# Towards structural models for the Ebola UTR regions using experimental SHAPE probing data

Afaf Saaidi, Delphine Allouche, Bruno Sargueil, Yann Ponty

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Afaf Saaidi, Delphine Allouche, Bruno Sargueil, Yann Ponty. Towards structural models for the Ebola UTR regions using experimental SHAPE probing data. JOBIM - Journées Ouvertes en Biologie, Informatique et Mathématiques - 2016, Jun 2016, Lyon, France. hal-01332469

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<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
Towards structural models for the Ebola UTR regions
using experimental SHAPE probing data

Afaf Saaidi¹,², Delphine Allouche³, Bruno Sargueil*,³ and Yann Ponty*,¹,²

¹CNRS/LIX, Ecole Polytechnique, Palaiseau

²AMIB team, Inria Saclay

³CNRS UMR 8015, Laboratoire de cristallographie et RMN Biologiques, Université Paris Descartes, Paris

Abstract

Next-Generation Sequencing (NGS) technologies have opened new perspectives to refine the pro-
cess of predicting the secondary structure(s) of structured non-coding RNAs. Herein, we describe an
integrated modeling strategy, based on the SHAPE chemistry, to infer structural insight from deep se-
quencing data. Our approach is based on a pseudo-energy minimization, incorporating additional
information from evolutionary data (compensatory mutations) and SHAPE experiments (reactiv-
ity scores) within an iterative procedure. Preliminary results reveal conserved and stable structures
within UTRs of the Ebola Genome, that are both thermodynamically-stable and highly supported by
SHAPE accessibility analysis.

Keywords RNA, Ebola, secondary structure prediction, SHAPE chemistry, High-throughput sequenc-
ing, compensatory mutations.

1 Context

The Ebola virus causes an acute, serious illness which is often fatal if untreated. A promising re-
search direction would be to selectively interfere with the expression of Ebola genes. This requires a
mechanical understanding, at a molecular level, of the processes underlying gene regulation. Many
such processes revolve around the presence of specific secondary structure elements [7] in untrans-
lated regions [2].

RNA structure prediction can either be performed computationally from thermodynamics principles in the presence of only the sequence [10], or via a comparative approach when an aligned set of homologous sequences is available [1].

Experimentally, such data can be supplemented by chemical probing data, leading to more ac-
curate predictions in the context of RNA structure determination [3].

Our approach is based on a pseudo-energy minimization, incorporating additional information from evolutionary data (compensatory mutations) and SHAPE experiments (reactivity scores) within an iterative procedure.

<footnote>*To whom correspondence should be addressed</footnote>
RNA folding can be approached in a less deterministic setting, by considering the outcome of the folding process as a dynamic ensemble of structures. In this vision, a sequence actively fluctuates between many alternative conformations through time, leading to a probability distribution over the set of structures.

The challenge is to take advantage from those probabilities to determine the most probable structures that verify the set of experimental constraints and thus help in revealing the Ebola virus replication mechanism.

SHAPE probing principles SHAPE (Selective 2'Hydroxyl Acylation analyzed by Primer Extension) chemistry is a prominent experimental method, when used in combination with structural modeling methods, it can lead to finding reliable structures [9]. This experimental technique is based on the addition of reagents that interact with the phosphate-sugar backbone on particularly flexible positions, inducing the formation of an adduct. Upon exposure to the reverse-transcriptase (RT), the adduct either causes the mutation of the nucleotide (SHAPE-MAP [9]), or causes the early detachment of the RT (SHAPE-CE [3], SHAPE-Seq [5]). A quantification of these effects allows to assign a Reactivity score to each position to refer to its ability to interact with the reagent. Unpaired nucleotides are predominantly reactive compared to the paired bases, the latter being constrained by their Hydrogen bonds. Hence, the reactivity of a nucleotide can be used to inform *ab initio* structure modeling, or to validate homology-based predictions.

SHAPE MAP reactivities In the context of SHAPE-Map, reactivities are computed by comparing three mutation rates observed in different experimental conditions: presence (Shape)/absence (Control) of SHAPE reagent, and in the absence of structure (Denatured). The reactivity of a position *n* is calculated as :

$$ \text{Reactivity}(n) = \frac{\text{mut}_{\text{Shape}}(n) - \text{mut}_{\text{Control}}(n)}{\text{mut}_{\text{Denatured}}(n)} $$

# 2 Material and methods

## 2.1 Dataset

The Ebola virus genome is 19 kb long, with seven open reading frames in the order 3' NP-VP35-VP40-GP-VP30-VP24-L 5', encoding for structural proteins, envelope glycoprotein, nucleoprotein, non structural proteins and viral polymerase. For each frame, we study consecutively the 5' and 3' non coding regions, this results into 14 sequences ranging from 3'NP to 5'L.

