MBNL proteins repress ES-cell-specific alternative splicing and reprogramming

Abstract

Previous investigations of the core gene regulatory circuitry that controls the pluripotency of embryonic stem (ES) cells have largely focused on the roles of transcription, chromatin and non-coding RNA regulators1,2,3. Alternative splicing represents a widely acting mode of gene regulation4,5,6,7,8, yet its role in regulating ES-cell pluripotency and differentiation is poorly understood. Here we identify the muscleblind-like RNA binding proteins, MBNL1 and MBNL2, as conserved and direct negative regulators of a large program of cassette exon alternative splicing events that are differentially regulated between ES cells and other cell types. Knockdown of MBNL proteins in differentiated cells causes switching to an ES-cell-like alternative splicing pattern for approximately half of these events, whereas overexpression of MBNL proteins in ES cells promotes differentiated-cell-like alternative splicing patterns. Among the MBNL-regulated events is an ES-cell-specific alternative splicing switch in the forkhead family transcription factor FOXP1 that controls pluripotency9. Consistent with a central and negative regulatory role for MBNL proteins in pluripotency, their knockdown significantly enhances the expression of key pluripotency genes and the formation of induced pluripotent stem cells during somatic cell reprogramming.

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Figure 1: Identification of regulators of ES-cell-differential alternative splicing.
Figure 2: MBNL proteins regulate ES-cell-specific alternative splicing.
Figure 3: MBNL proteins regulate approximately half of ES-cell-differential alternative splicing events.
Figure 4: Knockdown of MBNL proteins enhances reprogramming efficiency and kinetics.

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GEO accession numbers are provided in Supplementary Table 1.

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Acknowledgements

The authors thank U. Braunschweig, J. Ellis, S. Gueroussov and B. Raj for comments on the manuscript. We acknowledge D. Torti in the Donnelly Sequencing Centre for sequencing samples; L. Lee for assisting with the splicing code analysis; J. Garner (Hospital for Sick Children Embryonic Stem Cell Facility) for preparing feeder cells; A. Piekna for morphological examination of human iPSC colonies; M. Narimatsu for assisting with chimaerism analysis; and P. Mero for assisting with cell imaging. This work was supported by grants from the Canadian Institutes of Health Research (CIHR) (to B.J.B., J.L.W., A.N., J.E. and B.J.F.), the Ontario Research Fund (to J.L.W., B.J.B., A.N. and others), the Canadian Stem Cell Network (to A.N. and B.J.B.), and by a grant from the National Institutes of Health (R33MH087908) to J.E. H.H. was supported by a University of Toronto Open Fellowship. P.J.R., M.I. and N.L.B.-M. were supported by postdoctoral fellowships from the Ontario Stem Cell Initiative, Human Frontiers Science Program Organization, and the Marie Curie Actions, respectively.

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Authors

Contributions

H.H. performed experiments in Figs 14 and Supplementary Figs 2–9 and 11–13. M.I. performed bioinformatic analyses in Figs 14 and Supplementary Figs 1, 3, 7, 10 and 14, with input from N.L.B.-M. L.D. and A.G. assisted with secondary MEF reprogramming experiments and clone characterization, and D.T. generated secondary MEF lines and performed chimaerism testing. P.J.R., T.T. and M.G. performed human reprogramming experiments and iPSC characterization. H-K.S. performed teratoma assays. B.A. and B.J.F. generated splicing code data. I.P.M., H.-K.S. and D.O. assisted with ES-cell overexpression and differentiation experiments. E.W. and C.B.B. generated and analysed CLIP-seq data. E.N.N. and V.S. performed RT–PCR validation experiments. B.J.B., H.H. and M.I. designed the study, with input from J.L.W., J.E., A.N. and J.M. B.J.B., H.H. and M.I. wrote the manuscript, with input from the other authors.

Corresponding author

Correspondence to Benjamin J. Blencowe.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Methods, Supplementary References, full legends for Supplementary Tables 1-5 and Supplementary Figures 1-15. (PDF 8925 kb)

Supplementary Table 1

This file contains information on RNA-Seq datasets and samples – see Supplementary Information for full legend. (XLSX 48 kb)

Supplementary Table 2

This file contains information on Human and mouse ESC-differential AS events - Supplementary Information for full legend. (XLSX 195 kb)

Supplementary Table 3

This file contains DAVID (http://david.abcc.ncifcrf.gov/) output for functional enrichment categories for human, mouse or conserved ESC-differential AS events – see Supplementary Information for full legend. (XLSX 168 kb)

Supplementary Table 4

This file contains expression levels of the human and mouse splicing factors analyzed by RNA-Seq - see Supplementary Information for full legend. (XLSX 199 kb)

Supplementary Table 5

This file contains information on mouse ESC-differential AS events plotted in Supplementary Figure 14- see Supplementary Information for full legend. (XLSX 63 kb)

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Han, H., Irimia, M., Ross, P. et al. MBNL proteins repress ES-cell-specific alternative splicing and reprogramming. Nature 498, 241–245 (2013). https://doi.org/10.1038/nature12270

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