SHAPE-Reactivity and RNA Structure · Overview RNA-Probing RNA Probing Di erent methodological...
Transcript of SHAPE-Reactivity and RNA Structure · Overview RNA-Probing RNA Probing Di erent methodological...
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SHAPE-Reactivity and RNA StructureWinterseminar 2015
Roman Ochsenreiter, TBI
17.2.2015
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Overview
Table of Contents
1 OverviewRNA-ProbingSHAPERMDBEteRNA
2 SHAPE - AnalysisEteRNA ScoresEnsembles and ProbingBase-dependent ReactivityLoop-Type and Reactivity
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Overview RNA-Probing
RNA ProbingDifferent methodological approches
Modification rates depend on local RNA structure
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Overview RNA-Probing
RNA ProbingDifferent methodological approches
Modification rates depend on local RNA structure
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Overview SHAPE
SHAPEInvestigating nucleotide flexibility
Selective 2’-Hydroxyl Acylation analyzed by Primer Extension
A B
C D
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Overview SHAPE
SHAPEWhy use SHAPE?
Simple protocolCan be performed via high-throughputNo intrinsically different reactivity of different basesReactivity can be converted to Pseudo Free Energies for usage asfolding constraints:
∆GSHAPE (i) = m ln(SHAPE reactivity(i) + 1) + b
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Overview RMDB
SHAPE - Databases
RMDB - Stanford RNA Mapping Database (> 100000 probingexperiments)
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Overview EteRNA
“High-troughput” sequence design - by (many) hands
Each design is scored by SHAPE-Probing
Successful design rules are used for inverse folding algorithm
EteRNA Score:
SEteRNA =
∑Ni=1 ∆
N, ∆
1 if ri > 0.25 (designed to be unpaired)
1 if ri < 0.5 (designed to be paired)
0 otherwise
ri : Reactivity of Nucleotide i
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Overview EteRNA
EteRNAEteRNA - “Workflow”
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Overview EteRNA
EteRNAEteRNA - “Workflow”
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Overview EteRNA
EteRNAEteRNA - “Workflow”
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Overview EteRNA
EteRNAEteRNA - “Workflow”
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Overview EteRNA
EteRNAEteRNA - “Workflow”
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Overview EteRNA
EteRNAEteRNA - “Workflow”
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Overview EteRNA
EteRNAEteRNA - “Workflow”
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Overview EteRNA
EteRNAEteRNA - “Workflow”
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SHAPE - Analysis
Table of Contents
1 OverviewRNA-ProbingSHAPERMDBEteRNA
2 SHAPE - AnalysisEteRNA ScoresEnsembles and ProbingBase-dependent ReactivityLoop-Type and Reactivity
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SHAPE - Analysis EteRNA Scores
Scoring EteRNA Scores(Linear) Correlation of P(i is paired) vs. Reactivity(i)
EteRNA Score
Corr
ela
tion
Interesting drop in correlation with lower EteRNA-Scores
Change of temperature has no effect
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SHAPE - Analysis Ensembles and Probing
Structural Ensembles and ProbingEquilibrium Population vs. Probing Quality
100 90 80 70 60
0.2
0.4
0.6
0.8
1.0
EteRNA Score
MFE
bas
in p
opul
atio
n
Lower EteRNA-scores are correlated to low MFE population rates
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SHAPE - Analysis Ensembles and Probing
Dominant structuresHow well scores the dominant structure?
50 60 70 80 90 100
5060
7080
9010
0
EteR
NA
Scor
e (M
ost p
opul
ated
bas
in)
EteRNA Score (MFE basin)
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SHAPE - Analysis Base-dependent Reactivity
Base-dependent Reactivity
Reactivity of all 4 bases in different structural contexts:
A C G U
0.0
1.0
2.0
3.0
UNPAIRED
A C G U
0.0
0.5
1.0
1.5
2.0
HELIX-END
A C G U
0.0
0.5
1.0
1.5
2.0
STACKED
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SHAPE - Analysis Loop-Type and Reactivity
Loop-Type and Reactivity
Reactivity of Nucleotides in all possible Loop-types:
Paired Exterior Hairpin Interior Bulge Multi
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Loop Type
Rea
ctiv
ity
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Summary
Take-Home message
Large amounts of SHAPE data sets are freely available
Reactivity reflects more than just nucleotide flexibility - alsoLoop-Types and Ensemble structures are important
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Summary
Thanks to..
Ivo HofackerChristoph Flamm
Sven Findeissand
YOU for your attention!
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