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Turnip Yellow Mosaic Virus 3’UTR as a translational enhancer in Saccharomyces cerevisiae

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. TLS.

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Turnip Yellow Mosaic Virus 3’UTR as a translational enhancer in Saccharomyces cerevisiae

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  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 5’UTR 3’UTR DraI XmnI p69 p206 TLS • 3 overlapping reading frames (ORFs): • -p69: Overlapping Protein • -p206: Replication Protein • -Coat Protein (CP) CP

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

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

  9. RNA constructs Controls: Cap + Cap GLG GLG-pA GLG Tail + TY 3’ UTR: Cap vec-L-TYsg Cap vec-L-Bam 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

  10. Poly A & Cap Effects Poly A Effect Cap Effect 1 83.0 GLG 1 68.3 1 27.0 1 22.2 0 5 10 15 20 25 Light Units (x108)

  11. 3’ UTR & Cap Effects July 27 August 6 1 5.29 1 33.55 1 3.31 1 20.3 vec-L-Bam vec-L-TYsg Cap vec-L-Bam Cap vec-L-TYsg 0 5 10 15 20 0 5 10 15 20 Light Units (x109) Light Units (x109) TY 3’ and Cap Synergy: 33.55/5.29= 6.34 20.3/3.31= 6.12 Synergy in plant cells: ~10

  12. 3’UTR Effects August 17 August 20 2.10 1 0.09 0.24 0.08 2.24 1 0.07 0.2 0.07 2.18 1 0.21 0.13 0.09 TY3’sg TY3’g TY3’Dra TY3’Pvu TY3’Bam 0 2 4 6 8 10 12 0 2 4 6 8 10 12 Light Units (x109) Light Units (x109) Plant Cell Data Yeast Cell Data

  13. C C A U A U A U A C A-U U-A C-G GAUU G-C UCUUGAAU C TY3’ Valylation Effect 3´ CUCU C -Val A U 1 CCCG CCC UCGGA A C A TY3’sg 1 GGGC GGG AGCCU U C G UCA U U G A 0.14 0.46 A U A TY3’sg(CGC) 0.46 0.46 A C U 0.6 G-C 0.6 C-G G-C A-U 0.61 G-C 0.49 G 0.49 TY3’sg(GAC) 0.49 U-A 0.8 C-G 0.8 U-A G-C U-A C-G C C 0 2 4 6 8 10 12 Plant cell data C A Light Units (x109) C Experiment 1 C C G A G Experiment 2

  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 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

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