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Dynamics of Alternative Splicing During Somatic Cell Reprogramming Reveals Functions for RNA-Binding Proteins CPSF3, Hnrnp UL1 and TIA1

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Dynamics of Alternative Splicing During Somatic Cell Reprogramming Reveals Functions for RNA-Binding Proteins CPSF3, Hnrnp UL1 and TIA1 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.17.299867; this version posted September 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Dynamics of alternative splicing during somatic cell reprogramming reveals functions for RNA-binding proteins CPSF3, hnRNP UL1 and TIA1 Claudia Vivori1,2, Panagiotis Papasaikas1,#, Ralph Stadhouders1,##, Bruno Di Stefano1,%, Clara Berenguer Balaguer1,%%, Serena Generoso1,2, Anna Mallol1, José Luis Sardina1,%%, Bernhard Payer1,2, Thomas Graf1,2 and Juan Valcárcel1,2,3* 1 Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Carrer del Dr. Aiguader 88, 08003 Barcelona, Spain 2 Universitat Pompeu Fabra (UPF), Carrer del Dr. Aiguader 88, 08003 Barcelona, Spain 3 Institució Catalana de Recerca i Estudis Avançats (ICREA), Passeig Lluís Companys 23, 08010 Barcelona, Spain * Correspondence to [email protected] # Current address: Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66 / Swiss Institute of Bioinformatics, 4058 Basel, Switzerland ## Current address: Departments of Pulmonary Medicine and Cell Biology, Erasmus MC, Rotterdam, The Netherlands % Current address: Department of Molecular Biology, Massachusetts General Hospital / Center for Regenera- tive Medicine / Center for Cancer Research, Massachusetts General Hospital / Harvard Medical School, Boston, MA, USA / Department of Stem Cell and Regenerative Biology / Harvard Stem Cell Institute, Harvard University, Cambridge, MA, USA %% Current address: Josep Carreras Leukaemia Research Institute, Carretera de Can Ruti, Camí de les Escoles, s/n, 08916 Badalona, Spain 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.17.299867; this version posted September 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Abstract in coding capacity, stability or translational efficiency, and that can be translated into pro- In contrast to the extensively studied teins with distinct structural and functional rewiring of epigenetic and transcriptional pro- properties (Pan et al. 2008; E. T. Wang et al. grams required for cell reprogramming, the 2008). AS contributes to the regulation of dynamics of post-transcriptional changes and many biological processes in multicellular eu- their associated regulatory mechanisms re- karyotes, including embryonic development - main poorly understood. Here we have stud and tissue specification (reviewed in Baralle ied the dynamics of alternative splicing (AS) and Giudice, 2017). During the last decade, changes occurring during efficient repro- progress has been made to understand the gramming of mouse B cells into induced role of post-transcriptional regulation (includ- pluripotent stem (iPS) cells. These changes, ing AS) in the maintenance of cellular pluripo- generally uncoupled from transcriptional reg- tency and cell fate decisions, discovering ulation, significantly overlapped with splicing genes differentially spliced between stem cells programs reported during reprogramming of and differentiated cells and splicing regulators mouse embryonic fibroblasts (MEFs). Correla- that control these choices (G. W. Yeo et al. tion between gene expression of potential 2009; Salomonis et al. 2010; Gabut et al. 2011; regulators and specific clusters of AS changes Han et al. 2013; Venables et al. 2013; Lu et al. enabled the identification and subsequent 2014; Yamazaki et al. 2018; Solana et al. 2016a). validation of CPSF3 and hnRNP UL1 as facilita- tors, and TIA1 as repressor of MEFs repro- During reprogramming into induced gramming. These RNA-binding proteins con- pluripotent stem (iPS) cells, somatic cells revert trol partially overlapping programs of splicing to a pluripotent state after overexpression of regulation affecting genes involved in devel- the transcription factors OCT4, SOX2, KLF4 and opmental and morphogenetic processes. Our MYC (OSKM) (Takahashi and Yamanaka 2006). results reveal common programs of splicing Substantial progress has been made to under- regulation during reprogramming of different stand the process at the transcriptional and cell types and identify three novel regulators epigenetic level, such as by identifying nu- of this process. merous roadblocks and some facilitators, but comparatively little is known about how post- transcriptional regulation impacts cell fate de- Keywords cisions. Recent work has revealed the func- tional relevance and conservation of splicing Alternative splicing, Somatic cell repro- regulation during reprogramming (Han et al. gramming, CPSF3, hnRNP UL1, TIA1, Pluripo- 2013; Ohta et al. 2013; Toh et al. 2016; Kanitz et tency al. 2019; reviewed in Zavolan and Kanitz, 2018). Previously, a conserved functional splic- Introduction ing program associated with pluripotency and repressed in differentiated cells by the RNA- Alternative splicing (AS) is a widespread binding proteins MBNL1 and MBNL2 was pre- mechanism of gene regulation that generates viously reported (Han et al. 2013). This splicing multiple mRNA isoforms from a single gene, program includes a mutually exclusive exon dramatically diversifying the transcriptome event in the transcription factor FOXP1: a (and proteome) of eukaryotic cells. 