MYC regulates the core pre-mRNA splicing machinery as an essential step in lymphomagenesis

Abstract

Deregulated expression of the MYC transcription factor occurs in most human cancers and correlates with high proliferation, reprogrammed cellular metabolism and poor prognosis1. Overexpressed MYC binds to virtually all active promoters within a cell, although with different binding affinities2,3,4, and modulates the expression of distinct subsets of genes1,2,4,5. However, the critical effectors of MYC in tumorigenesis remain largely unknown. Here we show that during lymphomagenesis in Eµ-myc transgenic mice, MYC directly upregulates the transcription of the core small nuclear ribonucleoprotein particle assembly genes, including Prmt5, an arginine methyltransferase that methylates Sm proteins6,7. This coordinated regulatory effect is critical for the core biogenesis of small nuclear ribonucleoprotein particles, effective pre-messenger-RNA splicing, cell survival and proliferation. Our results demonstrate that MYC maintains the splicing fidelity of exons with a weak 5′ donor site. Additionally, we identify pre-messenger-RNAs that are particularly sensitive to the perturbation of the MYC–PRMT5 axis, resulting in either intron retention (for example, Dvl1) or exon skipping (for example, Atr, Ep400). Using antisense oligonucleotides, we demonstrate the contribution of these splicing defects to the anti-proliferative/apoptotic phenotype observed in PRMT5-depleted Eµ-myc B cells. We conclude that, in addition to its well-documented oncogenic functions in transcription2,3,4,5 and translation8, MYC also safeguards proper pre-messenger-RNA splicing as an essential step in lymphomagenesis.

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Figure 1: MYC directly upregulates the core snRNP assembly genes, including PRMT5.
Figure 2: PRMT5 is essential for maintaining splicing fidelity.
Figure 3: Acute MYC depletion in murine and human lymphoma cells results in aberrant splicing.
Figure 4: Antisense oligonucleotides targeting ATR-, EP400- and DVL1-alternative splicing mimic the cell-cycle arrest/apoptotic response induced by MYC/PRMT5 depletion.

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Acknowledgements

We thank S. Campaner, M. Pelizzola, D. Messerschmidt and P. R. Kaldis for sharing protocols and for discussions; F. B. M. Ibrahim and V. S. Y. Tay for help with histopathology work; BRC Shared Facilities for technical support; D. Bararia for help in the fluorescence-activated cell sorting analysis. We are grateful to X. Ruan, W. Soon and the GIS Genome Sequencing Team for help with the Solexa high-throughput sequencing, and to the entire E.G. laboratory for discussion. This work was supported by an AGA-SINGA (SINgapore Graduate Award) fellowship to M.B. and by IMCB, A-STAR. E.G. acknowledges support from JCO-ASTAR grants 1134c001 and 11/03/FG/07/04. B.A. acknowledges support from the European Research Council, the Italian health ministry and the Italian Association for Cancer Research (AIRC).

Author information

C.M.K., M.B., E.G.: overall design of the project. C.M.K., M.B., W.X.A., S.X.T., A.S., F.P.H.G.: acquisition of samples. C.M.K., M.B., E.G., D.L.: detailed molecular and genomics approaches. D.L.: bioinformatic analysis. C.M.K., M.B., BA, E.G.: organization of experiments and figures. M.B., M.O.: fluorescence-activated cell sorting analysis. K.B.W.: antisense oligonucleotide design. M.A.H., T.S.Y.: histopathology. C.M.K. and E.G. wrote the manuscript with comments from all authors.

Correspondence to Ernesto Guccione.

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Competing interests

The authors declare no competing financial interests.

Additional information

RNA-seq and microarray datasets generated in this study have been deposited in Gene Expression Omnibus database in the Superseries GSE61638.

