The Role of the 3’ UTR of Dulcamara mottle virus RNA in Translation
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Transcript of The Role of the 3’ UTR of Dulcamara mottle virus RNA in Translation
The Role of the 3’ UTR of Dulcamara mottle virus
RNA in Translation
Alma Laney
Dr. Yannis Tzanetakis
Dr. Theo Dreher
mRNA Translation
mRNA structure5’ cap 3’ UTR
60s
40s
An
An
Enzyme
mRNA translation scheme
Positive Strand RNA Viruses
VPg
mRNA, Flexiviruses,Togaviruses
TLS Tymo-, Tobamoviruses
Flaviviruses, Closteroviruses
AnPicornaviruses,
Potyviruses
Barley yellowdwarf virus
5´cap: m7G(5´)ppp(5´)-N
An
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
DuMV
MP (62 kD) CP (20 kD)
RP (196 kD)* MTR PRO HEL POL
129 nt5´-UTR
248 nt3´-UTR
RNAi suppressor
MP
* R HE
Pr teolyticMaturat oCleava e
OL
(69 kD)
p141 p66
CP (20 kD)
RP (206 kD)TLS (Val)MTR P O L
oi n
g
P
RNAi suppressor
TYMV
87 nt5´-UTR
109 nt3´-UTR
TYMV and DuMV 3’ termini
CUC
GA
UC
C
AA
C C
GAGCCUCG
CGUCGCAG
U
GCGACGCU
UUAAA
UUC
U
5´ Dulcamara mottle virus3´- pseudoknotA
AU
G-CC-GG-CA-UG-CGU-AC-GU-AG-CU-AC-G
AC
UA
CCCC A C
A
UU G
AA
C U
C G U
CCCGGGGC
CCCGGG
CUCU
UCGGAAGCCU
UCA
T
D
A/C
ACC(A)
5´
TYMV TLS
Quic
kTim
e™
and
aTIF
F (
Un
com
pre
ssed
) d
ecom
pre
ssor
are
ne
ede
d t
o s
ee t
his
pic
ture
.
The 3’ UTR of DuMV
DuMV 248 nt
44 nt 59 nt 145 nt
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGAAAAAAAAAAAAAACAAAACAAAACAAA
CUC
GA
UC
C
AA
C C
GAGCCUCG
CGUCGCAG
U
GCGACGCU
UUAAA
UUC
U
5´ Dulcamara mottle virus
3´- pseudoknot
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 145 nt
(Pseudoknot)( Poly A Tail)( Poly A Tail)
Methodology
• Six plasmid constructs were created with the luciferase gene.
• The plasmids will be tested to see luciferase expression.
luciferase 3’ UTR luciferase reaction Light generated
Luciferin + ATP
Plasmid constructs
LuciferaseLuciferase Spacer
LuciferaseLuciferase Spacer A Track
LuciferaseLuciferase A Track
LuciferaseLuciferase Pseudoknot
LuciferaseLuciferase A Track Pseudoknot
LuciferaseLuciferase A TrackSpacer Pseudoknot
2. Ligation of UTR fragment to pLUC
1. PCR of UTR section
Creating the plasmid constructs
3. Transformation 4. Restriction Enzyme Digest
Creating the plasmid constructs cont.
5. Gel Electrophoresis 6. DNA sequencing
Creating the plasmid constructs cont.
2. In vitro transcription1. Linearize plasmid
3. RNA transfection
5. Cell lysis
6. Luciferase reaction
LUC
Cowpea protoplasts
*
4. Incubation under light
+/- DuMV UTR fragments
Luciferase assay to test for translational enhancement
Results
XXLuc + A-tail
XXLuc + Spacer + A Tail
XXLuc + Spacer
AssayedSequencedCloned
Luc + complete
Luc + A-tail + Pseudoknot
Luc + Pseudoknot
X
x
X
X
X
0
2 104
4 104
6 104
8 104
1 105
1.2 105
1.4 105
RLU
0 1 2 3 4 76 85 9
Time (hrs)
Example of Luciferase Assay
Luc
Luc
Luc
Luc
LucSpacer A Track Pseudoknot
Future Work
• The next step is to create several genome chimeras to test infectivity in plants.
DuMV Complete Genome TYMV TLS
TYMV Complete Genome DuMV 3’ UTR
Acknowledgements
• Thanks to the Howard Hughes Medical Institute.
• Dr. Yannis Tzanetakis
• The Theo Dreher Lab