knowledge prior to this research?
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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?. - PowerPoint PPT PresentationTRANSCRIPT
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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.
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Rambaut Nature Genet 5:53, 2004
LIFE CYCLE OF HIV RETROVIRUS
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Trkola Curr Opin Microbiol. 7:407, 2004
HIV-1 retroviral genome: > 40 different RNAs & ~16 proteins from one primary transcript
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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)
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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)
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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 …
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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)
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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
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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)?
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Figure 1
RNA constructs used in this study
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RNA affinity chromatography
Alberts Fig. 7-30
ESS2 RNA
ESS2 RNA
ESS2 RNA
RNA immobilized on beads
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Figure 2
C
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Figure 3
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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
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Figure 4RNase T1 cleaves after Gs
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Figure 5
Figure 6
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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”