1. The Role of the 3’ UTR of Dulcamara mottle virus RNA in Translation Alma Laney Dr. Yannis Tzanetakis Dr. Theo Dreher

2. mRNA Translation mRNA structure 5’ cap 3’ UTR 60s 40s AnAn AnAn Enzyme mRNA translation scheme

3. Positive Strand RNA Viruses VPg mRNA, Flexiviruses, Togaviruses TLS Tymo-, Tobamoviruses Flaviviruses, Closteroviruses AnAn Picornaviruses, Potyviruses Barley yellow dwarf virus 5´cap: m 7 G(5´)ppp(5´)-N AnAn

4. RNA virus translation How do the untranslated regions (UTR) of RNA viruses enhance translation?

5. TYMV translation The TLS of TYMV mimics a tRNA.

6. TYMV and DuMV genomes DuMV MP (62 kD) CP (20 kD) RP (196 kD) * MTRPROHELPOL 129 nt 5´-UTR 248 nt 3´-UTR RNAi suppressor MP * RHE Prteolytic Maturato Cleavae OL (69 kD) p141 p66 CP (20 kD) RP (206 kD) TLS (Val) MTR POL o in g P RNAi suppressor TYMV 87 nt 5´-UTR 109 nt 3´-UTR

7. TYMV and DuMV 3’ termini C U C G A U C C A A C C GAGC CUCG CGUC GCAG U GCGA CGCU UUAAA UUC U 5´ Dulcamara mottle virus 3´- pseudoknot 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 CC C C A C A U U G A A C U C G U CCCG GGGC CCC GGG CUCU UCGGA AGCCU UCA T D A/C ACC(A) 5´ TYMV TLS

8. The 3’ UTR of DuMV DuMV 248 nt 44 nt 59 nt 145 nt AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGAAAAAAAAAAAAAACAAAACAAAACAAA C U C G A U C C A A C C GAGC CUCG CGUC GCAG U GCGA CGCU UUAAA UUC U 5´ Dulcamara mottle virus 3´- pseudoknot

9. The Question Does the 3’ UTR of DuMV enhance translation? 44 nt 59 nt 145 nt

10. The Hypothesis The combination of the 59 nt and 145 nt sequences are responsible for translational enhancement. 59 nt145 nt (Pseudoknot) ( Poly A Tail)

11. Methodology Six plasmid constructs were created with the luciferase gene. The plasmids will be tested to see luciferase expression. luciferase 3’ UTRluciferase reaction Light generated Luciferin + ATP

12. Plasmid constructsLuciferase Spacer Luciferase A Track Luciferase Luciferase Pseudoknot Luciferase A TrackPseudoknot Luciferase A Track Spacer Pseudoknot

13. 2. Ligation of UTR fragment to pLUC 1. PCR of UTR section Creating the plasmid constructs

14. 3. Transformation4. Restriction Enzyme Digest Creating the plasmid constructs cont.

15. 5. Gel Electrophoresis 6. DNA sequencing Creating the plasmid constructs cont.

16. 2. In vitro transcription 1. 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

17. Results XX Luc + A-tail XX Luc + Spacer + A Tail XX Luc + Spacer AssayedSequencedCloned Luc + complete Luc + A-tail + Pseudoknot Luc + Pseudoknot X x X X X

18. Example of Luciferase Assay Luc SpacerA TrackPseudoknot

19. Future Work The next step is to create several genome chimeras to test infectivity in plants. DuMV Complete GenomeTYMV TLS TYMV Complete GenomeDuMV 3’ UTR

20. Acknowledgements Thanks to the Howard Hughes Medical Institute. Dr. Yannis Tzanetakis The Theo Dreher Lab