RNA Secondary Structure Modeling Following the IPANEMAP Workflow.

The structure of RNA molecules and their complexes are crucial for understanding biology at the molecular level. Resolving these structures holds the key to understanding their manifold structure-mediated functions ranging from regulating gene expression to catalyzing biochemical processes. Predicting RNA secondary structure is a prerequisite and a key step to accurately model their three dimensional structure. Although dedicated modelling software are making fast and significant progresses, predicting an accurate secondary structure from the sequence remains a challenge. Their performance can be significantly improved by the incorporation of experimental RNA structure probing data. Many different chemical and enzymatic probes have been developed; however, only one set of quantitative data can be incorporated as constraints for computer-assisted modelling. IPANEMAP is a recent workflow based on RNAfold that can take into account several quantitative or qualitative data sets to model RNA secondary structure. This chapter details the methods for popular chemical probing (DMS, CMCT, SHAPE-CE, and SHAPE-Map) and the subsequent analysis and structure prediction using IPANEMAP.

Progress toward SHAPE Constrained Computational Prediction of Tertiary Interactions in RNA Structure.

As more sequencing data accumulate and novel puzzling genetic regulations are discovered, the need for accurate automated modeling of RNA structure increases. RNA structure modeling from chemical probing experiments has made tremendous progress, however accurately predicting large RNA structures is still challenging for several reasons: RNA are inherently flexible and often adopt many energetically similar structures, which are not reliably distinguished by the available, incomplete thermodynamic model. Moreover, computationally, the problem is aggravated by the relevance of pseudoknots and non-canonical base pairs, which are hardly predicted efficiently. To identify nucleotides involved in pseudoknots and non-canonical interactions, we scrutinized the SHAPE reactivity of each nucleotide of the 188 nt long lariat-capping ribozyme under multiple conditions. Reactivities analyzed in the light of the X-ray structure were shown to report accurately the nucleotide status. Those that seemed paradoxical were rationalized by the nucleotide behavior along molecular dynamic simulations. We show that valuable information on intricate interactions can be deduced from probing with different reagents, and in the presence or absence of Mg2+. Furthermore, probing at increasing temperature was remarkably efficient at pointing to non-canonical interactions and pseudoknot pairings. The possibilities of following such strategies to inform structure modeling software are discussed.

RNA Footprinting Using Small Chemical Reagents.

RNA is a pivotal element of the cell which is most of the time found in complex with protein(s) in a cellular environment. RNA can adopt three-dimensional structures that may form specific binding sites not only for proteins but for all sorts of molecules. Since the early days of molecular biology, strategies to probe RNA structure have been developed. Such probes are small molecules or RNases that most of the time specifically react with single strand nucleotides. The precise reaction or cleavage site can be mapped by reverse transcription. It appears that nucleotides in close contact or in proximity of a ligand are no longer reactive to these probes. Carrying the RNA probing experiment in parallel in presence and absence of a ligand yield differences that are known as the ligand "footprint." Such footprints allow for the identification of the precise site of the ligand interaction, but also reveals RNA structural rearrangement upon ligand binding. Here we provide an experimental and analytical workflow to carry RNA footprinting experiments.

HIV-1 gRNA, a biological substrate, uncovers the potency of DDX3X biochemical activity.

DEAD-box helicases play central roles in the metabolism of many RNAs and ribonucleoproteins by assisting their synthesis, folding, function and even their degradation or disassembly. They have been implicated in various phenomena, and it is often difficult to rationalize their molecular roles from in vivo studies. Once purified in vitro, most of them only exhibit a marginal activity and poor specificity. The current model is that they gain specificity and activity through interaction of their intrinsically disordered domains with specific RNA or proteins. DDX3 is a DEAD-box cellular helicase that has been involved in several steps of the HIV viral cycle, including transcription, RNA export to the cytoplasm and translation. In this study, we investigated DDX3 biochemical properties in the context of a biological substrate. DDX3 was overexpressed, purified and its enzymatic activities as well as its RNA binding properties were characterized using both model substrates and a biological substrate, HIV-1 gRNA. Biochemical characterization of DDX3 in the context of a biological substrate identifies HIV-1 gRNA as a rare example of specific substrate and unravels the extent of DDX3 ATPase activity. Analysis of DDX3 binding capacity indicates an unexpected dissociation between its binding capacity and its biochemical activity. We further demonstrate that interaction of DDX3 with HIV-1 gRNA relies both on specific RNA determinants and on the disordered N- and C-terminal regions of the protein. These findings shed a new light regarding the potentiality of DDX3 biochemical activity supporting its multiple cellular functions.