A Novel Method to Quantify RNA-Protein Interactions In Situ Using FMTRIP and Proximity Ligation.
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.
Wallace H Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, 313 Ferst Drive, UA Whitaker Blgd, Atlanta, GA, 30332, USA.
C
C
Zurla
Wallace H Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, 313 Ferst Drive, UA Whitaker Blgd, Atlanta, GA, 30332, USA.
J
J
Jung
Wallace H Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, 313 Ferst Drive, UA Whitaker Blgd, Atlanta, GA, 30332, USA.
E L
EL
Blanchard
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.
P J
PJ
Santangelo
D000818:Animals; D055592:Biophysical Phenomena; D002522:Chlorocebus aethiops; D006367:HeLa Cells; D006801:Humans; D061986:MCF-7 Cells; D051379:Mice; D009842:Oligopeptides / Q000378:metabolism; D025941:Protein Interaction Mapping / Q000379:methods*; D000067996:RAW 264.7 Cells; D012313:RNA / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D014709:Vero Cells
D009842:Oligopeptides; D016601:RNA-Binding Proteins; D012313:RNA; C085706:FLAG peptide
FMTRIPS; PLA; Posttranscriptional regulation; RCA; mRNA binding proteins
F1000 Biol Rep. 2012;4:16
22912650
Nat Methods. 2006 Dec;3(12):995-1000
17072308
Nat Commun. 2015 Jun 12;6:7294
26065580
Semin Cell Dev Biol. 2007 Apr;18(2):186-93
17459736
Biol Cell. 2005 Jan;97(1):97-110
15601261
J Virol. 2012 Aug;86(15):8245-58
22623778
Bioconjug Chem. 2010 Mar 17;21(3):483-8
20141184
Mol Cell. 2002 Jun;9(6):1161-7
12086614
RNA. 2009 Jan;15(1):97-103
19033374
Cell Mol Life Sci. 2008 Mar;65(5):798-813
18043867
Nat Rev Genet. 2007 Jul;8(7):533-43
17572691
Methods. 2008 Jul;45(3):227-32
18620061
Brief Funct Genomics. 2015 Jan;14(1):74-89
25504152
Chem Rev. 2014 Mar 26;114(6):3224-65
24417544
Nat Methods. 2011 Oct 28;8(11):892-3
22036742
Methods Enzymol. 2012;505:383-99
22289464
Science. 1991 Sep 13;253(5025):1275-7
1891715
Nucleic Acids Res. 2013 Jan 7;41(1):e12
22952158
ACS Nano. 2014 Jan 28;8(1):302-15
24351207
Curr Protoc Cytom. 2011 Apr;Chapter 9:Unit 9.36
21455970
J Biol Chem. 2015 Feb 6;290(6):3468-87
25519906
Wiley Interdiscip Rev RNA. 2014 Jan-Feb;5(1):69-86
24124109
PLoS One. 2011 May 05;6(5):e19727
21573130
Nat Methods. 2009 May;6(5):347-9
19349979
Genome Biol. 2014 Jan 27;15(1):203
24467948
Traffic. 2011 Aug;12 (8):1000-11
21518164
J Cell Biol. 1994 Aug;126(4):863-76
7914201
PLoS One. 2013 Sep 11;8(9):e74598
24040294
Wiley Interdiscip Rev Dev Biol. 2015 Mar-Apr;4(2):135-50
25645249