Bridging between in silico prediction and in vitro functional characterisation of RNA secondary structures

Tissue-specific long non-coding RNAs (lncRNAs) play pivotal roles in cellular physiology and differentiation, although the mechanisms of their action remain largely elusive. In particular, their ability to interact with RNA-binding proteins (RBPs) enables them to coordinate crucial cellular functions. We are currently investigating the human lncRNA HSCHARME (Buonaiuto et al., 2025), which appears to play a critical role during human myogenesis. In particular, we are focusing on disentangling MATR3 binding along the HSCHARME intron 1, which spans over 12 kb in length, and on understanding the structural determinants underlying this interaction. We predicted secondary structures along the pCHARME sequence by using different web-based prediction tools (such as Ipknot++, RNAFold, RNAStructure, Pkiss), using sliding windows of various sizes. Focusing, then, on the primary sequence between nucleotide 3874 and 7914 of the intron (which contains a big proportion of the consensus motifs for MATR3 binding), we obtained a consensus secondary structure from the predictions for pCHARME intron 1 using a Python script. After mapping all the motifs for MATR3 binding, we predicted a 3D structure of these regions to perform docking simulations. Meanwhile, we are moving in vitro to remove, through CRISPR/Cas9 technology, pCharme intron1 and analyse how this affects its binding to MATR3 and its role in myogenesis. Then, we plan to refine the analysis by selectively deleting the above-identified region of the intron using CRISPR/Cas9 and subsequently confirm its role in directing the pCHARME-MATR3 interaction in vitro.