Turnip Yellow Mosaic Virus 3’UTR as a
translational enhancer in
Saccharomyces cerevisiae
Lisa Bauer
Microbiology
Mentors: Daiki Matsuda
Dr. Theo Dreher
Background
Turnip Yellow Mosaic Virus (TYMV)

Single-stranded positive-sense RNA virus
5’UTR
3’UTR
DraI XmnI
p69
p206
3 overlapping reading frames (ORFs):
-p69: Overlapping Protein
-p206: Replication Protein
-Coat Protein (CP)
TLS
CP
3’ tRNA-like structure
A
C
CUCU
U
A
C
G
U G
U
CCCG
CCC
UCGGA
GGGC
GGG
AGCCU
U
A
A
U
Enhanced translation with 3’UTR
3´
seen in plant cells (Matsuda et al.,
AC C A -Val
2002)
UCA
A
C
G-C
AU U
A
U
C-G
A
C
G-C
A-U
U-A
A-U
C-G
GAUU G-C UCUUGAAU C
G-C
G
U-A
C-G
U-A

Major valine
G-C
U-A
identity nts in
C-G
C
the anticodon
C
A
C
loop
C A C
U
A
eIF4E
Saccharomyces cerevisiae

Fungi



Eukaryotic
Unicellular
Why is yeast ideal?




Small genome
Entire genome known
Genetic system with characterized
mutants
Simple system to use
Goal

The primary goal was to simulate the
same translational phenomenon seen
in plant cells of pre-existing RNA
constructs in yeast cells

Used pre-existing RNA constructs from
Daiki Matsuda and Wei Wei Chiu
Methodology of Yeast Electroporation

Gallie et al. (1992)
Development of yeast electroporation
system for expressing luciferase protein
 Cap and Poly A tail essential for efficient
translation


Searfoss et al. (2004)

Yeast electroporation method used
Experimental Procedure

Preparation of yeast spheroplasts

Strain BY4741
98 mins doubling time in YEPD medium
 Grow to 0.6 OD


Suspend in Buffer A


Lyticase treatment


(Sorbitol, TrisCl, MgCl2, DTT, ßmercaptoethanol)
BY4741 (18 mins)
90 minute recovery
Experimental Process
1. Linearize plasmid
LUC
2. In vitro run-off transcription
by T7 RNA polymerase
(with/ without cap analog)*
*Daiki
Matsuda
3. RNA transfection
vs.
Protoplasts of
cowpea leaves
S. cerevisiae
spheroplasts
4. Translation at RT
5. Cell lysis
6. Luciferase reaction
RNA constructs
Controls:
Cap
GLG-pA
GLG
GLG
Cap
GLG-pA
Cap +
Tail +
TY 3’ UTR:
Cap
Cap
vec-L-Bam
vec-L-TYsg
vec-L-TYsg
vec-L-Bam
TY3’sg(CGC)
TY3’sg(GAC)
TY3’Bam
TY3’Dra
TY3’sg
TY3’Pvu
TY3’g
genomic
subgenomic
Thanks to Wei Wei Chiu and Daiki Matsuda for use of constructs
Poly A & Cap Effects
Poly ACap
Effect
Effect
GLG
1
27.0
GLG-pA
1
1
1
Cap GLG
83.0
22.2
Cap GLG-pA
0
5
10
15
20
Light Units (x108)
25
68.3
3’ UTR & Cap Effects
July 27
August 6
vec-L-Bam
vec-L-TYsg
Cap vec-L-Bam
Cap vec-L-TYsg
0
5
10
15
Light Units (x109)
1
1
5.29
3.31
1
1
33.55
20.3
20
TY 3’ and Cap Synergy: 33.55/5.29= 6.34
Synergy in plant cells: ~10
0
5
10
15
20
Light Units (x109)
20.3/3.31= 6.12
3’UTR Effects
August 17
August 20
TY3’sg
2.10
2.24 2.18
TY3’g
1
1
0.09
0.07 0.21
0.24
0.2
0.08
0.07 0.09
TY3’Dra
TY3’Pvu
TY3’Bam
0
2
4
6
8
10
Light Units (x109)
12
0
2
4
6
8
10
1
0.13
12
Light Units (x109)
Plant Cell Data
Yeast Cell Data
TY3’ Valylation Effect
A
C
CUCU
U
A
C
G
U G
U
3´
CCCG
CCC
UCGGA AC C A -Val
GGGC
GGG
AGCCU
U
A
A
U
TY3’sg
UCA
A
C
TY3’sg(CGC)
G-C
AU U
A
U
C-G
A
C
G-C
A-U
U-A
A-U
C-G
GAUU G-C UCUUGAAU C
G-C
G
U-A
TY3’sg(GAC)
C-G
U-A
G-C
U-A
C-G
C
C
Plant cell data
A
C
Experiment 1
C
C
G
U
A
1
1
A
G
Experiment 2
0.14
0.46
0.46
0.46
0.6
0.6
0.49 0.61
0.49
0.49
0
2
4
6
Light Units (x109)
8
0.8
0.8
10
12
Conclusions

3’TYMV:
30 fold 3’ effect; similar to poly A effect
 ~27 fold TY cap effect
 3’ TY synergy with cap ~ 6 fold


3’UTR:
Subgenomic 2x genomic 3’end
 Dra and Bam cuts both ~7% of wild type;
TLS important factor
 Non-valylation
Non-valylation less
less effect
effect than
than expected
expected

Next Steps

Electroporation with W303 strain
 Utilize mutant yeast strains
 RNA turnover
 Initiation factor mutants
1.4E+09
TY 3'sg
Light Units
1.2E+09
TY 3' CGC
1.0E+09
TY Bam
8.0E+08
GAC(wobble)
6.0E+08
TY 3'g
4.0E+08
TY 3'Dra1
2.0E+08
TY Pvu
0.0E+00
0
20
40
60
Time (mins)
80
100
Acknowledgements





Dr. Theo Dreher
Daiki Matsuda
Kevin Ahern
Howard Hughes Medical
Institute
National Science
Foundation