Uncovering RNA-Protein Interactions with RNA Antisense Purification and Mass Spectrometry (RAP-MS)

What is RAP-MS?

RNA molecules play a vital role in cellular functions, ranging from gene expression regulation to protein synthesis. Understanding the interactions between RNA and proteins is crucial to comprehend the intricate mechanisms of cellular biology. However, identifying RNA-protein interactions can be challenging, especially for long noncoding RNAs (lncRNAs) that do not encode proteins. RNA antisense purification coupled with mass spectrometry (RAP-MS) is a method that enables the identification of direct and specific protein interaction partners of a specific RNA molecule, including lncRNAs.

RAP-MS is an antisense-mediated affinity purification method that involves the cross-linking of RNA molecules with their interacting proteins in vivo. The RNA-protein complexes are then purified under highly denaturing conditions, followed by the identification of the interacting proteins using mass spectrometry. RAP-MS has several advantages over traditional affinity purification methods, such as cross-linking and immunoprecipitation (CLIP) and RNA immunoprecipitation (RIP). For instance, RAP-MS allows the identification of direct and specific RNA-protein interactions without the need for antibody validation or RNA overexpression.

Schematic of RAP-MS purification procedure from SILAC labeled mouse ES cells (McHugh et al., 2018).

Why RAP-MS a Powerful Tool for RNA-Protein Interaction Studies?

One of the significant advantages of RAP-MS is its ability to identify protein interaction partners of lncRNAs. LncRNAs are a newly discovered class of RNA molecules that are longer than 200 nucleotides and do not encode proteins. LncRNAs are known to interact with various proteins, and their dysregulation has been implicated in several diseases, including cancer. However, identifying the binding partners of lncRNAs has been challenging, primarily due to their low abundance and lack of protein-coding potential. RAP-MS can overcome these challenges and provide insights into the function of lncRNAs.

Applications of RAP-MS

RAP-MS has been used to study RNA-Protein interactions in various biological systems. One of the key applications of RAP-MS is the identification of RBPs that are involved in alternative splicing in various systems, including human cells, plants, and fruit flies. Alternative splicing is a process that generates multiple protein isoforms from a single gene by selectively splicing exons from the precursor messenger RNA (pre-mRNA). RAP-MS was used to identify RBPs that are involved in alternative splicing of the CD44 pre-mRNA in human cells. The study revealed that the RBPs hnRNP L and hnRNP LL are critical for regulating CD44 alternative splicing by binding to specific RNA sequences in the CD44 pre-mRNA.

RAP-MS can be used to identify RBPs involved in the RNA degradation pathway. the RNA degradation pathway is essential for maintaining RNA homeostasis and regulating gene expression. the RBPs promote or inhibit RNA degradation by binding to specific RNA sequences. RAP-MS can identify RBPs involved in the replication of various viruses, including hepatitis C virus (HCV), Zika virus (ZIKV), and human immunodeficiency virus (HIV ). In addition, RAP-MS can study RNA-protein interactions involved in disease mechanistic analysis, identify RBPs involved in the pathology of these diseases, and investigate the mechanisms by which RBPs contribute to disease pathology.

Example of interacting proteins captured by RAP-MS for 18S rRNA (McHugh et al., 2018).

At Creative Proteomics, we offer RAP-MS services to help researchers uncover the RNA-Protein interactions in their specific biological system of interest.

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