Multiple Oligo assisted RNA Pulldown via Hybridization followed by Mass Spectrometry (MORPH-MS) for exploring the RNA-Protein interactions

ABSTRACT

Understanding RNA-protein interactions is crucial for deciphering the cellular functions and molecular mechanisms of regulatory RNAs. Consequently, there is a constant need to develop innovative and cost-effective methods to uncover such interactions. We developed a simple and cost-effective technique called Multiple Oligo assisted RNA Pulldown via Hybridization (MORPH) to identify proteins interacting with a specific RNA. MORPH employs a tiling array of antisense oligos (ASOs) to efficiently capture the RNA of interest along with proteins associated with it. Unlike existing techniques that rely on multiple individually biotinylated oligos spanning the entire RNA length, MORPH stands out by utilizing a single biotinylated oligo to capture all the ASOs. To evaluate MORPH’s efficacy, we applied this technique combined with mass spectrometry to identify proteins interacting with lncRNA NEAT1, which has previously been studied using various methods. Our results demonstrate that despite being a simple and inexpensive procedure, MORPH performs on par with existing methods.

Abbreviations: ASO, Antisense oligo; lncRNA, long non-coding RNA; MORPH, Multiple Oligo assisted RNA Pulldown via Hybridization

Graphical abstract

KEYWORDS:

• RNA-pulldown
• long non-coding RNA
• RNA-protein interaction
• ASO pulldown
• affinity purification-mass spectrometry (AP-MS)

Acknowledgments

This study was supported by DBT/Wellcome Trust India Alliance (India Alliance) fellowship grant to RK (fellowship No. IA/I/16/1/502357). PP received fellowship from University Grants Commission (UGC), India. We thank Proteomics facility at CSIR-Centre for Cellular and Molecular Biology for their technical assistance. We thank Shreyas Sakharwade for his assistance with cell culture.

Disclosure statement

No potential conflict of interest was reported by the authors.

Authorship contribution statement

Priyanka Pant: Conceptualization, Investigation, Methodology, Formal analysis, Visualization, Validation, Writing – original draft, Regalla Kumarswamy: Funding aquisition, Validation, Writing – review & editing, Visualization, Supervision, Resources, Project administration.

Data availability statement

Processed mass spectrometry data are included as supporting files. Raw data were generated at the CSIR-Centre for Cellular and Molecular Biology. Raw data supporting the findings of this study are available from the corresponding author RK on request.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/15476286.2023.2287302.

Additional information

Funding

This study was supported by DBT/Wellcome Trust India Alliance (India Alliance) fellowship grant to Kumarswamy Regalla [fellowship No. IA/I/16/1/502357]. Priyanka Pant received fellowship from University Grants Commission (UGC), India.