The Shape experiments are performed using 1M7 as reagent (weeks et al [8]). We also evaluated the differential SHAPE experiment that uses an additional reagent NMIA, in order to increase the accuracy of RNA structural models.

The experiments are followed by a sequencing and mapping process [8]. Each position in the RNA sequence is characterized by its occurrence from the throughput sequencing and thus by a mutation rate. ShapeMapper program(weeks et al [8]), after being adapted to Single End reads input, is subsequently called to calculate reactivity scores.

## 2.2 Modeling tools

We use SHAPE reactivities within an iterative modeling strategy based on a combination of predictive methods:
1. Free-Energy minimization. RNAfold predicts the most thermodynamically stable structure compatible with an RNA sequence [4]. It performs an exact optimization, using dynamic programming, of the free-energy within the Turner energy model (Zuker-Stiegler algorithm [10]).

2. Partition function analysis. RNAfold also allows analyzing the folding landscape at the ther-
modynamic equilibrium. An efficient dynamic-programming scheme (MCcaskill algorithm
[6]) produces the base-pairing probability matrix, at the Boltzmann equilibrium. Those prob-
abilities are used to provide a support for predicting base-pairs. Our current method is based
on finding the more reliable pairwises according to their probabilities, then use them as struc-
tural constraints to recalculate the energy values.

3. Comparative analysis. RNAalifold computes the consensus structure for a set of – previously aligned – homologous sequences. It optimizes a credibility score (Hofacker algorithm [1]), which primarily depends on compensatory mutations.

2.3 Main workflow

The present workflow relies on a conservative integrated approach where sets of structures are re-
trieved from different methods in order to detect similar substructures.
Methods 1, 2 are called to build a first set of structures.

The method 3 is used to reveal the most credible base pairs according to the internal scoring
scheme of RNAalifold. These base-pairs are then considered as constraints within a new run of
RNAfold, thus a second ensemble of structures is built.

Substructures that are found in the both datasets are kept and the pairing probabilities that are
higher than a certain threshold are introduced as structural constraints for the next run. This process
is performed iteratively until no additional bases is predicted. Contrasting with typical methods,
which include SHAPE data in the iterative modeling strategy, we chose to keep such a data for a final
validation step. In this last step, the compatibility between predicted single/double stranded regions
and SHAPE induced accessibilities is asessed. The resulting substructures are eminently supported
by the SHAPE reactivities, as shown in Figure 1.

3 Preliminary results and discussion

Our preliminary suggest the existence of conserved and stable hairpin loops in the UTR regions of
the Ebola genome are structured into hairpin-loop substructures stemming from the exterior region.
Moreover, we found little evidence for complex tree-like structures that are the landmark of struc-
tural ncRNAs. One such example is shown in Figure 1. The method proposed features the most
conserved stem-loop substructures. The first stem-loop of the 5'-UTR is highly reminiscent of typi-
cal structures found in viral genomes. By analogy, we hypothesize that the role of this substructure is
to protect the RNA from being degraded by nucleases. The SHAPE data is generally consistent with
our predictions, and indeed labels as accessible most single-stranded regions in predicted hairpins.
Conversely, helices are labeled as inaccessible.

We plan to further improve our modeling strategy by including Boltzmann sampling and structure-
based clustering instead of our current voting mechanisms. We also wish to include additional in-
formation, such as SHAPE experimental data using different reagents, possibly including probing on
different homologous sequences to establish a SHAPE-informed structural consensus.
<figure><img src="image_2.png" /><figcaption>Figure 1: Secondary structure inferred for the 5'UTR of the VP24 gene colored by SHAPE reactivity.</figcaption></figure>

## Acknowledgment

This work is supported by FRM Fondation de la Recherche Medicale. The authors would like to thank Steven Busan (Department of Chemistry, University of North Carolina), Alice Heliou (AMIB, INRIA saclay), Vladimir Reinharz (McGill University, Montreal) and Jules Desforges (Faculté de biologie et médecine, Lausanne) for their valuable feedback and support.

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