IPANEMAP: integrative probing analysis of nucleic acids empowered by multiple accessibility profiles.

The manual production of reliable RNA structure models from chemical probing experiments benefits from the integration of information derived from multiple protocols and reagents. However, the interpretation of multiple probing profiles remains a complex task, hindering the quality and reproducibility of modeling efforts. We introduce IPANEMAP, the first automated method for the modeling of RNA structure from multiple probing reactivity profiles. Input profiles can result from experiments based on diverse protocols, reagents, or collection of variants, and are jointly analyzed to predict the dominant conformations of an RNA. IPANEMAP combines sampling, clustering and multi-optimization, to produce secondary structure models that are both stable and well-supported by experimental evidences. The analysis of multiple reactivity profiles, both publicly available and produced in our study, demonstrates the good performances of IPANEMAP, even in a mono probing setting. It confirms the potential of integrating multiple sources of probing data, informing the design of informative probing assays.

Two ribosome recruitment sites direct multiple translation events within HIV1 Gag open reading frame.

In the late phase of the HIV virus cycle, the unspliced genomic RNA is exported to the cytoplasm for the necessary translation of the Gag and Gag-pol polyproteins. Three distinct translation initiation mechanisms ensuring Gag production have been described with little rationale for their multiplicity. The Gag-IRES has the singularity to be located within Gag ORF and to directly interact with ribosomal 40S. Aiming at elucidating the specificity and the relevance of this interaction, we probed HIV-1 Gag-IRES structure and developed an innovative integrative modelling strategy to take into account all the gathered information. We propose a novel Gag-IRES secondary structure strongly supported by all experimental data. We further demonstrate the presence of two regions within Gag-IRES that independently and directly interact with the ribosome. Importantly, these binding sites are functionally relevant to Gag translation both in vitro and ex vivo. This work provides insight into the Gag-IRES molecular mechanism and gives compelling evidence for its physiological importance. It allows us to propose original hypotheses about the IRES physiological role and conservation among primate lentiviruses.

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.