Abstract
RNA–protein proximity ligation assay (RNA-PLA) enables the detection of specific RNA–protein interactions in fixed cells. In RNA-PLA, bridging and ligation of a circular DNA template occurs if the target RNA and protein are within 40 nanometers of each other. The resulting circular template is amplified by rolling circle amplification and abundantly recognized by fluorescent antisense DNA oligonucleotides. This strategy therefore enables localization of RNA–protein interactions in situ with high specificity and sensitivity. Here, we describe the use of RNA-PLA to detect interactions between a nuclear viral RNA and a host RNA-binding protein in Epstein-Barr virus (EBV)-infected B cells.
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References
Söderberg O, Gullberg M, Jarvius M et al (2006) Direct observation of individual endogenous protein complexes in situ by proximity ligation. Nat Methods 3:995–1000
Zhang W, Xie M, Shu M-D et al (2016) A proximity-dependent assay for specific RNA-protein interactions in intact cells. RNA 22:1785–1792
Xie M, Zhang W, Shu M-D et al (2015) The host integrator complex acts in transcription-independent maturation of herpesvirus microRNA 3′ ends. Genes Dev 29:1552–1564
Brown JA, Kinzig CG, DeGregorio SJ et al (2016) Methyltransferase-like protein 16 binds the 3′-terminal triple helix of MALAT1 long noncoding RNA. Proc Natl Acad Sci 113:14013–14018
Lerner MR, Andrews NC, Miller G et al (1981) Two small RNAs encoded by Epstein-Barr virus and complexed with protein are precipitated by antibodies from patients with systemic lupus erythematosus. Proc Natl Acad Sci U S A 78:805–809
Glickman JN, Howe JG, Steitz JA (1988) Structural analyses of EBER1 and EBER2 ribonucleoprotein particles present in Epstein-Barr virus-infected cells. J Virol 62:902–911
Riley KJ, Rabinowitz GS, Yario TA et al (2012) EBV and human microRNAs co-target oncogenic and apoptotic viral and human genes during latency. EMBO J 31:2207–2221
Navin N, Grubor V, Hicks J et al (2006) PROBER: oligonucleotide FISH probe design software. Bioinformatics 22:2437–2438
Lee N, Moss WN, Yario TA et al (2015) EBV noncoding RNA binds nascent RNA to drive host PAX5 to viral DNA. Cell 160:607–618
Lipovsky A, Zhang W, Iwasaki A et al (2015) Chapter 30: Application of the proximity-dependent assay and fluorescence imaging approaches to study viral entry pathways. Methods Mol Biol 1270:437
Allalou A, Wählby C (2009) BlobFinder, a tool for fluorescence microscopy image cytometry. Comput Methods Prog Biomed 94:58–65
Acknowledgments
We would like to thank members of the Xie laboratory for helpful discussion. Initial data in this manuscript were acquired in the laboratory of Daniel DiMaio, and in the laboratory of Joan Steitz at Yale University, with the support of a grant from the National Institute of Health (P01-CA16038). The Xie lab is supported by the National Institute of Health (R35-GM128753 to M.X.).
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Li, T., Zhang, W., Xie, M. (2023). Fluorescent In Situ Detection of RNA–Protein Interactions in Intact Cells by RNA-PLA. In: Lin, RJ. (eds) RNA-Protein Complexes and Interactions. Methods in Molecular Biology, vol 2666. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3191-1_13
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DOI: https://doi.org/10.1007/978-1-0716-3191-1_13
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Publisher Name: Humana, New York, NY
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Online ISBN: 978-1-0716-3191-1
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