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: 325-342, 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, 1994

5’ splice sites (donor sites) = 5

3’ splice sites (acceptor sites) = 8 - 9

~ 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. 7-30

ESS2 RNA

ESS2 RNA

ESS2 RNA

RNA immobilized on beads

Figure 2

C

Figure 3

RNA footprinting

Alberts Fig.8-54

... 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 4RNase 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”