Desthiobiotin-Streptavidin-Affinity Mediated Purification of RNA-Interacting Proteins in Mesothelioma Cells

Summary

Desthiobiotin labeling of a synthetic 25-nucleotide RNA oligo, which contains an adenine-rich element (ARE) motif, allows specific binding of cytosolic ARE-binding protein.

Abstract

The in vitro RNA-pulldown is still largely used in the first steps of protocols aimed at identifying RNA-binding proteins that recognize specific RNA structures and motifs. In this RNA-pulldown protocol, commercially synthesized RNA probes are labeled with a modified form of biotin, desthiobiotin, at the 3' terminus of the RNA strand, which reversibly binds to streptavidin and thus allows elution of proteins under more physiological conditions. The RNA-desthiobiotin is immobilized through interaction with streptavidin on magnetic beads, which are used to pull down proteins that specifically interact with the RNA of interest. Non-denatured and active proteins from the cytosolic fraction of mesothelioma cells are used as the source of proteins. The method described here can be applied to detect the interaction between known RNA binding proteins and a 25-nucleotide (nt) long RNA probe containing a sequence of interest. This is useful to complete the functional characterization of stabilizing or destabilizing elements present in RNA molecules achieved using a reporter vector assay.

Introduction

Gene expression and the final level of the gene product can be tightly regulated by affecting the mRNA stability and mRNA translation rate¹. These post-transcriptional regulatory mechanisms are exerted through the interactions of non-coding RNA and/or RNA-binding proteins (RBPs) with targeted mRNA. It is usually the 3' untranslated region of mRNA (3' UTR - belonging to the non-coding portion of the genome²) that contains specific cis-regulatory elements (CRE), which are recognized by trans-acting factors such as miRNA or RBPs³. The best-studied cis-element within....

Protocol

1. Preparation of Cytosolic and Nuclear Protein Fraction

1. 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 fraction.
   NOTE: ACC-MESO-4 cell line was obtained from the RIKEN BioResource Centre¹¹.
2. Aspirate medium, wash the cells by adding 15 mL of 1x PBS, tilt the plate gently a few times and aspirate the 1x PBS. Add 3 mL of 0.25% Trypsin-EDTA and incu....

Results

In this experiment, a 25-nt long fragment of calretinin 3' UTR harboring ARE motif (CALB2 3' UTR (ARE) 25-nt) was used to test whether it binds specifically to the Human-antigen R (HuR) protein, a known mRNA stabilizer. To test the specificity of the ARE element, a 25-nt RNA probe CALB2 3' UTR (mtARE) containing an ARE-motif mutation, which was previously shown to abolish the stabilization effect of the ARE motif, was used⁶. The third RNA probe represents the negat.......

Discussion

3' UTRs belong to the non-coding genome³, and all non-coding RNAs can interact with proteins in order to exert their function⁷. When the mammalian genome was found to be pervasively transcribed and produced a significant portion of long noncoding RNAs¹⁶, emerging evidences demonstrated that these long-noncoding RNAs function in regulating gene expression as they interact with chromatin-remodeling complexes¹⁷. This knowledg.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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
Anti - HuR (MOUSE) monoclonal antibody	Thermo Fisher Scientific	-	The antibody is included in the Pierce Magnetic RNA-protein Pull-Down Kit  - 1:1000 in 5% BSA 1x TTBS - rabbit anti-mouse secondary antibody 1:10,000 in 5% BSA 1x TTBS
Anti - α - tubulin (MOUSE) monoclonal antibody	Santa Cruz	8035	1:1000 in 5% BSA 1x TTBS - rabbit anti-mouse secondary antibody 1:10,000 in 5% BSA 1x TTBS
Anti - PARP (RABBIT) Polyclonal antibody	Cell Signaling	9542	1:1000 in 5% BSA 1x TTBS - goat anti-rabbit secondary antibody 1:10,000 in 5% BSA 1x TTBS
Anti - Mesothelin (MOUSE) Monoclonal Antibody	Rockland	200-301-A87
Secondary goat anti-rabbit antibody	Cell Signaling	7074
Secondary rabbit anti-mouse antibody	Sigma-Aldrich	A-9044
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
Mini-PROTEAN 3 Cell	Bio-Rad	1653301
Mini Protean System Glass Plates	Bio-Rad	1653308
Mini-PROTEAN Spacer Plates with 1.5 mm integrated spacer	Bio-Rad	1653312
Mini-PROTEAN Comb, 10-well, 1.5 mm, 66 µL	Bio-Rad	1653365
Mini-PROTEAN Tetra Cell Casting Modules	Bio-Rad	1658050
RNaseZap RNase Decontamination Solution	Thermo Fisher Scientific	AM9780
Rotiphorese gel 30 - aqueous 30% acrylamide and bisacrylamide stock solution at a ration of 37.5:1	Roth	3029
20% SDS	PanReac AppliChem	A3942
PVDF transfer membrane	Perkin Elmer	NEF1002	Need to be activated by incubating in pure methanol for 1 min followed by washing in water for 2 min
Trans-Blot SD semi-dry transfer cell	Bio-Rad	1703940
Clarity Western ECL Substrate	Bio-Rad	1705061
FusionFX  Digital Imager	Vilber	-
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.
TEMED	Sigma-Aldrich	T9281
Ammonium persulfate	Sigma-Aldrich	A3678
Coomassie G-250	Applichem	A3480
Aluminiumsulfate-(14-18)-hydrate	Sigma-Aldrich	368458
ortho-phosphoric acid	Applichem	A0637