RNA-Protein Pull-Down Assay to Isolate RNA-Binding Proteins via Affinity Extraction

Abstract

Source: Kresoja-Rakic, J., et al. Desthiobiotin-Streptavidin-Affinity Mediated Purification of RNA-Interacting Proteins in Mesothelioma Cells. J. Vis. Exp. (2018)

This video demonstrates an in vitro RNA pull-down assay to identify RNA-binding proteins (RBPs), which interact with the adenylate-uridylate-rich element (ARE) sequences in mRNA. The target RBPs from a cell lysate are mixed with an RNA probe to form RNA-protein complexes. The complexes are isolated via affinity purification utilizing the affinity of the desthiobiotin label of the RNA probe to a streptavidin-labeled magnetic bead.

Protocol

1. Preparation of Cytosolic and Nuclear Protein Fraction Plate 4 x 106 ACC-MESO-4 cells in a T150 cell culture flask grown in RPMI – 1640 medium supplemented with 10% FBS, 1x penicillin/streptomycin (100x), and 2 mM L-Glutamine (200 mM). When cells reach a confluence of 80-90% (~5-5.5 x 106 cells), proceed with protein extraction of nuclear and cytosolic fractions. NOTE: ACC-MESO-4 cell line was obtained from the RIKEN BioResource Centre. <l…

Representative Results

Figure 1: Western blot analysis of 5 µg of cytosolic and nuclear protein fraction. PARP protein is present in the nuclear fraction (N) but absent from the cytosolic (C) fraction. α-tubulin is present in the cytosolic fraction (C) but absent in the nuclear fraction (N). Table 1: Sequences of RNA probes <table border="1" fo:keep-together.within-page="1" fo:keep…

Materials

NE-PER Nuclear and cytoplasmic extraction kit	Thermo Fisher Scientific	78833
Pierce Protease Inhibitor Tablets, EDTA-free	Thermo Fisher Scientific	A32965
Pierce BCA Protein Assay Kit	Thermo Fisher Scientific	23225
Pierce Magnetic RNA-protein Pull-Down Kit	Thermo Fisher Scientific	20164	Includes magnetic beads and therefore the kit need to be stored at 4 °C
Pierce RNA 3’ End Desthiobiotinylation Kit	Thermo Fisher Scientific	20163	The kit is a part of the "Pierce Magnetic RNA-protein Pull-Down Kit", and needs to be stored at -20 °C unlike the pull-down kit (4 °C).
DynaMag-2, magnetic stand	Thermo Fisher Scientific	12321D
RPMI – 1640 medium	Sigma-Aldrich	R8758
FBS – Filtrated Bovine Serum	Pan Biotech	P40-37500
Penicillin – streptomycin (100x)	Sigma-Aldrich	P4333
L-Glutamine solution (200 mM)	Sigma-Aldrich	G7513
0.25% Trypsin-EDTA (1x)	Gibco by Life technologies	25200-056
RNaseZap RNase Decontamination Solution	Thermo Fisher Scientific	AM9780
RNA oligo synthesis	Mycrosynth AG, Switzerland	–	The synthesis scale – 0.04 µmol – HPLC-purified
Bovine serum albumin	Sigma-Aldrich	A7030
Hydrochloric Acid (HCl) 6 M	PanReac AppliChem	182883.1211
Ultra Pure 1 M Tris, pH 7.5	Thermo Fisher Scientific	15567027
Glycerol	Thermo Fisher Scientific	17904	50% sterile aliquots to be stored at -20°C
Pierce Streptavidin magnetic beads	Thermo Fisher Scientific	88817	Please note that these magnetic beads have an average diameter of 1 µm (range from 0.5 – 1.5 µm). Furthermore, Dynabeads-M280 Streptavidin, which are beads of homogenouse size 2.8 µm, are also used in RNA-pulldown but be aware that this may affect purification process.
Aluminiumsulfate-(14-18)-hydrate	Sigma-Aldrich	368458
Ortho-phosphoric acid	Applichem	A0637