Abstract
Long noncoding RNAs (lncRNAs) have important roles in shaping chromatin by targeting chromatin-modifying enzymes to distinct genomic sites. This section covers two methods to analyze lncRNA-protein interactions. The RNA-protein pull-down assays use either bead-bound proteins to capture in vitro transcripts, or immobilized synthetic RNAs to bind proteins from cell lysates. In the RNA immunoprecipitation (RIP) assay, endogenous RNAs are co-immunoprecipitated with a protein of interest. Both the methods can be applied to material from proliferating and quiescent cells, thus providing insights into how lncRNA-protein interactions are altered between these two cellular states.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Guttman M, Rinn JL (2012) Modular regulatory principles of large non-coding RNAs. Nature 482(7385):339–346. doi:10.1038/nature10887
Rinn JL, Chang HY (2012) Genome regulation by long noncoding RNAs. Annu Rev Biochem 81:145–166. doi:10.1146/annurev-biochem-051410-092902
Quinodoz S, Guttman M (2014) Long noncoding RNAs: an emerging link between gene regulation and nuclear organization. Trends Cell Biol 24(11):651–663. doi:10.1016/j.tcb.2014.08.009
Roberts TC, Morris KV, Weinberg MS (2014) Perspectives on the mechanism of transcriptional regulation by long non-coding RNAs. Epigenetics 9(1):13–20. doi:10.4161/epi.26700
Bierhoff H, Schmitz K, Maass F, Ye J, Grummt I (2010) Noncoding transcripts in sense and antisense orientation regulate the epigenetic state of ribosomal RNA genes. Cold Spring Harb Symp Quant Biol 75:357–364. doi:10.1101/sqb.2010.75.060
Bierhoff H, Dammert MA, Brocks D, Dambacher S, Schotta G, Grummt I (2014) Quiescence-induced LncRNAs trigger H4K20 trimethylation and transcriptional silencing. Mol Cell 54(4):675–682. doi:10.1016/j.molcel.2014.03.032
Hammerle M, Gutschner T, Uckelmann H, Ozgur S, Fiskin E, Gross M, Skawran B, Geffers R, Longerich T, Breuhahn K, Schirmacher P, Stoecklin G, Diederichs S (2013) Posttranscriptional destabilization of the liver-specific long noncoding RNA HULC by the IGF2 mRNA-binding protein 1 (IGF2BP1). Hepatology 58(5):1703–1712. doi:10.1002/hep.26537
Hendrickson D, Kelley DR, Tenen D, Bernstein B, Rinn JL (2016) Widespread RNA binding by chromatin-associated proteins. Genome Biol 17:28. doi:10.1186/s13059-016-0878-3
Zhao J, Ohsumi TK, Kung JT, Ogawa Y, Grau DJ, Sarma K, Song JJ, Kingston RE, Borowsky M, Lee JT (2010) Genome-wide identification of polycomb-associated RNAs by RIP-seq. Mol Cell 40(6):939–953. doi:10.1016/j.molcel.2010.12.011
Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162(1):156–159. doi:10.1006/abio.1987.9999
Ascano M, Hafner M, Cekan P, Gerstberger S, Tuschl T (2012) Identification of RNA-protein interaction networks using PAR-CLIP. Wiley Interdiscip Rev RNA 3(2):159–177. doi:10.1002/wrna.1103
Acknowledgments
This work was supported by the Thuringian country program ProExzellenz (RegenerAging—FSU-I-03/14) of the Thuringian Ministry for Research (TMWWDG).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Bierhoff, H. (2018). Analysis of lncRNA-Protein Interactions by RNA-Protein Pull-Down Assays and RNA Immunoprecipitation (RIP). In: Lacorazza, H. (eds) Cellular Quiescence. Methods in Molecular Biology, vol 1686. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7371-2_17
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7371-2_17
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7370-5
Online ISBN: 978-1-4939-7371-2
eBook Packages: Springer Protocols