RNA2Fun is a research project funded by the European Union - Next-GenerationEU through the Italian National Recovery and Resilience Plan (NRRP) - PRIN 2022 PNRR.
RNA2Fun focuses on an innovative usage of RNA-related knowledge, which integrates advanced bioinformatic tools and biological state-of-the-art wet lab techniques for developing new strategies to predict potential RNA-mediated circuitries.
The long-term goal of the study is to precisely address strategies for targeting pathological aggregation processes caused by aberrant protein-RNA interactions and associated with several human diseases, including neurodegenerative disorders.
On this line, RNA2Fun will develop and test, biologically and computationally, the defined new strategies on the activity of pCharme, a lncRNA involved in the differentiation of muscle cells by forming nuclear condensates with the multifunctional RNA/DNA binding protein MATRIN3 (MATR3).
By applying state-of-the-art CRISPR-Cas9 methodologies, we will produce pCharme deletion mutants through single-guide RNAs designed to target the predicted pCharme 2NDSs. By this strategy, we will be able to test in vivo the need of predicted 2NDSs to the lncRNA function, as proof-of-principle for future applications to other RNAs.
Structure of the Project
The WP1 focuses on the creation of a suited RNA dataset containing some hundreds of experimentally validated ncRNA primary sequences and secondary structures of different types, associated with information about role/function and interaction with other molecules. Construction of different representations of the secondary structures at different levels of abstraction and computation of relevant features is performed. Particular attention will be given to the inclusion of molecules that have interactions with MATR3, and to the inclusion of domains of pCHARME.
The WP2 focuses on the creation of methodologies for assessing the quality of predicted secondary structures by the selection of folding methods, including pseudoknots, and the prediction of some tens of ncRNAs, selected from different families included in the dataset. Moreover, comparison methods, including pseudoknots, and evaluation of similarity of the predicted structures with the validated ones is considered. An analysis of relationships between primary and secondary structures in selected types of ncRNAs in the dataset is performed.
The WP3 focuses on the prediction and analysis of RNA secondary structures of lncRNAs with attention to their protein binding properties. Tasks include comparative genomics of the lncRNA pCharme across organisms to identify conserved secondary structures preserving function (protein binding) to transfer information from in-vivo mouse data to in-silico and in-vitro human case; selection of intronic portions of pCharme using different temporal techniques for the prediction of candidates associated secondary structures; analysis of the structural properties based on the dataset; quantitative evaluation of deletions to generate the array of pCharme-DEL hiPSC clones; analysis of predicted pCharme domain.