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Transcriptional profiling and mRNA stability – don’t shoot the messenger

Transcriptional profiling and mRNA stability – don’t shoot the messenger. David R. Sherman Seattle Biomedical Research Institute Grand Challenge of Latent TB Mtg. Cape Town February 25, 2012. Landscape of TB latency. 1.5 million deaths. 16 million active infections. 1.8 billion

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Transcriptional profiling and mRNA stability – don’t shoot the messenger

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  1. Transcriptional profiling and mRNA stability – don’t shoot the messenger David R. Sherman Seattle Biomedical Research Institute Grand Challenge of Latent TB Mtg. Cape Town February 25, 2012

  2. Landscape of TB latency 1.5 million deaths 16 million active infections 1.8 billion PPD(+) 4.4 billion PPD(-) CONFIDENTIAL

  3. Landscape of TB latency 1.5 million deaths Active disease 16 million active infections Evolving lesions Bacterial replication Latency to Disease Caseouslesions Low numbers of bacteria 1.8 billion PPD(+) Calcified lesions Few viable bacteria 4.4 billion PPD(-) CONFIDENTIAL

  4. Landscape of TB latency 1.5 million deaths Active disease 16 million active infections Evolving lesions Bacterial replication Latency to Disease Caseouslesions Low numbers of bacteria 1.8 billion PPD(+) Calcified lesions Few viable bacteria 4.4 billion PPD(-) GC-11 CONFIDENTIAL

  5. Using gene expression to probe latent TB Infected tissue Gene regulatory network Concept – TB gene expression in vivo will reveal the conditions that it experiences. - Physiology - Drug targets

  6. Using gene expression to probe latent TB Infected tissue Gene regulatory network Assumes TF binding = Tx initiation = mRNA abundance

  7. mRNA abundance balance Transcription Rate Degradation Rate Shalem et al. 2008

  8. Talk outline • Characterize MTB mRNA decay • Global mRNA half life (T1/2) • Most stable/labile messages • Factors affecting stability • mRNA decay in stress response • Mild cold shock • Hypoxia • Summary

  9. Measuring mRNA degradation 0 5 10 15 20 30 60 Rifampicin Log Phase Cy dye label RNA Microarray Custom array design: 100,000 TB oligos 30,000 control oligos

  10. mRNA decay by microarray T10 T5 T0 T0 T15 T20 T0 T0

  11. Individual decay curve Rifampicin (rif)

  12. Individual decay curve T1/2 Rifampicin (rif)

  13. Data filtered for reproducibility and R2 • Inclusion criteria: • T1/2with R2>0.7 • Starting [RNA] > 4x background • Valid measures at > half replicates • 2139 genes met criteria

  14. TB mRNA is very stable Average MTB T1/2 = 9.5 minutes

  15. TB mRNA is very stable

  16. mRNA T1/2 by functional category

  17. Physical characteristics and mRNA stability

  18. mRNA abundance and stability R2 = 0.8

  19. mRNA abundance and stability • Inherent to the mRNA? • Inherent to abundance? • Test: • DosRregulon: ~48 genes induced by hypoxia, etc. • Place dosR under tet control. • Induce regulon in log phase

  20. Induced transcripts degrade faster >2000 transcripts did not change stability

  21. Modified mRNA decay in response to stress conditions • Are specific transcripts (de)stabilized? • Does the global mRNA ½ life change? • Is mRNA decay regulated to change transcript abundance? Temperature Hypoxia

  22. mRNA degradation at 20C After 5 hrs: Only 55 genes decayed 2x or more. Degradation not measurable. T1/2 very sensitive to temp. T = 5 hrs

  23. mRNA stability in hypoxia 0 10 20 30 120+ 60 Log Phase Rifampicin Hypoxia Cy dye label RNA Microarray

  24. mRNA stability in altered O2

  25. MTB mRNA decay characterization • Reliable mRNA half lives for >2000 genes. • Average half life ~9.5 minutes. • Half life inversely correlated with transcript concentration. • Transcripts stabilized by cold, hypooxia.

  26. Questions to explore • Why is TB mRNA very stable? • RNases or mRNA secondary structure? • Hypoxia and low temp: Transcription decreased to balance decay? Mechanism? • How to repress genes? • What are the consequences? • Systems modeling

  27. Thank you! Bill Brabant Kyle Minch Tige Rustad Jessica Winkler Debbie Whitley Paul G Allen Family Foundation Bill and Melinda Gates Foundation NIAID contract # HHSN272200800059C

  28. Rifampicinis stopping transcription • No rif induced genes • Lux assay to look for induction after rif treatment • Hypoxia sensitive promoter driving lux • Total degradation by array

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