The Role of the 3’ UTR
of Dulcamara mottle virus
RNA in Translation
Alma Laney
Dr. Yannis Tzanetakis
Dr. Theo Dreher
mRNA Translation
An
mRNA structure
5’ cap
60s
Enzyme
40s
An
mRNA translation scheme
3’ UTR
Positive Strand RNA Viruses
mRNA, Flexiviruses,
An Togaviruses
TLS
Tymo-,
Tobamoviruses
Flaviviruses,
Closteroviruses
An Picornaviruses,
Potyviruses
VPg
Barley yellow
dwarf virus
5´cap: m7G(5´)ppp(5´)-N
RNA virus translation
• How do the untranslated regions (UTR) of RNA
viruses enhance translation?
TYMV translation
• The TLS of TYMV mimics a tRNA.
TYMV and DuMV genomes
TYMV
RNAi suppressor
MP (69 kD)
*
MTR
87 nt
5´-UTR
Proteolytic
Maturat ion
Cleava ge
PRO
p141
HEL
RP (206 kD)
CP (20 kD)
POL
p66
TLS (Val)
109 nt
3´-UTR
DuMV
RNAi suppressor
MP (62 kD)
*
129 nt
5´-UTR
MTR
CP (20 kD)
PRO
HEL
RP (196 kD)
POL
248 nt
3´-UTR
TYMV and DuMV 3’ termini
A
A
U
A
D
C
A
U
G-C
C-G
G-C
A-U
G-C
G
5´
U-A
C-G
U-A
G-C
U-A
C-G
C
C
C A/C A
C A C
CUCU
CCC UCGGA ACC(A)
GGG AGCCU
UCA
U
5´ C
TYMV TLS
QuickTime™ and a
TIFF (Uncompress ed) dec ompres sor
are needed t o s ee this pic ture.
A C U CCCG
A T
GGGC
C G U
U
U G
U
A A
C
G
GAGC CGUC GCGA UUC
A
CUCG GCAG CGCU
C
UUC
UUAAA
C
C
Dulcamara mottle virus
3´- pseudoknot
The 3’ UTR of DuMV
44 nt
59 nt
145 nt
DuMV
248 nt
U
A A
C
G
GAGC CGUC GCGA UUC
A
CUCG GCAG CGCU
C
UUC
UUAAA
C
C
U
5´ C
Dulcamara mottle
virus
3´- pseudoknot
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGAAAAAAAAAAAAAACAAAACAAAACAAA
The Question
• Does the 3’ UTR of DuMV enhance
translation?
44 nt
59 nt
145 nt
The Hypothesis
• The combination of the 59 nt and 145 nt
sequences are responsible for
translational enhancement.
59 nt
( Poly A Tail)
145 nt
(Pseudoknot)
Methodology
• Six plasmid constructs were created with
the luciferase gene.
• The plasmids will be tested to see
luciferase expression.
luciferase
3’ UTR
luciferase reaction
Luciferin + ATP
Light generated
Plasmid constructs
Luciferase
Spacer
Luciferase
A Track
Luciferase
Luciferase
A Track
Pseudoknot
A Track
Pseudoknot
Luciferase
Spacer
Luciferase
Spacer
Pseudoknot
A Track
Creating the plasmid constructs
1. PCR of UTR
section
2. Ligation of UTR
fragment to pLUC
Creating the plasmid constructs
cont.
3. Transformation
4. Restriction Enzyme Digest
Creating the plasmid constructs
cont.
5. Gel Electrophoresis
6. DNA sequencing
Luciferase assay to test for
translational enhancement
1. Linearize plasmid
2. In vitro transcription
LUC
*
3. RNA transfection
+/- DuMV UTR fragments
Cowpea protoplasts
5. Cell lysis
6. Luciferase
reaction
4. Incubation
under light
Results
Cloned Sequenced Assayed
Luc +
Spacer
X
X
Luc +
Spacer + A
Tail
Luc + A-tail
X
X
X
X
X
X
Luc +
Pseudoknot
Luc + A-tail
+
Pseudoknot
Luc +
complete
x
X
X
Example of Luciferase Assay
Luc
Spacer A Track
5
1.4 10
5
1.2 10
Luc
5
1 10
RLU
4
8 10
4
6 10
4
4 10
Luc
4
2 10
Luc
0
0
1
2
3
4
5
6
Time (hrs)
7
8
9
Luc
Pseudoknot
Future Work
• The next step is to create several genome
chimeras to test infectivity in plants.
TYMV Complete Genome
DuMV 3’ UTR
DuMV Complete Genome
TYMV TLS
Acknowledgements
• Thanks to the Howard Hughes Medical
Institute.
• Dr. Yannis Tzanetakis
• The Theo Dreher Lab