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 HIV1 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?

Rambaut Nature Genet 5:53, 2004

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

“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

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

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

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

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

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

RNA constructs used in this study
Figure 1

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

C
Figure 2

Figure 3

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

RNase T1 cleaves after Gs Figure 4

Figure 5
Figure 6

“Possible physical and functional interactions between the identified protein partners of SLS2-A7 RNA and their possible links with HIV1 RNA biology”
Figure 7