Knowledge prior to this research?
description
Transcript of Knowledge prior to this research?
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”