Extended data figures and tables

Extended Data Figure 1 MYC directly upregulates the core snRNP assembly genes.

a, RNA-seq expression of all Refseq genes, core Snrnp genes, spliceosome genes and RNA splicing genes in wild-type, Eµ-myc pre-tumoural B cells and Eµ-myc tumour B cells. b, ChIP-seq analysis of MYC binding (top lanes) and RNA-seq (bottom lanes) showing the expression of ‘core snRNPs assembly’ genes in control (C), Eµ-myc pre-tumoural (P) and Eµ-myc tumours (T). Student’s t-test (two-tailed) was used; *P < 0.05, **P < 0.01. c, Quantitative PCR validation of the expression of representative ‘core snRNPs assembly’ genes in wild-type B cells (n = 7), Eµ-myc pre-tumoural B cells (n = 4) and Eµ-myc tumour cells (n = 16). d, Immunoblots of representative ‘core snRNPs assembly’ proteins in wild-type B cells (n = 4) and Eµ-myc tumour cells (n = 4). e, Spearman’s r2 and P values for the correlation between the expression of MYC and PRMT5, WDR77, SNRPB, SNRPD1 and SNRPD3 in publicly available lymphoma data sets. f, Correlation between the expression of PRMT5 and MYC in 29 samples from patients with primary leukaemia and lymphoma. g, Quantification of PRMT5 staining in normal human lymph nodes and lymphoma. A representative image for IHC staining of PRMT5 in normal human tonsil and follicular lymphoma. h, Survival of patients with lymphoma stratified for PRMT5 and MYC expression, individually and combined.

Extended Data Figure 2 Reduced disease burden in Eμ-myc;Prmt5+/− mice.

a, Analysis of WBC counts in 8-week-old wild-type (n = 20), Prmt5+/− (n = 13), Eµ-myc (n = 21) and Eµ-myc;Prmt5+/− (n = 19) littermates. Each point represents one animal. b, Analysis of tumour burden in 8-week-old Eµ-myc (n = 15) and Eµ-myc;Prmt5+/− (n = 15) littermates. Each point represents one animal. c, Representative images showing haematoxylin and eosin and IHC for PRMT5, B220, Ki67 and Y12 in the spleens of 8-week-old, age-matched wild-type, Prmt5+/−, Eµ-myc and Eµ-myc;Prmt5+/− littermates.

Extended Data Figure 3 PRMT5 depletion leads to a reduction in functioning of the core splicing machinery.

a, A representative immunoblot showing the reduction of PRMT5 protein levels after OHT treatment, and a corresponding decrease in Y12 levels (methylated Sm proteins). b, Validation of alternative splicing of Mdm4 mRNA in Eμ-myc B cells after PRMT5 deletion. c, Functional annotation of genes affected by alternative splicing events (either exon skipping or intron retention). d, Distribution of reads within exons and introns (ratio of OHT/EtOH). e, MATS output: quantification of skipped exons (SE), retained introns (RI), mutually exclusive exons (MXE), A5SS and A3SS (alternative 5′ or 3′ splice site). f, Shapiro score of the 5′ donor sites of retained intron events identified by MATS. A smooth density estimate is drawn as calculated by a Gaussian kernel. Top: sequence logo of the 5′donor of retained intron events (left) compared with the 5′donor of the downstream exon (right), detected in PRMT5-depleted cells. Bottom: sequence logo of the 5′donor sites of the skipped exon events (right), compared with the 5′donor of the upstream exon (left).

Extended Data Figure 4 Alternative splicing events in Eµ-myc;Prmt5F/FCreER bone marrow pre-B cells lead to reduction in full length protein levels.

a, Validation of additional alternatively spliced transcripts in Eµ-myc;Prmt5F/FCreER and Prmt5F/FCreER bone marrow B cells after Prmt5 deletion (OHT) by semi-quantitative PCR. Quantification of three independent biological replicates is shown on top, while a representative example is shown in the bottom panel. b, Quantification of selected proteins by immunoblotting or flow cytometry in Eµ-myc;Prmt5F/FCreER bone marrow B cells after Prmt5 deletion (OHT).

