Overview
Protein-RNA interactions are central mechanisms of gene expression regulation, governing RNA processing, transport, translation, and degradation. RNA-binding proteins control mRNA fate by recognizing specific sequences or structures, while non-coding RNAs dynamically engage with proteins in chromatin remodeling and signaling; dysregulation of these interactions is implicated in cancer, neurodegeneration, and viral infection, representing a critical frontier in functional genomics. The principal techniques for studying PRIs are RNA pull-down and RIP: RNA pull-down utilizes in vitro synthesized labeled RNA (e.g., biotinylated) as bait to capture interacting proteins from cell lysates for identification by mass spectrometry or Western blot, making it ideal for screening proteins bound to a specific RNA; RIP employs specific antibodies to immunoprecipitate a target protein and detects associated RNAs (by RT-qPCR or RIP-seq), reflecting endogenous physiological interactions within cells. These two approaches complement each other from “RNA-to-protein” and “protein-to-RNA” perspectives, collectively mapping the functional landscape of protein-RNA interaction networks.
Subcategories
RNA Pull-downRNA Pull-downRNA pull-down is an in vitro affinity-based technique leveraging the biotin-streptavidin interaction system for the efficient enrichment and identification of RNA-binding proteins (RBPs). A biotin-labeled RNA probe is generated by in vitro transcription and subsequently incubated with whole-cell protein extracts, enabling the formation of sequence-specific RNA-protein complexes. These complexes are then captured by streptavidin-coated magnetic beads and magnetically separated from unbound components in the incubation mixture. Following stringent washes to eliminate non-specifically adsorbed contaminants, the target proteins are recovered via competitive elution (using free biotin) or denaturing elution (for downstream proteomic analysis). The eluted proteins are ultimately identified by Western blot (for validation of known RBPs) or liquid chromatography-tandem mass spectrometry (for discovery of novel interacting proteins), thereby enabling systematic profiling of the protein repertoire associated with the RNA of interest.
>> View productsRIP (RNA Immunoprecipitation)RIP (RNA Immunoprecipitation)RNA Immunoprecipitation (RIP) is a core molecular biology technique for studying intracellular RNA-protein interactions, serving as a powerful tool for deciphering the dynamic processes of post-transcriptional regulatory networks and revealing the functional roles of RNA-binding proteins (RBPs). RIP leverages the antigen-antibody specific recognition principle to capture and identify RNA bound by target proteins. Cells are lysed under mild conditions (containing RNase inhibitors) to maintain the native binding state of RNA-protein complexes. A specific antibody against the target RNA-binding protein or protein of interest is added to bind the protein-RNA complex; Protein A/G magnetic beads are then used to capture the antibody-antigen-RNA complex. Multiple wash steps remove non-specifically bound RNA and proteins. Proteinase K digestion of the protein components to release the RNA bound to the target protein. DNase I treatment is performed to eliminate genomic DNA contamination, followed by extraction and purification of the enriched RNA fragments. The recovered RNA is then subjected to RT-qPCR analysis for quantitative validation of candidate target RNAs, or to RIP-seq (high-throughput RNA sequencing) for systematic identification of the complete RNA repertoire bound to the target protein at the whole-transcriptome level.
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