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Transcriptional profiling and mRNA stability dont shoot the messengerDavid R. ShermanSeattle Biomedical Research InstituteGrand Challenge of Latent TB Mtg.Cape TownFebruary 25, 2012Landscape of TB latencyCONFIDENTIAL4.4 billionPPD(-)1.8 billionPPD(+)16 millionactive infections1.5 milliondeathsLandscape of TB latencyCONFIDENTIAL4.4 billionPPD(-)1.8 billionPPD(+)16 millionactive infections1.5 milliondeathsCalcified lesionsFew viable bacteriaCaseous lesionsLow numbers of bacteriaEvolving lesionsBacterial replicationLatency to DiseaseActive diseaseLandscape of TB latencyCONFIDENTIAL4.4 billionPPD(-)1.8 billionPPD(+)16 millionactive infections1.5 milliondeathsCalcified lesionsFew viable bacteriaCaseous lesionsLow numbers of bacteriaEvolving lesionsBacterial replicationLatency to DiseaseActive diseaseGC-11

Using gene expression to probe latent TBConcept TB gene expression in vivo will reveal the conditions that it experiences.- Physiology- Drug targets

Infected tissueGene regulatory network5

Using gene expression to probe latent TB

Infected tissueGene regulatory networkAssumes TF binding = Tx initiation = mRNA abundance6

mRNA abundance balanceShalem et al. 2008Transcription RateDegradation RateHow does mRNA decay in MTB? Does mRNA decay play a role in MTB transcriptional regulation?7Talk outlineCharacterize MTB mRNA decayGlobal mRNA half life (T1/2)Most stable/labile messagesFactors affecting stability

mRNA decay in stress responseMild cold shockHypoxia

Summary

8Measuring mRNA degradationRifampicinLog Phase01060RNACy dye labelMicroarray2030515Custom array design:100,000 TB oligos30,000 control oligosSame samples analyzed by qPCR and array. Other time points sort of available9

T0T0T0T0T20T5T10T15mRNA decay by microarrayEach spot represents a single gene as it decays. The stable spots are (mostly) tRNAs, which can be used to normalize the arrays.10Individual decay curveRifampicin (rif)One example transcript as it decays.11Individual decay curveRifampicin (rif)T1/2To calculate the life, simply flip the axes and take the negative slope of the line. You can also get an R2 for that line as an indicator of how well those spots describe a logarithmic decay (i.e. how well that T1/2 is described by the measurement taken.12Data filtered for reproducibility and R2Inclusion criteria:T1/2 with R2>0.7Starting [RNA] > 4x backgroundValid measures at > half replicates

2139 genes met criteriaTB mRNA is very stableAverage MTB T1/2 = 9.5 minutes14TB mRNA is very stable15Functional CategoryMean half-life (minutes)information pathways8.7*virulence, detoxification, and adaptation9.3lipid metabolism9.4cell wall and cell processes9.4metabolism and respiration9.5hypothetical protein9.6Regulatory proteins9.6insertion seqs and phages10.4**PE/PPE11.1***=significantly lower**=significantly highermRNA T1/2 by functional categoryInformation pathways are somewhat oddly named, and contain many ribosomal proteins and housekeeping genes. Foreshadow abundance vs. T figure?16Functional subcategorySpecified Gene %Whole Genome %P ValueFunctional Enrichment for the genes with the shortest 1/2 lifePosttranslational modification, protein turnover, chaperones12.82.30Translation, ribosomal structure and biogenesis21.43.00Energy production and conversion14.25.20.002Intracellular trafficking, secretion, and vesicular transport2.80.30.025Functional Enrichment for the genes with the longest 1/2 lifeReplication, recombination and repair12.84.80.004Amino acid transport and metabolism11.44.50.01PE/PPE2.80.20.014Energy production and conversion11.45.20.019Still thinking how to orally summarize these results17Physical characteristics and mRNA stability

As seen in other organisms.18mRNA abundance and stabilityR2 = 0.8This data strongly implies that the decay rate, which should be a constant independent of abundance, needs to be treated differently from other enzymatic reactions.19mRNA abundance and stabilityInherent to the mRNA?

Inherent to abundance?

Test:DosR regulon: ~48 genes induced by hypoxia, etc.Place dosR under tet control.Induce regulon in log phaseThis data strongly implies that the decay rate, which should be a constant independent of abundance, needs to be treated differently from other enzymatic reactions.20Induced transcripts degrade faster>2000 transcripts did not change stabilityModified mRNA decay in response to stress conditionsAre specific transcripts (de)stabilized?Does the global mRNA life change?

Is mRNA decay regulated to change transcript abundance?

Temperature Hypoxia

mRNA degradation at 20CAfter 5 hrs:Only 55 genes decayed 2x or more.

Degradation not measurable.

T1/2 very sensitive to temp.T = 5 hrsmRNA stability in hypoxiaRifampicinLogPhase010602030Hypoxia120+RNACy dye labelMicroarraySame samples analyzed by qPCR and array. Other time points sort of available24mRNA stability in altered O2MTB mRNA decay characterizationReliable mRNA half lives for >2000 genes.Average half life ~9.5 minutes.Half life inversely correlated with transcript concentration.Transcripts stabilized by cold, hypooxia.

i.e. the average t of MTB is equal to the 90th percentile of e. coli26Questions to exploreWhy 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

Thank you!

Kyle MinchTige RustadDebbie WhitleyBill BrabantJessicaWinkler

Paul G Allen Family FoundationBill and Melinda Gates Foundation NIAID contract # HHSN272200800059CRifampicin is stopping transcriptionNo rif induced genesLux assay to look for induction after rif treatmentHypoxia sensitive promoter driving lux

Total degradation by array