1 / 17

Knowledge prior to this research?

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?.

tudor
Download Presentation

Knowledge prior to this research?

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  1. 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? Experimental approaches used? and what outcome? Impact of these findings? Future experiments?

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

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

  4. “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

  5. 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

  6. 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

  7. 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

  8. 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

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

  10. RNA constructs used in this study Figure 1

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

  12. C Figure 2

  13. Figure 3

  14. 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

  15. Figure 4 RNase T1 cleaves after Gs

  16. Figure 6 Figure 5

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

More Related