RNA-Protein Interactions

RNA-binding proteins (RBPs) play critical roles in gene expression regulation through a diverse array of post-transcriptional mechanisms. From the moment an RNA is transcribed until its eventual degradation, RBPs physically associate with it to control every aspect of its life cycle: splicing, export, localization, translation, storage, and turnover. The mRNA coding sequence guides protein synthesis while untranslated regions (UTRs) and regulatory sequences modulate the fate of the encoded protein through translational control, subcellular positioning, and interaction with other macromolecules. Conversely, proteins bind and regulate RNA expression and function in a reciprocal relationship essential for maintaining cellular homeostasis.

Disruption of RNA-protein interactions has been implicated in a broad spectrum of human diseases, including neurodegeneration, cancer, metabolic disorders, and viral pathogenesis. Understanding these interactions at molecular and genomic scales is therefore a research priority with significant therapeutic implications. Profacgen offers a comprehensive platform for RNA-protein interaction analysis, providing both RNA-centric approaches (starting from an RNA of interest to discover its binding proteins) and protein-centric approaches (starting from a protein of interest to identify all RNAs it binds), spanning CLIP-based and non-CLIP methodologies.

Background: The RNA-Protein Interactome

The importance of RNA-protein interactions has been recognized since the 1960s, when ribosomes were first shown to be ribonucleoprotein complexes. However, the full scope of the RNA-binding protein repertoire remained obscure until the development of systematic, genome-wide methods in the 2010s. The landmark study by Castello et al. (2012) used quantitative mass spectrometry to identify over 800 proteins with RNA-binding activity in human cells—nearly double the number previously recognized—including many metabolic enzymes and proteins with no known RNA-related function. This "expanded RBPome" fundamentally changed our understanding of how extensively RNA and protein worlds intersect.

Today, RNA-protein interaction research is driven by two complementary conceptual frameworks. The RNA-centric approach starts with an RNA molecule of interest and asks: which proteins bind to it? Methods such as RNA pull-down, ChIRP, and RNA-centric CLIP variants are designed to answer this question. The protein-centric approach starts with an RBP and asks: which RNAs does it bind? CLIP-seq and its derivatives (iCLIP, eCLIP, miCLIP) are the primary tools for this question. By combining both approaches, researchers can build comprehensive interaction networks that reveal how post-transcriptional regulation shapes gene expression in health and disease.

A special dimension of RNA-protein interaction analysis concerns RNA modifications—particularly N⁶-methyladenosine (m⁶A), the most abundant internal modification on eukaryotic mRNA. m⁶A functions through a dynamic cycle of deposition (by the METTL3/METTL14 writer complex), recognition (by YTH-domain reader proteins), and removal (by FTO and ALKBH5 erasers). Mapping m⁶A sites at single-nucleotide resolution and identifying their associated reader proteins has become a major frontier in epigenetics research. Profacgen's miCLIP-seq platform addresses this need directly.

Figure 1. Schematic representation of experimental methods for the identification of protein–RNA interactions. (Left) Protein-centric approaches; (right) RNA-centric approaches. (Marchese et al., 2016)

Our RNA-Protein Interaction Services

Profacgen provides a comprehensive portfolio of CLIP-based and non-CLIP methods for RNA-protein interaction analysis:

Common Principles of CLIP-Based Methods

All CLIP-family methods share a core experimental workflow that distinguishes them from non-crosslinking approaches:

• In vivo UV crosslinking: Living cells are irradiated with 254 nm UV light, creating covalent bonds between proteins and RNA molecules at direct contact sites. This eliminates non-specific associations that can arise during cell lysis.
• Stringent purification: Crosslinked protein-RNA complexes are solubilized, partially digested with RNase, and immunoprecipitated with an antibody specific to the target RBP. SDS-PAGE separation and nitrocellulose membrane transfer remove unbound RNA and protein contaminants.
• Radioactive labeling and visualization: Protein-RNA complexes are visualized by autoradiography after ³²P-labeling, enabling precise excision of the correct molecular weight species from the membrane.
• RNA isolation and sequencing library preparation: Proteinase K digestion releases RNA fragments with peptide remnants at crosslink sites. Linker ligation, reverse transcription, and PCR amplification generate sequencing libraries compatible with Illumina platforms.
• Bioinformatic analysis: Sequencing reads are mapped to the transcriptome, and peak calling algorithms identify statistically significant binding sites or modification positions.

Applications

• RNA-protein interaction mapping: Genome-wide identification of the RNA targets bound by a specific RBP, revealing its regulatory network and functional scope
• Post-transcriptional regulation: Determine how RBPs control mRNA splicing, stability, localization, and translation through direct binding to regulatory elements
• RNA modification mapping: Profile the distribution of m⁶A and other epitranscriptomic marks across the transcriptome at single-nucleotide resolution
• Non-coding RNA function: Identify the protein partners of lncRNAs, circRNAs, and miRNAs to elucidate their molecular mechanisms of action
• Disease mechanism research: Discover how mutations in RBPs or their RNA targets disrupt normal post-transcriptional regulation in disease
• Viral RNA biology: Map how viral RNAs hijack host RBPs or how viral RBPs manipulate host RNA metabolism

Why Choose Profacgen?

Profacgen has an experienced team specializing in RNA–protein interaction research. We are committed to providing high-quality technical support and customized solutions for researchers worldwide. We welcome opportunities to collaborate with academic laboratories and industry partners. Contact us now!