MORC2 regulates RBM39-mediated CDK5RAP2 alternative splicing to promote EMT and metastasis in colon cancer

Colorectal carcinogenesis and progression are associated with aberrant alternative splicing, yet its molecular mechanisms remain largely unexplored. Here, we find that Microrchidia family CW-type zinc finger 2 (MORC2) binds to RRM1 domain of RNA binding motif protein 39 (RBM39), and RBM39 interacts with site 1 of pre-CDK5RAP2 exon 32 via its UHM domain, resulting in a splicing switch of cyclin-dependent kinase 5 regulatory subunit associated protein 2 (CDK5RAP2) L to CDK5RAP2 S. CDK5RAP2 S promotes invasion of colorectal cancer cells in vitro and metastasis in vivo. Mechanistically, CDK5RAP2 S specifically recruits the PHD finger protein 8 to promote Slug transcription by removing repressive histone marks at the Slug promoter. Moreover, CDK5RAP2 S, but not CDK5RAP2 L, is essential for the promotion of epithelial-mesenchymal transition induced by MORC2 or RBM39. Importantly, high protein levels of MORC2, RBM39 and Slug are strongly associated with metastasis and poor clinical outcomes of colorectal cancer patients. Taken together, our findings uncover a novel mechanism by which MORC2 promotes colorectal cancer metastasis, through RBM39-mediated pre-CDK5RAP2 alternative splicing and highlight the MORC2/RBM39/CDK5RAP2 axis as a potential therapeutic target for colorectal cancer.

After washing with ultrapure water, the SDS-PAGE gel was cut into small pieces of 1 mm square and put in an Eppendorf tube.500 μL of destaining solution which was consisted of 50 mM ammonium bicarbonate (NH4HCO3) and 50% acetonitrile (ACN) was used for decolorizing until the gel pieces became colorless at 37 ℃, 600 rpm.After washing with 50 mM NH4HCO3, the gel pieces were solidified with ACN.Then the gel pieces were reduced with 10 mM dithiothreitol (DTT) which was dissolved with 50 mM NH4HCO3 at 37 ℃, 500 rpm for 1 h.After cooling to room temperature, ACN was added to solidify the gel.55 mM iodoacetamide (IAA) dissolved with 50 mM NH4HCO3 was added to gel to alkylate sample at room temperature for 40 min in darkness.After washing with 50 mM NH4HCO3, the gel pieces were solidified with ACN.Appropriate volume of 13 ng/μL trypsin solution dissolved with 50 mM NH4HCO3 was added to digest gel pieces and incubated at 37 ℃ overnight.Trifluoroacetic acid (TFA) was added to the sample to the final concentration of 1% to terminate the digestion process.At last, 0.1% TFA with 60% ACN and 0.1% TFA with 90% ACN were used to extract peptides from gel pieces.Peptides were desalted by Pierce C18 Spin Tips, and dried in a speed vacuum concentrator.
The peptides were re-dissolved in solvent A (A: 0.1% formic acid in water) and analyzed by Q-Exactive Plus coupled to an EASY-nanoLC 1200 system (Thermo Fisher Scientific, MA, USA). 4 μL peptide sample was loaded onto a 25 cm analytical column (75 μm inner diameter, 1.9 μm resin (Dr.Maisch) and separated with 60 min gradient starting at 2% buffer B (80% ACN with 0.1% FA) followed by a stepwise increase to 30% in 47 min, 100% in 1 min and stayed there for 12 min.The column flow rate was maintained at 300 nL/min with a column temperature of 40 ℃.The electrospray voltage was set to 2 kV.The mass spectrometer was run under data dependent acquisition (DDA) mode, and automatically switched between MS and MS/MS mode.The survey of full scan MS spectra (m/z 350-1,800) was acquired in the Orbitrap with 70,000 resolution.The automatic gain control (AGC) target of 3e6 and the maximum injection time of 50 ms.Then the precursor ions were selected into collision cell for fragmentation by higher-energy collision dissociation (HCD), the normalized collection energy was 28.The MS/MS resolution was set at 17,500, the automatic gain control target of 1e5, the maximum injection time of 45 ms, and dynamic exclusion was 30 seconds.
Trypsin was set as the digestion enzyme, and Semi-specific was specified as the digest type.PEAKS DB was searched with a fragment ion mass tolerance of 0.02 Da and a parent ion tolerance of 10 ppm.The max missed cleavages was 2.
Carbamidomethyl on Cysteine was specified as the fixed modification.Oxidation on Methionine, Deamination on Asparagine and Glutamine, and Acetylation on protein N-term were specified as the variable modifications.The peptides with 1% FDR and the proteins with 1% FDR and containing at least 1 unique peptide were filtered.
Flag-vector, Flag-CDK5RAP2 L or Flag-CDK5RAP2 S were transfected into SW620 cells, and the Co-IP experimental process was the same as above.The bands were excised from the gel and subjected to mass spectrometry analysis.The mass spectrometry experiments and data analysis were supported by Shanghai Applied Protein Technology Co., Ltd (Shanghai, China).The method of the mass spectrometry experiments has been described previously in detail [61].
The primers used for qPCR are shown in Supplementary Table 4.

Supplemental Tables
Supplementary Table 1： Information of 26 skipped exon events regulated upon RBM39 and MORC2 in shRBM39 RNA-seq.

RT-primers
Primer sequence

Fig. S1
Fig. S1 Analysis of MORC2-binding proteins and the expression of RBM39 regulated by MORC2.A,B KOG (A) and GO (B) analysis of MORC2-binding proteins.C Top 20 splicing factors interacting with MORC2 by MS analysis were ranked by intensity.D Western blot analysis of the indicated protein expression in SW480 and SW620 cells after knockdown of MORC2.E Western blot analysis of the indicated protein expression in HCT116 and SW480 cells transfected with Flag-vector and Flag-MORC2.F RT-qPCR was used to detect the effect of MORC2 knockdown on RBM39 mRNA expression.n=3, *P<0.05.G SW480 cells were transfected with Flag-vector and Flag-MORC2, followed by RT-qPCR to detect the mRNA level of RBM39.n=3, *P<0.05,**P<0.01.

Fig. S2
Fig. S2 Alternative splicing genes regulated by both RBM39 and MORC2.A and C RNA-seq data analysis showed alternative splicing events regulated by RBM39 silencing (A) and MORC2 silencing (C), respectively.Volcano plot on the left and statistical plot of the number of splicing events on the right, with three independent replicate experiments.B Volcano plot showing RNA bound to RBM39 in RIP-seq data (red).D Schematic representation of pre-MARK2, pre-OBSL1, pre-ZNF35 and pre-FRMD8 alternative splicing.Exons were represented by green, red and blue rectangles, introns by black lines, and splicing patterns by dashed lines.E,F RT-qPCR was used to detect the effect of RBM39 knockdown (E) or MORC2 knockdown (F) on MARK2 L, MARK2 S, OBSL1 L and OBSL1 S mRNA expression.n=3, *P<0.05,n.s indicates no significance.G,H RT-qPCR was used to detect the effect of RBM39 knockdown (G) or MORC2 knockdown (H) on ZNF35 L, ZNF35 S, FRMD8 L and FRMD8 S mRNA expression.n=3, *P<0.05,n.s indicates no significance.