A Novel Method to Quantify RNA-Protein Interactions In Situ Using FMTRIP and Proximity Ligation

C Zurla; J Jung; E L Blanchard; P J Santangelo

doi:10.1007/978-1-4939-4035-6_12

A Novel Method to Quantify RNA-Protein Interactions In Situ Using FMTRIP and Proximity Ligation

Methods Mol Biol. 2017:1468:155-70. doi: 10.1007/978-1-4939-4035-6_12.

Authors

C Zurla¹, J Jung¹, E L Blanchard¹, P J Santangelo²

Affiliations

¹ Wallace H Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, 313 Ferst Drive, UA Whitaker Blgd, Atlanta, GA, 30332, USA.
² Wallace H Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, 313 Ferst Drive, UA Whitaker Blgd, Atlanta, GA, 30332, USA. philip.santangelo@bme.gatech.edu.

Abstract

RNA binding proteins (RBP) and small RNAs regulate the editing, localization, stabilization, translation, and degradation of ribonucleic acids (RNAs) through their interactions with specific cis-acting elements within target RNAs. Here, we describe a novel method to detect protein-mRNA interactions, which combines FLAG-peptide modified, multiply-labeled tetravalent RNA imaging probes (FMTRIPs) with proximity ligation (PLA), and rolling circle amplification (RCA). This assay detects native RNA in a sequence specific and single RNA sensitive manner, and PLA allows for the quantification and localization of protein-mRNA interactions with single-interaction sensitivity.

Keywords: FMTRIPS; PLA; Posttranscriptional regulation; RCA; mRNA binding proteins.

MeSH terms

Animals
Biophysical Phenomena
Chlorocebus aethiops
HeLa Cells
Humans
MCF-7 Cells
Mice
Oligopeptides / metabolism
Protein Interaction Mapping / methods*
RAW 264.7 Cells
RNA / metabolism*
RNA-Binding Proteins / metabolism*
Vero Cells

Substances

Oligopeptides
RNA-Binding Proteins
RNA
FLAG peptide

Abstract

MeSH terms

Substances

Grants and funding