Extended Data Figure 5 Alternative splicing events in Eµ-myc;Prmt5F/FCreER lymphoma cells.

a, Disease burden of recipient mice (n = 5 for each group), as assessed by white blood cell (WBC) counts (left panel), spleen weight (middle panel) and tumour weight (right panel), 3 weeks after transplantation. b, Representative images showing the haematoxylin and eosin and IHC for B220, Ki67, PRMT5 and Y12 in the spleens of recipient mice. c, Validation of additional alternatively spliced transcripts in Eµ-myc;Prmt5F/FCreER lymphoma B cells after Prmt5 deletion (OHT) by semi-quantitative PCR. Representative gel images are shown (n = 5). d, Expression of p53 target genes after Prmt5 deletion in Eµ-myc;Prmt5F/FCreER lymphoma cells, as a demonstration of functional/inactive p53 response. Of the 22 lymphomas, each isolated from independent tumour-bearing Eµ-myc;Prmt5F/FCreER mice, five (22.72%) showed the upregulation of classical p53 target genes in response to PRMT5 deletion (indicating a functional p53 pathway), while 17 (77.27%) did not (indicating an inactive p53 pathway), rates that are similar to previous reports26. e, Validation of alternatively spliced transcripts in Eµ-myc;Prmt5+/FCreER lymphoma B cells after Prmt5 deletion (OHT) by semi-quantitative PCR. Representative gel images are shown (n = 3). f, Validation of additional alternatively spliced transcripts in after Myc knockdown in Eµ-myc lymphoma B cells after Prmt5 deletion (OHT) by semi-quantitative PCR. Representative gel images are shown (n = 4).

Extended Data Figure 6 PRMT5 and SmB depletions in human Burkitt lymphoma lines lead to reduced viability and splicing defects.

a, Relative viability of Raji and Daudi cells upon PRMT5 depletion with four independent shRNAs (shPrmt5-1 to shPRMT5-4) (n = 4). b, Apoptosis profile of Raji and Daudi cells after PRMT5 knockdown (shPrmt5-1 to shPRMT5-4) (n = 4). c, Cell-cycle profile of Raji and Daudi cells after PRMT5 knockdown (shPrmt5-1 and shPRMT5-4) (n = 4). d, Control and PRMT5 depleted (shPrmt5-1 and shPRMT5-2) Daudi and Raji cells were xenografted into SCID recipients. The Kaplan–Meier analysis of tumour-free survival of the recipient mice is shown; n, number of recipient mice analysed. e, PRMT5 and Y12 (methylated Sm proteins) levels upon PRMT5 depletion in Raji and Daudi cells. A representative blot is shown. f, Validation of additional alternatively spliced transcripts (retained introns and skipped exons) after PRMT5 knockdown in Daudi and Raji cells by semi-quantitative PCR (n = 4). g, Validation of SMB knockdown in Daudi cells (upper left panel) and relative viability after SMB knockdown (lower left panel) (n = 3). Validation of additional alternatively spliced transcripts (retained introns and skipped exons) after SMB knockdown in Daudi cells by semi-quantitative PCR and quantification of gels by Image J. The data presented in this figure are the average and s.d. Student’s t-test (two sided) was used; *P < 0.05, **P < 0.01.

