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Protein‑RNA interaction-Co-IP
1.1 RIP (RNA Immunoprecipitation)
1.1.1 Introduction
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.
1.1.2 Products
1.1.3 Applications
| Application | Description |
|---|---|
| Mapping the full spectrum of target RNAs for RNA‑binding proteins | Systematically identify the transcriptome‑wide target RNAs of an RNA‑binding protein using specific antibody immunoprecipitation combined with high‑throughput sequencing (RIP‑seq) to construct RBP‑RNA interaction networks. |
| Genome‑wide identification of miRNA target genes | Immunoprecipitate Argonaute protein to capture bound miRNAs and their target mRNAs, combined with sequencing to identify genome‑wide miRNA regulatory targets. |
| Functional mechanism analysis of lncRNAs/circRNAs | Identify non‑coding RNAs interacting with functional protein complexes via RIP to reveal regulatory mechanisms in gene silencing and chromatin remodeling. |
| Transcriptome‑wide mapping of RNA modifications (like m⁶A) | Use m⁶A‑specific antibodies for immunoprecipitation (meRIP‑seq/m⁶A‑seq) to map m⁶A modifications transcriptome‑wide and decipher epitranscriptomic regulatory mechanisms. |
| Studying disease‑related RBP‑RNA regulatory networks | Identify aberrant RBP‑RNA interactions in disease models via RIP‑seq to uncover pathogenic mechanisms and discover potential therapeutic targets. |
References:
- Zhao J, Ohsumi TK, Kung JT, Ogawa Y, Grau DJ, Sarma K, Song JJ, Kingston RE, Borowsky M, Lee JT. Genome‑wide identification of polycomb‑associated RNAs by RIP‑seq. Mol Cell. 2010 Dec 22;40(6):939‑53. doi: 10.1016/j.molcel.2010.12.011. PMID: 21172659; PMCID: PMC3021903.
- Chi SW, Zang JB, Mele A, Darnell RB. Argonaute HITS‑CLIP decodes microRNA‑mRNA interaction maps. Nature. 2009 Jul 23;460(7254):479‑86. doi: 10.1038/nature08170. Epub 2009 Jun 17. PMID: 19536157; PMCID: PMC2733940.
- Khalil AM, Guttman M, Huarte M, Garber M, Raj A, Rivea Morales D, Thomas K, Presser A, Bernstein BE, van Oudenaarden A, Regev A, Lander ES, Rinn JL. Many human large intergenic noncoding RNAs associate with chromatin‑modifying complexes and affect gene expression. Proc Natl Acad Sci USA. 2009 Jul 14;106(28):11667‑72. doi: 10.1073/pnas.0904715106. Epub 2009 Jul 1. PMID: 19571010; PMCID: PMC2704857.
- Dominissini D, Moshitch‑Moshkovitz S, Schwartz S, Salmon‑Divon M, Ungar L, Osenberg S, Cesarkas K, Jacob‑Hirsch J, Amariglio N, Kupiec M, Sorek R, Rechavi G. Topology of the human and mouse m6A RNA methylomes revealed by m6A‑seq. Nature. 2012 Apr 29;485(7397):201‑6. doi: 10.1038/nature11112. PMID: 22575960.
- Wang Y, Li Y, Toth JI, Petroski MD, Zhang Z, Zhao JC. N6‑methyladenosine modification destabilizes developmental regulators in embryonic stem cells. Nat Cell Biol. 2014 Feb;16(2):191‑8. doi: 10.1038/ncb2902. Epub 2014 Jan 7. PMID: 24394384; PMCID: PMC4640932.