Knowledge prior to this research?

1. Research paper for class discussion: Marchand et al. “Identification of protein partners of the HIV-1 tat/rev exon3 leads to the discovery of a new HIV-1 splicing regulator hnRNP K” RNA Biol. 8: 325-342, 2011. Knowledge prior to this research? Questions addressed? Experimental approaches used? and what outcome? Impact of these findings? Future experiments?

2. LIFE CYCLE OF HIV RETROVIRUS Rambaut Nature Genet 5:53, 2004

3. HIV-1 retroviral genome: > 40 different RNAs & ~16 proteins from one primary transcript Trkola Curr Opin Microbiol. 7:407, 2004

4. “Global landscape of HIV-human protein complexes” Network representation of protein-protein interactions using 2 different cell lines (HEK293 and Jurkat) Jager et al. Nature 481:365, 2012

5. Early RNA processing events - multiply-spliced mRNAs (for regulatory proteins like Tat, Rev) exported to cytoplasm Frankel Ann Rev Genet 67:1, 1998 Later expression events - singly-spliced & unspliced transcripts exported for translation (eg gal-pol mRNA) or packaging (full-length RNA genome into virion) Alberts Fig.7-97

6. Role of Tat at transcriptional level Tat - transcription activator which binds to Tar (response element) downstream of promoter in 5’ LTR (hairpin with 3 nt bulge) - recruitment or activation of factors which hyperphosphorylate CTD of RNA pol II … Brady Retrovirol. 2:69, 2005

7. 5’ splice sites (donor sites) = 5 (strong) 3’ splice sites (acceptor sites) = 8 - 9 (weak, non-consensus) ~ 40 different processed RNA species Amendt Mol Cell Biol 14: 3960, 1994

8. HIV-1 mRNAs generated by alternative splicing “The acceptor site A7 plays an essential role for tat and rev mRNA production.” Marchand paper Abstract Karn & Stoltzfus Cold Spring Harb Perspect Med 2:a006916, 2012

9. “Five splicing inhibitory sequences in HIV-1 pre-mRNA have been identified...” Zahler (2004) Approaches to find such cis-elements (& trans factors)? Tange EMBO J 20: 5748, 2001

10. RNA constructs used in this study Figure 1

11. RNA affinity chromatography ESS2 RNA ESS2 RNA ESS2 RNA RNA immobilized on beads Alberts Fig. 7-30

12. C Figure 2

13. Figure 3

14. RNA footprinting (conceptually similar to DNA footprinting) - single stranded RNA, tagged at one end - incubated with protein - treated ‘gently’ with RNase so that on average only one cleavage per molecule ... RNA region bound to protein will be protected from RNase attack Alberts Fig.8-54

15. Figure 4 RNase T1 cleaves after Gs

16. Figure 6 Figure 5

17. “Possible physical and functional interactions between the identified protein partners of SLS2-A7 RNA and their possible links with HIV-1 RNA biology” Figure 7