the role of the 3’ utr of dulcamara mottle virus rna in translation alma laney dr. yannis...
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![Page 1: The Role of the 3’ UTR of Dulcamara mottle virus RNA in Translation Alma Laney Dr. Yannis Tzanetakis Dr. Theo Dreher](https://reader036.vdocuments.site/reader036/viewer/2022062516/56649d485503460f94a245bf/html5/thumbnails/1.jpg)
The Role of the 3’ UTR of Dulcamara mottle virus
RNA in Translation
Alma Laney
Dr. Yannis Tzanetakis
Dr. Theo Dreher
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mRNA Translation
mRNA structure5’ cap 3’ UTR
60s
40s
An
An
Enzyme
mRNA translation scheme
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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
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RNA virus translation• How do the untranslated regions (UTR) of RNA
viruses enhance translation?
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TYMV translation
• The TLS of TYMV mimics a tRNA.
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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
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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
.
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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
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The Question
• Does the 3’ UTR of DuMV enhance translation?
44 nt 59 nt 145 nt
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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)
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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
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Plasmid constructs
LuciferaseLuciferase Spacer
LuciferaseLuciferase Spacer A Track
LuciferaseLuciferase A Track
LuciferaseLuciferase Pseudoknot
LuciferaseLuciferase A Track Pseudoknot
LuciferaseLuciferase A TrackSpacer Pseudoknot
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2. Ligation of UTR fragment to pLUC
1. PCR of UTR section
Creating the plasmid constructs
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3. Transformation 4. Restriction Enzyme Digest
Creating the plasmid constructs cont.
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5. Gel Electrophoresis 6. DNA sequencing
Creating the plasmid constructs cont.
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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
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Results
XXLuc + A-tail
XXLuc + Spacer + A Tail
XXLuc + Spacer
AssayedSequencedCloned
Luc + complete
Luc + A-tail + Pseudoknot
Luc + Pseudoknot
X
x
X
X
X
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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
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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
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Acknowledgements
• Thanks to the Howard Hughes Medical Institute.
• Dr. Yannis Tzanetakis
• The Theo Dreher Lab