RNA Structure modeling GDR MASIM Paris, 16-17 th November 2017 - - PowerPoint PPT Presentation

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RNA Structure modeling GDR MASIM Paris, 16-17 th November 2017 Bruno Sargueil, CNRS UMR 8015 Facult de pharmacie PARIS Bruno.sargueil@parisdescartes.fr RNA structure Primary structure 5

SLIDE 1

RNA Structure modeling

Bruno Sargueil, CNRS UMR 8015 Faculté de pharmacie – PARIS Bruno.sargueil@parisdescartes.fr GDR MASIM Paris, 16-17th November 2017

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RNA structure

Primary structure

5’ aaaaagcaaaaatgtgatcttgcttgtaaatacaattttgagaggttaataaattacaagtagtgcta tttttgtatttag gttagctatttagctttacgttccagg atgcctagtg gcagccccac aatatccagg aagccctctctgcggttttt 3’

Secondary structure
Tertiary structure

SLIDE 3

Nucleotide interactions

SLIDE 4

Some examples of tertiary motifs

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RNA structure modeling

Secondary structure modeling is a limiting step
Structure modeling software (Mfold, RNAfold …) are based on :
Thermodynamic – experimental data have defined a free energy for a bp in a

given context (nearest neighbour theory)

Probability (Boltzmann statistics)
Such modeling is often inexact if the RNA is over > 50(ish) nucleotide long
Thermodynamic model is incomplete
Does not predict non canonical base pairs – pseudoknots
Does not take into account folding kinetics
A single RNA may adopt several foldings
Yields several models – how to choose?

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Generating folding constraints

Goal: Experimentally define nucleotides that are in single strand conformation

Single stand RNAse : T1, A, S1 etc …
Small molecules: DMS, CMCT, SHAPE reagents

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Probing RNA structure

The reactivity map is used as (soft) constrains by the modeling software (Bonus/penalty)

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Is this enough ?

Modeling without constraint Modeling using probing structure data X-Ray structure Not predicting the tertiary structure impairs the 2D prediction

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Multiple probes

Each molecular probe brings different information The experimental process has been entirely automated

Use a multiprobing approach to improve modeling

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Multiprobing approach

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Multiprobing approach

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Multiprobing approach

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Multiprobing approach

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Developpement of a new model that takes into account all the probing results

Currently validating the approach on a « benchmark » RNA

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Detecting the tertiary structure

Reactivity low medium high Probing the structure in presence/absence of Mg2+ can reveal tertiary contacts We are currently developping approaches to predict pseudoknots using such data

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Combining Probing and NGS

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Naive probing of multiple mutants

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Loop IIId is crucial for HCV IRES/40S binding

BK H B

Loop IIId

Adapted from Hashem et al. 2014

(CryoEM envelop)

Base pairing (kissing complex) between loop IIId (HCV IRES) and ES7 (18S rRNA) favours the 40S recruitment, and is required for an efficient translation

SLIDE 20

IRES structure and structural rearrangment

WT

> 0.7 0,5 - 0,7 0,2 - 0,5 0 - 0,2 Undetermined

SHAPE reactivity

Less More Reactive in presence of 40S N N N N N

Loop IIId is protected from modification Domain IV unfolds

SLIDE 21

IRES footprinting on the18S rRNA

ES7 ES7 rRNA loop is protected Pattern modifications are observed in other sites

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Fitting the atoms in the envelop

Coordinates from Hashem et al. 2013 Model by Benoit Masquida

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3D model of the IRES-40S

Model by Benoit Masquida CryoEM envelop Hashem et al. 2013 Angulo et al. 2016

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Nathalie Chamond (CR CNRS) Christelle Vasnier (AI CNRS) Delphine Allouche (PhD Student) Grégoire de Bisschop (M2 student)

People involved

CIRI ENS Lyon T.Ohlmann

S. De Breyne
C. Herbreteau

Pontificia Universidad Católica de Chile M.Lopez-Lastra M.Vallejos

J. Angulo
F. Carvajal

CNRS UMRR7156 Strasbourg

B. Masquida

LIX – Ecole Polytechnique Yann Ponty (CR CNRS) Afaf Saaidi (PhD Student)