Decoding RNA–Protein Interactions: Methodological Advances and Emerging Challenges

RNA-protein interactions are fundamental to cellular processes such as gene regulation and RNA metabolism. Over the past decade, significant advancements in methodologies have transformed the ability to study these interactions with unprecedented resolution and specificity. This review systematically compares RNA- and protein-centric approaches, highlighting their strengths, limitations, and optimal applications. RNA-centric methods, including hybridization-based pulldowns, proximity labeling, and CRISPR-assisted techniques, enable the identification of proteins interacting with specific RNAs, even low-abundance or transient partners. Protein-centric strategies, such as immunoprecipitation-based CLIP-seq, and emerging proximity-tagging methods, map RNA interactomes of RNA-binding proteins with nucleotide precision. This study evaluates key innovations like LACE-seq and ARTR-seq, which minimize cell input requirements, and HyPro-MS, which bypasses genetic modifications. Guidelines for method selection are provided, emphasizing experimental goals, RNA abundance, interaction dynamics, and technical constraints. Critical challenges are also discussed, including capturing low-affinity interactions, resolving RNA structural complexities, and integrating multi-omics data. This review underscores the importance of method-tailoring to biological contexts, offering a roadmap for researchers to navigate the evolving landscape of RNA-protein interaction studies. By bridging technical advancements with practical recommendations, this study aims to accelerate discoveries in RNA biology, therapeutic development, and precision medicine.