RNA secondary structure modelling following the IPANEMAP workflow
| Autor: | Allouche, Delphine, De Bisschop, Grégoire, Saaidi, Afaf, Ponty, Yann, Bruno, Sargueil |
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| Přispěvatelé: | Laboratoire de cristallographie et RMN biologiques (LCRB - UMR 8015), Université Paris Descartes - Paris 5 (UPD5)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Cibles Thérapeutiques et conception de médicaments (CiTCoM - UMR 8038), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5), Laboratoire d'informatique de l'École polytechnique [Palaiseau] (LIX), École polytechnique (X)-Centre National de la Recherche Scientifique (CNRS), Algorithms and Models for Integrative BIOlogy (AMIBIO), École polytechnique (X)-Centre National de la Recherche Scientifique (CNRS)-École polytechnique (X)-Centre National de la Recherche Scientifique (CNRS), Ponty, Yann, Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5), Université Paris Descartes - Paris 5 (UPD5)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-École polytechnique (X), Inria Saclay - Ile de France, Institut National de Recherche en Informatique et en Automatique (Inria)-Institut National de Recherche en Informatique et en Automatique (Inria) |
| Jazyk: | angličtina |
| Rok vydání: | 2019 |
| Předmět: | |
| Zdroj: | RNA Folding-Methods and Protocols RNA Folding-Methods and Protocols, Springer Nature, In press, Methods in Molecular Biology |
| Popis: | International audience; RNA secondary structure modelling has been a challenge since the early days of molecular biology. Although algorithms for RNA structure modelling are more and more efficient and accurate, they significantly benefit from the integration of experimental structure probing data. RNA structure probing consists in submitting an RNA to enzymes or small molecules that specifically react with individual nucleotides according to their pairing status. Most enzymes used are single strand specific RNAses (RNAses T1, U2, nuclease S1 …) with the notable exception of the double strand specific RNAse V1. Although they are low molecular weight proteins, they are too bulky to access some nucleotides of a folded RNA. Small molecules can essentially reach any nucleotide and most of them are also single-strand specific although psoralen has recently been successfully used a double strand probe (Lu et al., 2016). For the longest time, RNA probing experiments remained tedious and rather qualitative than quantitative. RNA structure probing recently reached the medium, and then high, throughput. Pioneered and mostly developed within the Weeks lab, the SHAPE technology uses small molecules that react with flexible ribose, thus essentially reporting single-stranded nucleotides with some subtleties (Frezza et al., 2019; Steen et al., 2012). A medium throughput version of the SHAPE protocol was first developed based on capillary electrophoresis, later to be transformed into a high throughput method using next generation sequencing. The same workflows can be applied to more traditional probes such as DiMethyl Sulfate (DMS) and N-Cyclohexyl-N′-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (CMCT) that reveal unpaired A,C and G,U respectively. It appeared that different probes provide complementary information that further improves RNA structure prediction. We therefore developed IPANEMAP, an experimental and computational workflow that models RNA secondary structure from different sets of RNA structure probing performed with different probes, and/or in different conditions and/or on mutants (Saaidi et al. Submitted). This workflow relies on medium or high throughput structure probing, and combines statistical sampling, clustering (Ding and Lawrence, 2003) and pseudo-potentials (Deigan et al, 2009). The method was shown to produce more accurate and stable predictions than other workflows developed to date, even when a single reactivity profile is available, while the availability of multiple reactivities was shown to increase robustness and, to a lesser extent, accuracy of the modeling (Saaidi et al. Submitted). Below, we detail a whole IPANEMAP workflow, starting with experimental probing with DMS and/or CMCT and/or SHAPE reagent. Such probing can be carried out in various relevant conditions (varying température, Mg2+ concentration, introducing point mutations in the RNA to be modeled etc) (Saaidi et al. Submitted). Two versions of the experimental procedure (medium throughput and high throughput) are proposed, DMS and CMCT probing were adapted from Ehresmann et al. and Brunel et al. while the SHAPE probing is described in K. Weeks team publications (Karabiber et al., 2013; Low and Weeks, 2010a; Mortimer and Weeks, 2007; Smola et al., 2015a; Wilkinson et al., 2006, 2008). We then detail instructions for executing the IPANEMAP algorithm to obtain the RNA secondary structure model. |
| Databáze: | OpenAIRE |
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