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 of proteins interacting with lncRNAs is the objective of various protocols, including different RNA-immunoprecipitation (RIP) procedures in which an antibody against the protein of interest is used to specifically pull down lncRNAs (for a review1). Other techniques such as Capture Hybridization Analysis of RNA Targets2 (CHART) or Chromatin Isolation by RNA Purification3 (ChIRP) allow to pull-down lncRNAs together with the associated protein complexes. In these latter techniques, the lncRNA is used as bait. CHART and ChIRP are also powerful techniques to identify genomic maps showing lncRNA occupancy2,3. In addition, these RNA pull-down methods make it possible to identify the RNA partners of a lncRNA. A procedure that allows the capture of RNAs targeted by a lncRNA has been described. Antisense DNA biotinylated oligonucleotide probes are designed against regions of low probability of internal base pairing as determined by bioinformatics modeling of the lncRNA secondary structure4. However, this procedure does not discriminate whether the RNA partners interact indirectly via a protein network or directly via direct RNA/RNA interactions with the lncRNA. It has been shown that cross-linking with psoralen derivatives is the method of choice to select direct RNA/RNA interactions mediated by base-pairing5. Psoralen derivatives are indeed able to intercalate into double-stranded DNA or RNA and to covalently link pyrimidines after irradiation with UV light (365 nm)6. Coupling the RNA pull-down using biotinylated oligonucleotide probes with the cross-linking with psoralen is an easy-to-use procedure proposed here for the identification of the RNAs engaged in direct RNA/RNA interactions with a lncRNA. Moreover, this procedure can allow delineating the RNA-interaction zones in the sequence of the lncRNA if different oligonucleotide probes designed along the length of the lncRNA are used.
Protocol
Representative Results
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by Aix-Marseille University and Centre National Recherche Scientifique and funded by a grant from Sandoz Laboratories.
Funding for open access charge: Aix-Marseille University and Centre National Recherche Scientifique
Materials
| 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 |
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