A Live-cell Assay for the Detection of Pre-microRNA-protein Interactions

Overview

Journal RSC Chem Biol

Publisher Royal Society of Chemistry

Specialty Biology

Date 2021 Apr 5

PMID 33817642

Citations 6

Authors

Sydney L Rosenblum

Daniel A Lorenz

Amanda L Garner

Affiliations

Soon will be listed here.

Abstract

Recent efforts in genome-wide sequencing and proteomics have revealed the fundamental roles that RNA-binding proteins (RBPs) play in the life cycle and function of coding and non-coding RNAs. While these methodologies provide a systems-level view of the networking of RNA and proteins, approaches to enable the cellular validation of discovered interactions are lacking. Leveraging the power of bioorthogonal chemistry- and split-luciferase-based assay technologies, we have devised a conceptually new assay for the live-cell detection of RNA-protein interactions (RPIs), RNA interaction with Protein-mediated Complementation Assay, or RiPCA. As proof-of-concept, we utilized the interaction of the pre-microRNA, pre-let-7, with its binding partner, Lin28. Using this system, we have demonstrated the selective detection of the pre-let-7-Lin28 RPI in both the cytoplasm and nucleus. Furthermore, we determined that this technology can be used to discern relative affinities for specific sequences as well as of individual RNA binding domains. Thus, RiPCA has the potential to serve as a useful tool in supporting the investigation of cellular RPIs.

Citing Articles

Protein recognition methods for diagnostics and therapy.

Montoya R, Deckerman P, Guler M BBA Adv. 2025; 7:100149.

PMID: 40060358 PMC: 11889627. DOI: 10.1016/j.bbadva.2025.100149.

Live cell screening to identify RNA-binding small molecule inhibitors of the pre-let-7-Lin28 RNA-protein interaction.

Rosenblum S, Soueid D, Giambasu G, Vander Roest S, Pasternak A, DiMauro E RSC Med Chem. 2024; 15(5):1539-1546.

PMID: 38784453 PMC: 11110735. DOI: 10.1039/d4md00123k.

Adaptation of RiPCA for the Live-Cell Detection of mRNA-Protein Interactions.

Soueid D, Garner A Biochemistry. 2023; 62(23):3323-3336.

PMID: 37963240 PMC: 11466511. DOI: 10.1021/acs.biochem.3c00334.

Optimization of RiPCA for the Live-Cell Detection of Pre-MicroRNA-Protein Interactions.

Rosenblum S, Garner A Chembiochem. 2022; 23(24):e202200508.

PMID: 36322053 PMC: 9831681. DOI: 10.1002/cbic.202200508.

RiPCA: An Assay for the Detection of RNA-Protein Interactions in Live Cells.

Rosenblum S, Garner A Curr Protoc. 2022; 2(2):e358.

PMID: 35113480 PMC: 8852372. DOI: 10.1002/cpz1.358.

References

Jazurek M, Ciesiolka A, Starega-Roslan J, Bilinska K, Krzyzosiak W . Identifying proteins that bind to specific RNAs - focus on simple repeat expansion diseases. Nucleic Acids Res. 2016; 44(19):9050-9070. PMC: 5100574. DOI: 10.1093/nar/gkw803. View

DAngelo M, Hetzer M . Structure, dynamics and function of nuclear pore complexes. Trends Cell Biol. 2008; 18(10):456-66. PMC: 2697926. DOI: 10.1016/j.tcb.2008.07.009. View

Marx V . Profiling the dress codes of RNA-binding proteins. Nat Methods. 2018; 15(9):655-658. DOI: 10.1038/s41592-018-0117-9. View

Mili S, Steitz J . Evidence for reassociation of RNA-binding proteins after cell lysis: implications for the interpretation of immunoprecipitation analyses. RNA. 2004; 10(11):1692-4. PMC: 1370654. DOI: 10.1261/rna.7151404. View

Lee F, Ule J . Advances in CLIP Technologies for Studies of Protein-RNA Interactions. Mol Cell. 2018; 69(3):354-369. DOI: 10.1016/j.molcel.2018.01.005. View

