 0
g e n o ty p e
%Topclimbingflies
2 5 C D 1 E L A V
e
la
v
/W
1
1
1
8
E
la
v
/e
g
fp
2
E
la
v
/1
2
X
3
0
2 0
4 0
6 0
8 0
1 0 0
2 5 C D 7 E L A V
g e n o ty p e
%Topclimbingflies
*
*
e
la
v
/W
1
1
1
8
E
la
v
/e
g
fp
2
E
la
v
/1
2
X
3
0
2 0
4 0
6 0
8 0
1 0 0
g e n o ty p e
%Topclimbingflies
2 5 C D 1 4 E L A V
***
**
e
la
v
/W
1
1
1
8
E
la
v
/e
g
fp
2
E
la
v
/1
2
X
3
-2 0
0
2 0
4 0
6 0
8 0
1 0 0
g e n o ty p e
%Topclimbingflies
2 5 C D 2 1 E L A V
****
****

68. Buratti and Baralle, 2012, TiBS 37: 237.
TAR DNA Binding Protein (TDP 43) is a splicing factor that belongs to the
hnRNP family and plays a role in many aspects of RNA metabolism.
Its aggregation/dysfunction is central to ALS and FTLD pathogenesis

69. RRM1
linker
C-terminus
N-terminus
RRM2
P187
L188
Q184
K102
N259
C244
M132
I253
Q134
I249
L139
E200
R151
D247
S254
Interactions between the TDP-43 RRMs

70. RRM1 RRM2GUA5
D105
F149
S254
GUGUGAAUGAAU
K145
R197
linker
GUA5 interacts at the interface of both RRMs

71. RRM1
RRM2
GUA1
N179
W113
D174
GUA3
URA2R171
URA4
L177
K176
F147
I107
GUGUGAAUGAAU
linker
Specific interactions on RRM1

72. RRM1
RRM2
E261
URA8
GUA9
N259
S258
F229
F194
K192
K263
F231
F221
GUGUGAAUGAAU
Specific interactions on RRM2

73. Functional studies on the CFTR exon 9 splicing model
of relevant TDP 43 amino acid variants
+ + + + + + + + + +
Add back of si-resistant TDP-43 wt and mutants (X>A)
+ +
GUA5 GUA1 GUA3
siRNA
Ex 9+
Ex 9-
URA4 URA8 GUA9
+ + + + + +
Single mutants
+
+ + + + + + + +
Double mutants Add back of si-resistant TDP-43 wt and mutants (X>A)
Strong impairement of splicing inhibition ability
Moderate impairement of splicing inhibition ability
No/Weak impairement of splicing inhibition ability
siRNA
Ex 9+
Ex 9-
WT F4
L
M132
I249
R151
D247
D174
R171
K176
N179
K192
E261
D105
S254
WT F4L D105 F149 K181 R197 S254 H256 W113 R171 D174 K176 N179 F194 S258 K192 E261 K263

74. TDPBR
7 87
6
6
AAAAAAA
6
Low TDP-43 concentrations
High TDP-43 concentrations
Efficient TDP-43
production
coding sequence non-coding sequence
AAAAAAA
TDPBR
Reduced TDP-43
production
AAAAAAA
+
AAAAAAA
Model of TDP-43 autoregulation:

75. A
B
7GFP
7GFP
28S
18S
Two identical mRNAs ending in pA2
have different cellular distribution if
they are generated from splicing or
from direct transcription of an
intronless gene

76. X7 7 8GFP
X7-sup5’-3’ 7 8GFP
gt ag
gt ag
A
C
Fig.4
gacg/gtgggtgt
gCAg/gtAAgtgt
tctttgttttgcag/ccctgaa
tTtttTttttgcag/Gcctgaa
- +Tet
X7- sup5’-3’
28S
18S
7GFP
7GFP
B
D
WMM=6.61
WMM=13.07
NNSp=0.86
NNSp=1.00
X7- sup5’-3’
+CHX
F
GFP
reporter
DiGFP
Tub.
Flag-TDP-43
TDP-43 end.
X7X7- sup5’-
3’
E
- +
- +Tet - +
7
Improved splice sites drive
splicing Independently of
TDP 43 levels but the mRNA
produced is inefficient in
protein production.

77. GFP Anti-digox-rhod merge
Fig.5
X7
X7-Δin7cDNA
X7-sup5’-3’
A
B
C
TO-PRO 3
9μ
m
9μ
m
9μ
m
9μ
m
9μm 9μm 9μm 9μm
9μ
m
9μ
m
9μ
m
9μ
m