95% of switch in inclusion of Foxp1 exons 16/16b multi-exonic mammalian genes undergo AS, modulates the functions of the transcription producing mRNA isoforms which often differ factor between pluripotent and differentiated 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.17.299867; this version posted September 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. cells (Gabut et al. 2011). Illustrating the com- Results plexity of such splicing program, dynamic changes of AS during cell reprogramming of mouse embryonic fibroblasts (MEFs) revealed Dynamics of alternative splicing sequential waves of exon inclusion and skip- in C/EBPα-enhanced B cell ping in reprogramming intermediates and the reprogramming occur independently from gene expression changes functional role of splicing regulators in modu- lating reprogramming, in particular during the To study the dynamics of changes in alter- initial mesenchymal-to-epithelial transition native splicing (AS) during cell reprogram- (MET) phase (Cieply et al. 2016). Given the lim- ming, primary mouse pre-B cells (hereafter ited efficiency of cell reprogramming in this referred to as “B cells”) were reprogrammed as system, subpopulations of reprogramming previously described (Di Stefano et al. 2016; intermediates had to be isolated through the Stadhouders et al. 2018; Di Stefano et al. 2014). expression of a pluripotency marker, biasing Briefly, B cells were isolated from bone marrow studies to the most prevalent and dominant of reprogrammable mice, containing a doxy- factors. cycline-inducible OSKM cassette and an OCT4- GFP reporter. These cells were infected with a Here we took advantage of the rapid, high- retroviral construct containing an inducible ly efficient and largely synchronous repro- version of C/EBPα fused to the estrogen recep- gramming of pre-B cells (hereafter referred to tor ligand-binding domain (ER). Infected cells as “B cell reprogramming”), obtained by a were selected and re-plated on a feeder layer pulse of the transcription factor C/EBPα fol- of inactivated mouse embryonic fibroblasts lowed by induced OSKM expression (Di Ste- (MEFs). This was followed by a 18h-long pulse fano et al. 2014, 2016) to study the dynamics of β-estradiol, triggering the translocation of of AS during this transition. The essentially C/EBPα-ER to the nucleus and poising the B homogeneous reprogramming of the cells in cells for efficient and homogeneous repro- this system allowed detailed temporal tran- gramming (Di Stefano et al. 2016, 2014). After scriptome analyses of the bulk population, washout of β-estradiol, reprogramming was without the need of selecting for reprogram- induced by growing the cells for 8 days in re- ming intermediates. We established clusters of programming medium containing doxycycline temporal regulation and compared these (see Methods and Stadhouders et al. 2018). changes with the ones differentially spliced in RNA was isolated every other day from dupli- MEF reprogramming (Cieply et al. 2016). Ana- cates and subjected to paired-end sequencing lyzing the dynamic expression of RNA-binding (RNA-seq), resulting in high coverage (more proteins (RBPs) during reprogramming, we than 100 million reads per sample) (Figure inferred potential AS regulators, of which three 1A). As positive controls, mouse embryonic were studied in detail. Characterization of stem (ES) cells and induced pluripotent stem these factors, namely CPSF3, hnRNP UL1 and (iPS) cells were also sequenced. TIA1, by perturbation experiments demon- strated their role as AS regulators in the induc- AS analysis was performed using vast-tools tion of pluripotency. (Tapial et al. 2017), an event-based software that quantifies Percent Spliced-In (PSI) values of annotated AS events and constitutive exons in all samples. These analyses revealed more than 14000 AS changes during the entire re- programming time course (for any possible 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.17.299867; this version posted September 18, 2020. The copyright holder for this
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