Extended Data Figure 7 Myc depletion leads to splicing defects both in mouse and in human lymphoma cells.

a, Relative expression of selected ‘core snRNPs assembly’ genes following MYC knockdown in Eµ-myc B cells, assessed by quantitative real-time PCR (n = 3). b, Validation of alternatively spliced transcripts (retained introns and skipped exons) after MYC knockdown in Eµ-myc B cells by semi-quantitative PCR (n = 3; representative gel images are shown). c, MYC, PRMT5, SmD1, SmD3 and β-actin protein expression in whole cell lysates from Daudi and Raji cells infected with viruses encoding non-targeting shRNA (shControl) and two independent sequences targeting MYC (shMyc-1, shMyc-2). d, Validation of alternatively spliced transcripts (retained introns and skipped exons) after MYC knockdown in Daudi and Raji cells by semi-quantitative PCR (n = 3). shControl, scramble control shRNA; shMyc-1, shMyc-2, shRNA sequences targeting MYC. The data presented in this figure are the average and s.d. Student’s t-test (two-sided) was used; *P < 0.05, **P < 0.01.

Extended Data Figure 8 PRMT5 is required for normal haematopoiesis.

a, Fetal liver cellularity at E14.5, after tamoxifen injection at E10.5 (Prmt5+/FCreER: n = 3, Prmt5F/FCreER, n = 5). b, Flow cytometry analysis of Kit+LinCD34+ (bottom panel), Kit+LinCD34 (top panel), using fetal liver cells from a. Total number of cells is indicated (Prmt5+/FCreER: n = 3, Prmt5F/FCreER, n = 5). c, Methocult M3434 colony formation assay using fetal liver cells from a. CFU-GEMM, colony-forming unit—granulocyte, erythrocyte, monocyte/macrophage, megakaryocyte; CFU-GM, colony-forming unit—granulocyte, macrophage; CFU-M, colony-forming unit—macrophage; CFU-G, colony-forming unit—granulocyte; BFU-E, burst-forming unit—erythrocyte. d, GO analysis of differentially expressed genes after PRMT5 deletion in fetal liver cells (n = 4). e, Bone marrow cellularity of adult mice after tamoxifen injection (n = 3 in each group). f, Methocult M3434 colony formation assay using bone marrow cells from e. g, Whole blood counts (WBC) 5 days after PRMT5 deletion in vivo (n = 5). NE, neutrophils; LY, lymphocytes; MO, monocytes; EO, eosinophils; BA; basophils; RBC, red blood cells. h, Bone marrow cellularity after selective deletion of PRMT5 in the haematopoietic system (n = 4 mice in each group). The data presented in this figure are the average and s.d. Student’s t-test (two-sided) was used; *P < 0.05, **P < 0.01.

Extended Data Figure 9 Antisense oligonucleotides targeting ATR, EP400 and DVL1 lead to the reduction of their full-length protein levels.

a, Sashimi plots showing alternatively spliced transcripts of DVL1, ATR and EP400 after PRMT5 depletion in Eµ-myc;Prmt5F/FCreER cells. b, Quantification of protein expression after electroporation of Eµ-myc B cells with the respective ASOs (n = 3). c, Upper panel: schematic representation of ASOs designed to block the intron retention in Dvl1 induced by PRMT5 knockout. Middle panel: validation of efficacies of ASOs in reversing the alternative splicing of Dvl1, after PRMT5 knockout (n = 3). Bottom panel: cell viability of Eµ-myc B cells 2 days after the electroporation with the respective ASOs. The data presented in this figure are the average and s.d. Student’s t-test (two-sided) was used; *P < 0.05, **P < 0.01.

Extended Data Figure 10 Graphical summary.

Top panel: black lines indicate MYC direct transcriptional upregulation of PRMT5 and other components of the core snRNP assembly machinery, which ensures splicing fidelity. Bottom panel: red arrows indicate the perturbation of the MYC–PRMT5 axis, which leads to a reduction in splicing fidelity within the cell, skipped exons and retained introns of genes, such as Ep400, Dvl1 and Atr (which harbour exons with weak 5′-donor sites), downregulation of their protein levels and, consequently, cell-cycle arrest and apoptosis.

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Koh, C., Bezzi, M., Low, D. et al. MYC regulates the core pre-mRNA splicing machinery as an essential step in lymphomagenesis. Nature 523, 96–100 (2015). https://doi.org/10.1038/nature14351

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