1. Turnip Yellow Mosaic Virus 3’UTR as a translational enhancer in Saccharomyces cerevisiae Lisa Bauer Microbiology Mentors: Daiki Matsuda Dr. Theo Dreher

2. Background Turnip Yellow Mosaic Virus (TYMV) Single-stranded positive-sense RNA virus p69 p206 XmnIDraI 3 overlapping reading frames (ORFs): -p69: Overlapping Protein -p206: Replication Protein -Coat Protein (CP) CP 5’UTR 3’UTR TLS

3. 3’ tRNA-like structure U A A U G-C C-G G-C A-U G-C G U-A C-G U-A G-C U-A C-G A C U A C C A A C U C G U CCCG GGGC CCC GGG CUCU UCGGAA AGCCU UCA UG 3´ GAUU C-G U-A G-C U U U A A A C A-U UCUUGAAU C CCAC l Major valine identity nts in the anticodon loop -Val Enhanced translation with 3’UTR seen in plant cells (Matsuda et al., 2002) eIF4E A A C C C C A C

4. Saccharomyces cerevisiae Fungi Eukaryotic Unicellular Why is yeast ideal? Small genome Entire genome known Genetic system with characterized mutants Simple system to use

5. 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

6. 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

7. Experimental Procedure Preparation of yeast spheroplasts Strain BY4741 98 mins doubling time in YEPD medium Grow to 0.6 OD Suspend in Buffer A (Sorbitol, TrisCl, MgCl2, DTT, ß- mercaptoethanol) Lyticase treatment BY4741 (18 mins) 90 minute recovery

8. Experimental Process 2. In vitro run-off transcription by T7 RNA polymerase (with/ without cap analog ) * 1. Linearize plasmid 3. RNA transfection 5. Cell lysis 6. Luciferase reaction LUC Protoplasts of cowpea leaves S. cerevisiae spheroplasts vs. 4. Translation at RT * Daiki Matsuda

9. RNA constructs Cap GLG-pA Cap + Tail + Controls: Cap GLGGLG-pAGLG TY 3’ UTR: Cap vec-L-TYsgCap vec-L-Bamvec-L-TYsgvec-L-Bam TY3’gTY3’sgTY3’sg(CGC)TY3’sg(GAC)TY3’BamTY3’PvuTY3’Dragenomic subgenomic Thanks to Wei Wei Chiu and Daiki Matsuda for use of constructs

10. Poly A & Cap Effects Cap GLG-pA GLG-pA Cap GLG GLG 1 22.2 0 5 10 15 20 25 Light Units (x10 8 ) Poly A EffectCap Effect 1 68.3 1 27.0 1 83.0

11. 3’ UTR & Cap Effects Cap vec-L-Bam vec-L-Bam vec-L-TYsg Cap vec-L-TYsg 0 5 10 15 20 Light Units (x10 9 ) 1 5.29 1 33.55 TY 3’ and Cap Synergy: 33.55/5.29= 6.34 20.3/3.31= 6.12 July 27August 6 1 3.31 1 20.3 0 5 10 15 20 Light Units (x10 9 ) Synergy in plant cells: ~10

12. 3’UTR Effects August 17August 20 TY3’Dra TY3’Bam TY3’Pvu TY3’g TY3’sg 0 2 4 6 8 10 12 Light Units (x10 9 ) 2.10 1 0.09 0.24 0.08 2.24 1 0.07 0.2 0.07 0 2 4 6 8 10 12 Light Units (x10 9 ) Plant Cell Data Yeast Cell Data 2.18 1 0.21 0.13 0.09

13. U A A U G-C C-G G-C A-U G-C G U-A C-G U-A G-C U-A C-G A C U A C C A A C U C G U CCCG GGGC CCC GGG CUCU UCGGAA AGCCU UCA UG 3´ GAUU C-G U-A G-C U U U A A A C A-U UCUUGAAU C CCAC -Val A A C C C TY3’ Valylation Effect TY3’sg(CGC) TY3’sg TY3’sg(GAC) Plant cell data Experiment 1 Experiment 2 0 2 4 6 8 10 12 Light Units (x10 9 ) G G 1 0.46 0.49 0.61 0.6 0.14 0.8 0.6 0.8 0.46 0.49 1 0.46 C

14. 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 less effect than expected

15. Next Steps Electroporation with W303 strain Utilize mutant yeast strains RNA turnover Initiation factor mutants

16. Acknowledgements Dr. Theo Dreher Daiki Matsuda Kevin Ahern Howard Hughes Medical Institute National Science Foundation