shape-reactivity and rna structure · overview rna-probing rna probing di erent methodological...

SHAPE-Reactivity and RNA StructureWinterseminar 2015

Roman Ochsenreiter, TBI

17.2.2015

Roman Ochsenreiter, TBI SHAPE-Reactivity and RNA Structure 17.2.2015 1 / 24

Overview

Table of Contents

1 OverviewRNA-ProbingSHAPERMDBEteRNA

2 SHAPE - AnalysisEteRNA ScoresEnsembles and ProbingBase-dependent ReactivityLoop-Type and Reactivity


Overview RNA-Probing

RNA ProbingDifferent methodological approches

Modification rates depend on local RNA structure


Overview SHAPE

SHAPEInvestigating nucleotide flexibility

Selective 2’-Hydroxyl Acylation analyzed by Primer Extension

A B

C D


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


Overview RMDB

SHAPE - Databases

RMDB - Stanford RNA Mapping Database (> 100000 probingexperiments)


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


Overview EteRNA

EteRNAEteRNA - “Workflow”


Overview EteRNA



SHAPE - Analysis

Table of Contents

1 OverviewRNA-ProbingSHAPERMDBEteRNA

2 SHAPE - AnalysisEteRNA ScoresEnsembles and ProbingBase-dependent ReactivityLoop-Type and Reactivity


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


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


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)


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


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


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


Summary

Thanks to..

Ivo HofackerChristoph Flamm

Sven Findeissand

YOU for your attention!


shape-reactivity and rna structure · overview rna-probing rna probing di erent methodological...

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