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 occupancy^2,3. 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

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.
   NOTE: Check for optimal parameters on the software as following. Percentage of GC: between 40 and 60%, 3' stability, complexity, self-end dimer: "good". Melting temperature (T_M) is not important because the affinity is performed at room temperature.
3. Using an alignment search tool (i.e., Blast, etc.), ensure that the selected antisense oligonucleotide probes do not recognize nucleotide sequences in any other RNA expressed in the cells of choice.
   1. Design a 25 bases long non-specific DNA oligonucleotide probe. Ensure this probe neither shows an affinity for the lncRNA of interest nor any other RNA sequences in the genome of interest.
4. Order all probes with a biotin modification added to their 3'-end along with a triethyleneglycerol spacer (TEG). This spacer increases the oligo-biotin distance in order to avoid steric hindrance.
   ​NOTE: To obtain an optimal result and assess the specificity of results of the pull-down, design a minimum of two different antisense oligonucleotide probes in the same region and then compare their efficiency experimentally.

2. Cross-linking

1. Grow cells (e.g., GH4C1 cells used in this experiment) until confluence in 78.5 cm² cell culture dishes. After removing the cell culture medium, rinse the cell layer with 5 mL of cold phosphate-buffered saline enriched with Ca²⁺ and Mg²⁺ (PBS⁺) and add 3 mL of psoralen-derived molecule (4′Aminomethyltrioxsalen hydrochloride) solution prepared in PBS⁺ at 0.1 mg/mL concentration.
2. Place the culture plate for 30 min in a mammalian cell culture incubator at 37 °C with 5% CO₂. After 30 min, remove the lid, place culture dishes on ice at 2.5 cm away from 365 nm UV tubes in an UV crosslinker for 10 min. Agitate the dishes delicately and restore the UV exposure for an additional 10 min period.
3. Discard media by aspiration, add 1 mL of PBS⁺ to collect cells with a cell scraper, and transfer to a micro-tube. Centrifuge at 4 °C, 400 x g for 5 min. Remove as much supernatant as possible and stock pellets at -80 °C.

3. Cell lysis

1. Prepare Proteinase K buffer by adding 100 mM NaCl, 10 mM Tris-HCl, pH 7.0, 1 mM EDTA, 0.5% SDS and 5 µL/mL RNase inhibitor.
2. Resuspend the cell pellets in this buffer (approximately 200 µL per 1 x 10⁷ cells). Add proteinase K to a final concentration of 0.1 µg/µL and incubate for 45 min at 50 °C. Then incubate for 10 min at 95 °C, to inactivate the proteinase K.

4. Sonication

1. Distribute 100 µL of this lysate (corresponding to 5.10⁶ cells) in two micro-tubes and add 200 µL of hybridization buffer (700 mM NaCl, 70 mM Tris-HCl, pH 7.0, 1 mM EDTA, 1.25% SDS, 5 µL/mL RNase inhibitor solution and 15% formamide) in each tube.
2. Place the tubes in the 4 °C water bath of a sonicator and start the sonication with 2 series of 30 s pulses (high intensity) separated by 30 s off.
3. Collect 20 µL of samples to serve as input control samples and store at -80 °C.
   ​NOTE: This sonication procedure was previously optimized for the GH4C1 cell line in order to obtain RNA fragments ranging in 2,000 nucleotides⁹. For others cell lines, the sonication procedure must be optimized. When this technique is performed to study the RNA partners of a very long non-coding RNA, the sonication step proves to be indispensable to obtain an adequate recovery of the lncRNA. In this case, it is, therefore, necessary to design several probes with a binding site located every 4,000 nucleotides maximum along the lncRNA length. To recover the full length of the lncRNA, use a pool of several specific probes. However, if the purpose is to identify the regions of the lncRNA involved in RNA-RNA binding, the specific probes must be used separately. Furthermore, if studying RNA partners of a smaller lncRNA, experiments should be performed to determine if the sonication step is required.

5. RNA pull-down

1. Day 1 - Hybridization step
   1. Add 150 pmol (1.5 µL of a 100 µM stock solution) of a biotinylated specific or non-specific probe to each sample tube. Incubate for 4 h under agitation on a tube rotator at room temperature (RT) (approximately 30 rpm).
   2. Take the necessary volume of magnetic beads (i.e., 40 µL of beads per 150 pmol of probes hybridization reaction). Use magnet support to separate beads from commercial media. Discard the supernatant and wash the beads with 900 µL of the Hybridization buffer. Once done, resuspended the magnetic beads in the same volume of the Hybridization buffer.
   3. Add magnetic beads in each sample tube (40 µL of beads per 150 pmol of probes hybridization reaction). Incubate overnight under agitation on a tube rotator at RT (approximately 30 rpm).
2. Day 2 - RNA isolation step
   1. Separate beads from the cell lysate using magnet support (2-3 min). Discard the supernatant and wash the beads with 900 µL of the Wash buffer (0.5% SDS, 2x SSC) with 5 min incubation under agitation on the rotator at RT. Repeat this wash 5 times.
   2. After removing the last wash, add 95 µL of Proteinase K buffer on the beads. Defrost the input samples and add 75 µL of Proteinase K buffer. Add 5 µL of proteinase K to a final concentration of 1 µg/µL in all tubes (samples and input samples). Incubate at 45 °C for 45 min and then at 95 °C for 10 min.
   3. Keep the sample on ice. Use magnetic support to separate beads from RNAs present in the supernatant. Purify RNAs with an RNA purification kit that includes a DNase digestion step. Store purified RNAs at -80 °C.
      NOTE: Eluted RNA can be subjected to RT-qPCR or high throughput RNA sequencing to detect enriched transcripts. For RNA sequencing analysis, the reads can then be aligned to the genome of interest using free software, such as STAR¹⁰ or HISAT2¹¹, and the quantification can be done using FeatureCounts¹² or HTSeq count¹³.

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