1. Ty3 lifecycle maturation Nuclear pore translocation txn assembly Nucleus integration translation Gag3 Gag3-Pol3 export uncoating reverse transcription cDNA VLP Pheromone induction Ste12 GLFG Nups TFIIIB, TFIIIC Imt1-4 Xrn1 Dhh1 Lsm1 Eap1 Pub1 Stm1 Tma20 Hcr1

2. Ty3 expression systems α α factor inducible endogenous promoter galactose inducible synthetic promoter LTR GAG3 POL3 GAL10 GAG3 POL3

3. Gag3 Mutagenesis Larsen and Zhang SP

4. wt Dhh1-GFP Ty3-RFP Merge Basic K271A K272A E273A  basic  NC Zinc C267AC280A C267A C280A ** NC (p7) C267A C280A NC determines Gag3-Pol3 association with P bodies IN-GFP m7Gm7G AAAAA Dhh1-GFP

5. Plasmid cDNA WTG207DH233V G207D/H233V WTPR - ΔAcidicΔacidic PR - ΔSP CA SP NC H233VG207D Gag3 ATTHLQEEYDSDYDSEDNLTQNGYVH ΔSP/ΔAcidic

7. Blocking SP-NC blocks subsequent processing and cDNA synthesis (G207D) Blocking CA-SP blocks transposition after cDNA synthesis (H233V) Neutralization of the acidic residues of SP causes failure to condense the immature particle (  acidic)  SP produces particles and cDNA, but fails to transpose Therefore particle structure requires a negative charge on SP, but the essential function is after cDNA synthesis, for example in uncoating

9. Localization with P body proteins does not insure packaging of GAG3- POL3 RNA

13. DDX6 Family of DExD/H RNA Helicases —Implicated in splicing, ribosome biogenesis, RNA transport, degradation, and translation —ATP binding and hydrolysis activities, RNA binding activity. — Facilitate unwinding of double stranded RNA; thought to act as a remodeler of mRNP complexes —Evolutionarily conserved p54 family: DDX6, p54, RCK (human), Xp54 (X. laevis), CGH-1 (C. elegans) dhh1 (S. cerevisae) and others —promotes RNA decapping and translational repression in yeast —component of both stress granule and P body mRNP complexes in yeast —Component of germ granules in C. elegans; protects maternal mRNAs during oogenesis. —Dhh1 and RCK/hDhh1 are required for efficient translation o f positive strand RNA viruses BMV and HCV, respectively. **Recent data has demonstrated that Dhh1p has weaker RNA-dependent ATPase activity than other DEAD-box helicases; Dhh1p helicase activity was not observed in vitro. Q I Ia Ib IIIIIIVVVI REC A-like Domain 1Rec A-like Domain 2

14. dhh1Δ lsm1Δ stm1Δ xrn1Δ uncoating translocation, integration Dhh1 Xrn1 Dhh1 Xrn1 Lsm1 Stm1 Proteins associated with Ty3 Gag3 affect retrotransposition intermediates