Treiber T, Treiber N, Meister G . Publisher Correction: Regulation of microRNA biogenesis and its crosstalk with other cellular pathways. Nat Rev Mol Cell Biol. 2019; 20(5):321. DOI: 10.1038/s41580-019-0106-6. View

Lorenz D, Garner A . A click chemistry-based microRNA maturation assay optimized for high-throughput screening. Chem Commun (Camb). 2016; 52(53):8267-70. PMC: 4919152. DOI: 10.1039/c6cc02894b. View

Brannan K, Jin W, Huelga S, Banks C, Gilmore J, Florens L . SONAR Discovers RNA-Binding Proteins from Analysis of Large-Scale Protein-Protein Interactomes. Mol Cell. 2016; 64(2):282-293. PMC: 5074894. DOI: 10.1016/j.molcel.2016.09.003. View

Sherman E, Lorenz D, Garner A . Click Chemistry-Mediated Complementation Assay for RNA-Protein Interactions. ACS Comb Sci. 2019; 21(7):522-527. DOI: 10.1021/acscombsci.9b00071. View

10.

Lorenz D, Song J, Garner A . High-throughput platform assay technology for the discovery of pre-microrna-selective small molecule probes. Bioconjug Chem. 2014; 26(1):19-23. DOI: 10.1021/bc500544v. View

11.

Treiber T, Treiber N, Plessmann U, Harlander S, Daiss J, Eichner N . A Compendium of RNA-Binding Proteins that Regulate MicroRNA Biogenesis. Mol Cell. 2017; 66(2):270-284.e13. DOI: 10.1016/j.molcel.2017.03.014. View

12.

Jankowsky E, Harris M . Specificity and nonspecificity in RNA-protein interactions. Nat Rev Mol Cell Biol. 2015; 16(9):533-44. PMC: 4744649. DOI: 10.1038/nrm4032. View

13.

Heinrich S, Sidler C, Azzalin C, Weis K . Stem-loop RNA labeling can affect nuclear and cytoplasmic mRNA processing. RNA. 2017; 23(2):134-141. PMC: 5238788. DOI: 10.1261/rna.057786.116. View

14.

Nam Y, Chen C, Gregory R, Chou J, Sliz P . Molecular basis for interaction of let-7 microRNAs with Lin28. Cell. 2011; 147(5):1080-91. PMC: 3277843. DOI: 10.1016/j.cell.2011.10.020. View

15.

Lorenz D, Kaur T, Kerk S, Gallagher E, Sandoval J, Garner A . Expansion of cat-ELCCA for the Discovery of Small Molecule Inhibitors of the Pre-let-7-Lin28 RNA-Protein Interaction. ACS Med Chem Lett. 2018; 9(6):517-521. PMC: 6004563. DOI: 10.1021/acsmedchemlett.8b00126. View

16.

Castello A, Fischer B, Frese C, Horos R, Alleaume A, Foehr S . Comprehensive Identification of RNA-Binding Domains in Human Cells. Mol Cell. 2016; 63(4):696-710. PMC: 5003815. DOI: 10.1016/j.molcel.2016.06.029. View

17.

Porter J, Stains C, Jester B, Ghosh I . A general and rapid cell-free approach for the interrogation of protein-protein, protein-DNA, and protein-RNA interactions and their antagonists utilizing split-protein reporters. J Am Chem Soc. 2008; 130(20):6488-97. DOI: 10.1021/ja7114579. View

18.

Wheeler E, Van Nostrand E, Yeo G . Advances and challenges in the detection of transcriptome-wide protein-RNA interactions. Wiley Interdiscip Rev RNA. 2017; 9(1). PMC: 5739989. DOI: 10.1002/wrna.1436. View

19.

Han Y, Wang S, Zhang Z, Ma X, Li W, Zhang X . In vivo imaging of protein-protein and RNA-protein interactions using novel far-red fluorescence complementation systems. Nucleic Acids Res. 2014; 42(13):e103. PMC: 4117741. DOI: 10.1093/nar/gku408. View

20.

Lorenz D, Vander Roest S, Larsen M, Garner A . Development and Implementation of an HTS-Compatible Assay for the Discovery of Selective Small-Molecule Ligands for Pre-microRNAs. SLAS Discov. 2017; 23(1):47-54. PMC: 5797708. DOI: 10.1177/2472555217717944. View