Knowledge prior to this research? Questions addressed? Experimental approaches used? and what outcome? Impact of these findings? Future experiments? 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: , 2011.

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

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

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

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

Role of Tat at transcriptional level Brady Retrovirol. 2:69, 2005 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 …

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

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

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

Figure 1 RNA constructs used in this study

RNA affinity chromatography Alberts Fig ESS2 RNA RNA immobilized on beads

Figure 2 C

Figure 3

RNA footprinting Alberts Fig RNA region bound to protein will be protected from RNase attack (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

Figure 4 RNase T1 cleaves after Gs

Figure 5 Figure 6

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