RNA in situ conformation sequencing reveals the effects of HNRNPD on RNA-RNA interactions in primary colorectal cancer cells
To elucidate the role of HNRNPD (AUF1) in orchestrating RNA spatial organisation and intermolecular interactions, we employed RNA in situ conformation sequencing (RIC-seq), a proximity-ligation-based technology that captures genome-wide RNA-RNA contacts and higher-order structural conformations. Using primary tumour cells derived from colorectal cancer patients, we generated two perturbed conditions: stable overexpression of HNRNPD via a eukaryotic expression vector, and targeted knockout using specific sgRNAs. Following these manipulations, we applied RIC-seq to preserve and sequence RNA duplexes and multi-way junctions that occur in their natural intracellular context. By comparing the interaction networks, contact frequencies, and structural rearrangements among RNA across the three groups, we aim to determine whether HNRNPD acts as a global or transcript-specific architectural factor-facilitating or disrupting RNA-RNA interactions, and altering RNA folding landscapes. This approach will not only reveal how HNRNPD dictate the three-dimensional RNA interactome in patient-derived cancer cells, but also provide mechanistic insights into its post-transcriptional regulatory functions. Ultimately, these findings may uncover novel HNRNPD-dependent RNA structural networks that contribute to colorectal cancer pathogenesis and could be exploited for RNA-targeted therapeutic strategies.