Multiple Oligo assisted RNA Pulldown via Hybridization followed by Mass Spectrometry (MORPH-MS) for exploring the RNA-Protein interactions

Figures & data

Figure 1. NEAT1 pulldown using MORPH (A) semi quantitative PCR using NEAT1 primers to assess enrichment of NEAT1 after MORPH-based pulldown in even, odd and RNaseA groups. Schematic indicate the relative position of primer binding (B) correlation plot of the Log₂FC of the proteins detected in the ODD and EVEN set of oligos. (Pearson’s correlation of 0.91). (C) subcellular distribution of the proteins detected by MORPH-MS.

Figure 2. Gene ontology of the NEAT1 interacting proteins (A) bar graph depicting the enrichment score of the mentioned GO terms under biological processes (BP), Cellular component (CC) and Molecular function (MF) category. (B) heatmap showing the enrichment of the RNA splicing proteins detected in the ODD and EVEN pulldowns (C) heatmap showing the enrichment of the paraspeckles proteins in the ODD and EVEN sets of pulldown.

Table 1. Comparing MORPH with different antisense oligo-based capture techniques.

Name	oligo design	Fixation	Hybridization	Elution	Remark	Citation
iDRiP-MS	Fewer number of 20–25 oligomers biotinylated DNA oligos	UV crosslinking	slowly decreasing temperature-based hybridization	Tris-Hcl at 70°C for elution	Requires a smaller number (20–25) of individually biotin labelled probes, high cost of synthesis	[26,27]
CHART	Single 20-25mers biotinylated oligo designed by RNaseH mapping	3% Formaldehyde for 30 min	urea and denhardt solution at room temperature	RNaseH elution	Requires RNAseH mapping to design a biotinylated oligo	[7]
RAP-MS	Tiling array of 90mer oligos across target RNA	UV crosslink at 254 nm	using detergents such as DDM and sodium deoxycholate at 67°C	Benzonase nuclease	longer biotinylated oligos (4–5) increases the chances of non-specific binding	[6]
ChiRP-MS	Array of 20–25 oligomers biotinylated oligo across target	3% formaldehyde for 30 min	formamide based at 37°C	excess biotin	Multiple (upto 40) biotinylated oligos increase the cost of the protocol	[8]
HyPR-MS	Biotinylated capture oligo complementary to target RNA. The oligo has a toehold sequence	1% Formaldehyde fixation	DTT buffer at 37°C for 3 h	toehold sequence mediated release	Multiple biotinylated oligos having toehold sequence, increasing the cost. Use of RNA specific toehold sequence for the release of probes	[9]
MORPH-MS	Array of 20–25 oligomers having a common sequence at 5’ end across all oligos which can be captured by a single biotin labelled Universal oligo	3% PFA for 30 min	formamide based at 37°C	Benzonase elution	Use of a universal biotinylated oligo for all the anti-sense probes. Low cost of synthesis	This study

Figure 3. Validation of MORPH (A) venn diagram showing the number of common NEAT1 interacting proteins detected using CHART and MORPH-MS (Fisher exact test, p = 4.833e-89). (B) Venn diagram showing the number of common NEAT1 interacting proteins detected using CHART and HyPR-MS (Fisher exact test, p = 1.25e-08). (C) Venn diagram showing the number of common paraspeckle proteins detected by MORPH-MS (Fisher exact test, p = 6.49e-34). (D) Western blot after performing RNA pulldown via MORPH (MORPH-WB) using anti-SFPQ, anti-RBM14, anti-HNRNPK, and anti-RALY. (E) qRT-PCR using NEAT1 primers after performing RNA-IP from AC16 cell lysates using antibodies against SFPQ, RBM14, HNRNPK, and RALY. IgG was used as negative control.

Figure 4. Validation of proteins by RNA-IP qPCR (A) schema depicting the RNA-IP experiment. (B) qRT-PCR using NEAT1 primers after performing RNA-IP from HEK293T cell lysates using anti-GFP antibody to enrich FUBP1 and NCL. IgG was used as the negative control.

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Data availability statement

Processed mass spectrometry data are included as supporting files. Raw data were generated at the CSIR-Centre for Cellular and Molecular Biology. Raw data supporting the findings of this study are available from the corresponding author RK on request.