Identification and Verification of Novel DANCR-Binding Proteins Using Limited Proteolysis Mass Spectrometry

• Elissa Kouemeni, Applied Molecular Biology & Biotechnology, University of Delaware

• Mona Batish, Medical Molecular Sciences, University of Delaware

RNA-binding proteins (RBPs) regulate essential cellular processes, including RNA splicing, translation, stability, and decay. Traditional methods for identifying RNA–protein interactions often rely on affinity pull-downs or chemical labeling. However, these approaches frequently miss low-abundance RBPs or small RNAs with few binding sites, and they typically require genetic modifications that are challenging to perform in primary cells. To overcome these limitations, we adapted a label-free, pull-down-free strategy combining limited proteolysis (LiP) with stability-based mass spectrometry to detect and validate dynamic RNA–protein interactions. Cell lysates were incubated with in vitro-transcribed RNA, followed by controlled enzymatic digestion with trypsin to yield small peptides. The resulting digested proteins were filtered and then analyzed via mass spectrometry. To test this approach, we used the long non-coding RNA (lncRNA) DANCR as the target RNA and identified 2,172 RBP candidates, comprising both known and novel interactors. We validated these findings using RNA immunoprecipitation (RIP) paired with PCR to confirm RNA enrichment, Western blotting to confirm target protein expression, and single-molecule fluorescence in situ hybridization combined with immunofluorescence (smFISH/IF) to visualize spatial interactions. Specifically, we evaluated DANCR’s interactions with its known interactors (EEF2 and EZRIN) alongside two newly identified RBPs (HNRNPD and EIF4A3). Both novel candidates demonstrated significant RNA enrichment, and smFISH/IF confirmed their spatial colocalization. Together, these findings confirm that our platform reliably identifies RBP–RNA interactions in unmodified primary cells, offering a promising tool for biomarker and drug target discovery across diverse clinical settings.