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LncRNA Pulldown Combined with Mass Spectrometry to Identify the Novel lncRNA-Associated Proteins Protocol

Experiment Summary

Long non-coding RNAs (LncRNAs) are non-protein coding transcripts longer than 200 nucleotides. Recent studies have revealed that nearly 80% of transcripts in human cells are lncRNA species. Based on their genomic location, most lncRNAs can be characterized as large intergenic non-coding RNAs, natural antisense transcripts, pseudogenes, and long intronic ncRNAs, as well as other divergent transcripts. However, despite mounting evidence suggesting that many lncRNAs are likely to be functional, only a small proportion has been demonstrated to be biologically and physiologically relevant due to their lower expression levels and current technique limitations. Here, we describe an open-ended method, LncRNA pulldown assay, which has been frequently used to identify lncRNA interacting protein partners in a cellular context. We provide a detailed protocol for this assay with hands-on tips based on our own experience working in the lncRNA field.

Materials and Reagents

A. For Preparation of In Vitro- Transcribed RNA (IVT)

pGEM-3Z VectorBiotin RNA labeling mix
T7 and SP6 RNA polymeraseTURBO DNase
Anti-RNaseRNA Clean and Concentrator
Nuclease-free H2OUltraPure Agarose
Formaldehyde10× MOPS Buffer
Ethidium bromideRNA loading dye
RNA marker

B. For Preparation of Cell Lysate

ProteaPrep Zwitterionic Cell Lysis KitProtease/phosphatase inhibitor cocktail.
PanobinostatMethylstat
1× Phosphate-buffered saline (PBS) buffer

C. For lncRNA Pulldown

BcMag™ Monomer Avidin Magnetic BeadsRNA structure buffer
RNA capture bufferNT2 buffer
Wash buffer

D. For In-Solution Tryptic Digestion

Digestion buffer, ammonium bicarbonate (50 mM)Reducing buffer, DTT (100 mM)
Alkylation buffer, iodoacetamide in NH 4HCO 3 (100 mM)Trifl uoroacetic acid (TFA), 10 %
AcetonitrolePeptide recovery buffer, 1 ml
Immobilized trypsin

Procedure

A. Construction of RNA overexpression plasmid

  1. First, identify the InRNA you are interested in by other means; search the database to find the corresponding RNA base sequence
  2. After confirming the sequence used for RNA pull down, we will design primers to synthesize the RNA sequence, construct it into pGEM-3Z vector (the vector can be flexibly replaced according to other experiments) for overexpression, and then sequence it to verify the accuracy of the sequence.
  3. Finally, extract the overexpression plasmid for spare parts.

B. In vitro transcription template preparation

  1. Experimental grouping:

Experimental group: sense strand i.e. target RNA sequence

Control group: antisense strand, i.e., complementary sequence of the target RNA.

2. Primer design method:

Add T7 promoter sequence in front of the forward primer; keep the reverse primer unchanged.

The constructed overexpression plasmid was used as the template and the primers were designed and synthesized to amplify the in vitro transcript template. The PCR products were detected by agarose gel electrophoresis, and the target bands were purified and recycled for later use.

C. In vitro transcription

  1. The purified and recovered PCR product is used as the transcription template, and the in vitro transcription kit is used to transcribe the target RNA (both sense and antisense).
  2. Transcription profiles (RNA size, amount of transcribed RNA, etc.) vary among kits.

Schematic diagram showing lncRNA pulldown.Fig. 1 Schematic diagram showing lncRNA pulldown.

D. lncRNA Pulldown

1. Pre-clearing the Lysate with Activated Beads to Reduce Nonspecific Binding

1) Remove the PBS from the activated avidin beads.

2) Add 1 ml of cell lysate to the beads and mix well.

3) Incubate for 1 h at 4 °C with end-to-end gentle rotation.

4) Place the tube on a magnetic separator and keep the supernatant for further use. Discard bead pellet.

2. Folding of IVT RNA

1) 20 μg of biotinylated RNA was heated to 90 °C for 2 min.

2) Chill on ice for 2 min and briefly centrifuge.

3) Bring the total volume to 100 μl with RNA structure buffer.

4) Incubate at room temperature for 20 min to allow proper secondary structure formation.

3. LncRNA Pulldown

1) Prepare the 50 μl activated avidin magnetic beads.

2) The beads were immediately subject to RNA (20 μg) capture in RNA capture buffer for 30 min at room temperature with gentle agitation.

3) Remove the RNA capture buffer and the RNA-captured beads were washed once with NT2 buffer and incubated with 30 mg pre-cleared cell lysate for 2 h at 4 °C with gentle rotation.

4) Briefly centrifuge the tube and place the tube on a magnetic separator. Remove the supernatant from the beads and discard. The complex of interest should now bind to the beads. The following steps are used to remove the nonspecific binding to the beads.

5) Wash the RNA-binding protein complexes with NT2 buffer twice.

6) Wash the RNA-binding protein complexes with NT2 high-salt buffer (500 mM NaCl) twice.

7) Wash the RNA-binding protein complexes with NT2 high-salt buffer (1 M NaCl) once.

8) Wash the RNA-binding protein complexes with NT2 high-salt buffer (750 mM KSCN) once.

9) Wash the RNA-binding protein complexes with PBS twice.

10) Elute the beads by incubating the sample with elution buffer for 20 min at 4 °C with frequent agitation. Collect the elute.

11) Repeat elution and pool the elute together.

E. MS Analysis

1. Concentrate and dry the eluted samples with Speedy Vacuum system.

2. Dissolve the dry elute in 10 μl ddH2O and prepare the reduction reaction.

1) 15 μl Digestion buffer.

2) 10 μl Dissolved eluted samples.

3) 1.5 μl Reducing buffer.

4) Bring the total volume to 27 μl with ddH2O and incubate the reaction at 95 °C for 5 min. Allow samples to cool to room temperature.

3. Perform alkylation reaction by adding 3 μl alkylation buffer to the above tube and incubate in the dark at room temperature for 20 min.

Schematic diagram showing sample preparation for MS analysis.Fig. 2 Schematic diagram showing sample preparation for MS analysis.

4. Perform the trypsin digestion overnight with the immobilized trypsin kit following the manufacturer's instructions.

5. The recovered digested peptides were subject to the LS-MS analysis.

Related Products and Services at Creative Biogene

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* For research use only. Not intended for any clinical use.
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