Direct lncRNA Interactome Mapping: Amplification-Based Proteomics

• Jadira Fuentes Bautista, Applied Molecular Biology & Biotechnology, University of Delaware

• Mona Batish, Medical & Molecular Sciences, University of Delaware

Direct lncRNA Interactome Mapping: Amplification-Based Proteomics

Jadira Fuentes Bautista, Brigette Romero, Mona Batish

Department of Medical & Molecular Sciences, College of Health Sciences

University of Delaware, Newark, DE

Once considered transcriptional noise, research has now shown that long non-coding RNAs (lncRNAs) act as key regulators in both nuclear and cytoplasmic processes. To perform these diverse functions, lncRNAs often interact with RNA-binding proteins (RBPs), forming ribonucleoprotein complexes (RNPs) that regulate processes like transcription, translation, RNA stability, and nuclear organization.

A conventional method to identify these proteins is the RNA pull-down assay. This technique uses biotinylated RNA probes as bait to capture interacting proteins for subsequent analysis. However, this approach has several limitations, including the detection of only high-affinity interactions, missing potentially important weaker interactions. Other drawbacks include RNA degradation and non-specific protein binding to the matrix used in the assay.

To overcome some of these challenges, we modified a recently developed pull-down-free method involving limited-proteolysis (LiP) and stability-based mass spectrometry. This technique detects RNA-protein interactions by taking advantage of the principle that ligand binding can alter proteolytic susceptibility. We replaced the ligand in the existing method with in vitro transcribed lncRNA. After incubating the cell lysate with the in vitro transcribed lncRNA, a large amount of trypsin is used to generate small peptides. The partially digested proteins (or the RNPs) were then separated from the peptides and analyzed by shotgun proteomics. The lncRNA binding regions and their corresponding binding proteins can be determined by comparing the peptide abundance in lysates incubated with and without lncRNA. Once fully optimized, this protocol has the potential to open new avenues for the label-free identification of RNA-protein interactions.