Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

Summary

An RNA pull-down protocol is optimized here for detection of interactions between RNA-binding proteins (RBPs) and noncoding as well as coding RNAs. An RNA fragment from androgen receptor (AR) was used as an example to demonstrate how to retrieve its RBP from lystate of primary brown adipocytes.

Abstract

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression regulation at posttranscriptional levels by affecting the stability and translational efficiency of target mRNAs. RNA pull-down technique has been widely used to study RNA-protein interaction, which is necessary to elucidate the mechanism underlying RBPs' as well as long non-coding RNAs' (lncRNAs) function. However, the high lipid abundance in adipocytes poses a technical challenge in conducting this experiment. Here a detailed RNA pull-down protocol is optimized for primary adipocyte culture. An RNA fragment from androgen receptor's (AR) 3' untranslated region (3'UTR) containing an adenylate-uridylate-rich elementwas used as an example to demonstrate how to retrieve its RBP partner, HuR protein, from adipocyte lystate. The method described here can be applied to detect the interactions between RBPs and noncoding RNAs, as well as between RBPs and coding RNAs.

Introduction

RBPs are proteins that bind to the double or single stranded RNA in cells and participate in forming RNA-protein complexes. RBPs may bind a variety of RNA species, including mRNA and long noncoding RNAs (lncRNAs), and exert their influence at post-transcriptional levels. Both RBPs and lncRNAs are emerging as novel regulators in adipose development and function^1,2,3. To understand the mechanism of RBP- and lncRNA-mediated regulation of cellular pathways, it is often necessary to detect the interaction between a specific RNA molecule and one or several RBPs, and, sometimes, to identify the full spectrum of protein partners of a RNA transcript. However, the ex....

Protocol

NOTE: The RNA of interest in the context of this study is a fragment of AR 3'UTR.

1. Preparation of Biotin-labeled RNA

1. To obtain the RNA, amplify T7-AR-oligo by PCR, followed by in vitro transcription using a commercial kit and following manufacturer's instructions¹¹.
   NOTE: Primers for PCR are listed in Table 4: T7-AR-F and T7-AR-R.
2. For non-specific binding control, amplify a partial sequence of the firefly luciferase (FL) DNA by PCR from psiCHECK-2 plasmid, followed by in vitro transcription.
   NOTE: Primers for PCR are listed in Table 4

Results

In this demo experiment, an AR RNA fragment was used as the bait to capture its binding protein HuR. Both a FL RNA bait that is non-relevant to HuR protein, and an aliquot of unconjugated blank streptavidin beads served as negative controls. RNA-protein interactions can occur either in nucleus or cytoplasm, and this pull-down protocol can be applied to either total or fractionated (nuclear or cytoplasmic) cell lysates. Analysis of proteins by western blotting shows that the sample of 15 &.......

Discussion

lncRNAs and RBPs have vital roles in health and disease, however the molecular mechanisms of these molecules are poorly understood. Identification of proteins that interact with lncRNA molecules is a key step towards elucidating the regulatory mechanisms. Enrichment of RBPs by the RNA pull-down assay system is based on in-solution capture of interacting complex so that proteins from a cell lysate can be selectively extracted using biotinylated RNA baits bound to streptavidin magnetic beads. Several other methods such as .......

Materials

Name	Company	Catalog Number	Comments
Major materials used in this study.
MEGAscript kit	Ambion
Biotin-14-CTP	Invitrogen
NucAway spin columns	Ambion
Dynabeads M-280 Streptavidin	Invitrogen
HuR (3A2) mouse monoclonal antibody (sc-5261)	Santa Cruz Biotechnology
Goat anti-mouse IgG-HRP(sc-2005)	Santa Cruz Biotechnology
Hypure Molecular Biology Grade Water (nuclease-free)	HyClone
Phosphate Buffer Saline (1×PBS)	HyClone
RNase inhibitor	Bioline
Protease inhibitor	Sigma
Pfu Turbo DNA polymerase	Agilent Technologies
TRIsure	Bioline
Name	Company	Catalog Number	Comments
Major equipment used in this study.
Sorvall Legend Micro 21R centrifuge	Thermo Scientific
Sorvall Legend X1R centrifuge	Thermo Scientific
Bio-Rad T100 thermal cycler	Bio-Rad
Nanodrop 2000	Thermo Scientific
BOECO rotator Multi Bio RS-24	BOECO,Germany
Protein gel electrophoresis and blotting apparatus	Bio-Rad
DynaMag-2 magnet	Life Technologies