A method for in situ visualization of Protein-Nascent RNA interactions in single cell using Proximity Ligation Assay (IPNR-PLA) in mammalian cells

ABSTRACTProximity ligation assay (PLA) is an immunofluorescence assay, which determines in situ interaction of two biomolecules present within 40 nm close proximity. Here, we describe a modification of PLA for visual detection of in situ protein interactions with nascent RNA in a single cell (IPNR-PLA). In IPNR-PLA, nascent RNA is labeled by incorporating 5-fluorouridine (FU), a uridine nucleotide analogue, followed by covalent cross-linking of the interacting partners in proximity to newly synthesized RNA. By using combination of anti-BrdU antibody, which specifically binds to FU, and primary antibody against a protein of interest, the IPNR reaction results in fluorescent puncta as a positive signal, only if the candidate proteins are in proximity to nascent RNA. We have validated this method by demonstrating known CDK9 and elongating RNA pol II interaction with nascent RNA. Finally, we used this method to test for the presence of DNA double strand breaks as well as Poly (ADP-ribose) polymerase 1 (PARP1), an RNA binding protein, in the vicinity of nascent RNA in cancer cells. The capability of performing parallel IF labeling and quantifiable multiparameter measurements within heterogeneous cell populations makes IPNR-PLA very attractive for use in biological studies. Overall, we have developed the IPNR-PLA method for analysis of protein association with nascent RNA with single-cell resolution, which is highly sensitive, quantitative, efficient, and requires little starting experimental material.KEYWORDS: Proteininteractionnascent RNAtranscriptiontranscription factorproximity ligation assay AcknowledgmentsWe acknowledge financial support from Bhabha Atomic Research Centre, Department of Atomic Energy, Mumbai, India. We are thankful to Dr. Tapan K Ghanty, Dr. Hari S Misra and Dr. Hema Rajaram for their support during this study. We are also thankful to Ms. Anita Prajapati for assistance with confocal imaging.Disclosure statementNo potential conflict of interest was reported by the authors.Author contributionsRD performed experiments, analyzed data, AD performed experiments, analyzed data, and wrote the manuscript, SU conceived the study, analyzed data, guided experiments and wrote the manuscript. All the authors approved the final version.Additional informationFundingThe work was supported by the Bhabha Atomic Research Centre.