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

<ns3:p> 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 <ns3:italic>HSCHARME</ns3:italic> (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 <ns3:italic>HSCHARME</ns3:italic> intron 1, which spans over 12 kb in length, and on understanding the structural determinants underlying this interaction. We predicted secondary structures along the <ns3:italic>pCHARME</ns3:italic> sequence by using different web-based prediction tools (such as Ipknot++, RNAFold, RNAStructure, Pkiss), using sliding windows of various sizes. </ns3:p> <ns3:p/> <ns3:p> 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 <ns3:italic>pCHARME</ns3:italic> 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. </ns3:p> <ns3:p/> <ns3:p> 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 <ns3:italic>pCHARME</ns3:italic> -MATR3 interaction in vitro. </ns3:p>