Identification of RNAs Engaged in Direct RNA-RNA Interaction with a Long Non-Coding RNA

Summary

An easy-to-use RNA pull-down protocol is designed for the identification of RNAs engaged in direct RNA/RNA interaction with a long non-coding RNA. The protocol uses psoralen as a fixative to cross-link only RNA/RNA interactions and provides the whole direct RNA interactome of a long non-coding RNA when coupled with RNA sequencing.

Abstract

The growing role attributed nowadays to long non-coding RNAs (lncRNA) in physiology and pathophysiology makes it crucial to characterize their interactome by identifying their molecular partners, DNA, proteins and/or RNAs. The latter can interact with lncRNA through networks involving proteins, but they can also be engaged in direct RNA/RNA interactions. We, therefore, developed an easy-to-use RNA pull-down procedure that allowed identification of RNAs engaged in direct RNA/RNA interaction with a lncRNA using psoralen, a molecule that cross-links only RNA/RNA interactions. Bioinformatics modeling of the lncRNA secondary structure allowed the selection of several specific antisense DNA oligonucleotide probes with a strong affinity for regions displaying a low probability of internal base pairing. Since the specific probes that were designed targeted accessible regions throughout the length of the lncRNA, the RNA-interaction zones could be delineated in the sequence of the lncRNA. When coupled with a high throughput RNA sequencing, this protocol can be used for the whole direct RNA interactome studies of a lncRNA of interest.

Introduction

Long non-coding RNAs (lncRNAs) are non-protein-coding transcripts longer than 200 nucleotides in length. Their number is ever increasing, more than 58,000 in humans. Furthermore, their crucial role in physiology and pathophysiology makes it essential to characterize their molecular partners that allow them to implement their regulatory functions. Actually, one approach to understand the functions of lncRNAs is the detection of the interacting molecular partners of each lncRNA.

The molecular targets of lncRNAs can be DNA, proteins, or RNAs, and various techniques have been developed to identify them. In this regard, the identification o....

Protocol

1. Probe design

1. Generate the secondary structure of the lncRNA using a specialized free web server software : RNAstructure software⁷ or Vienna RNA web suite⁸. Select regions that display a low probability of internal base pairing and design 25 bases long antisense oligonucleotide probes for different regions of this lncRNA.
2. Check all the oligonucleotides designed with the free academic software, AmplifX (https://inp.univ-amu.fr/en/amplifx-manage-test-and-design-your-primers-for-pcr), in order to obtain different information on these probes and to choose those with the best parameters.
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Results

The elucidation of the lncRNA interactome i.e., the cellular components that interact with lncRNAs, proteins, RNA, and DNA, is of key importance for understanding the functions of lncRNAs. Various techniques have been developed to characterize the lncRNA interactome, including RIP, CHART, ChIRP, and RNA pull-down. While the latter has been shown to be powerful in identifying RNA targets of lncRNAs, these procedures do not indicate whether the RNA partners interact indirectly via a protein network or directly via direct R.......

Discussion

Numerous lncRNAs carry out their function through complementary base pairing to mRNAs. It is, therefore, important to develop procedures that allow characterizing the direct RNA interactome of the lncRNAs. Therefore, a procedure was developed that combines the use of psoralen as cross-linking reagent with RNA pull-down technique.

In the RNA pull-down protocol described, the design and the selection of the antisense DNA biotinylated oligonucleotide probes are based on bioinformatics modeling of.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
4′-Aminomethyltrioxsalen hydrochloride	Sigma	A4330	Crosslinker reagent
Bioruptor Plus	Diagenode	B01020001	Sonicator
Biotynilated probes	IDT		Oligonucleotide probes
CFX96 Real Time System	BioRad	4351107	qPCR apparatus
DNA Olignucleotides	IDT		Primers for qPCR
Dynabeads My One	Thermo-Fisher	65001	Magnetic streptavidin beads
Formamide	Thermo-Fisher	15515-026	Formamide
iTaq Universal SYBR Green Supermix	BioRad	1725124	qPCR reagent
Proteinase K	Sigma	P2308	Proteinase K
RNA to DNA	Thermo-Fisher	4387405	Reverse transcription kit
RNA XS purification kit	Macherey-Nagel	740902	RNA purificationkit
RNAseOUT	Thermo-Fisher	10777-019	RNAse inhibitor
Tube Rotator	Stuart	SB2	Eppendorf tube rotator
UV Stratalinker 1800	Stratagene	#400072	UV crosslinker