The core spliceosomal factor U2AF1 controls cell-fate determination via the modulation of transcriptional networks

Abstract

Alternative splicing (AS) plays a central role during cell-fate determination. However, how the core spliceosomal factors (CSFs) are involved in this process is poorly understood. Here, we report the down-regulation of the U2AF1 CSF during stem cell differentiation. To investigate its function in stemness and differentiation, we downregulated U2AF1 in human induced pluripotent stem cells (hiPSCs), using an inducible-shRNA system, to the level found in differentiated ectodermal, mesodermal and endodermal cells. RNA sequencing and computational analysis reveal that U2AF1 down-regulation modulates the expression of development-regulating genes and regulates transcriptional networks involved in cell-fate determination. Furthermore, U2AF1 down-regulation induces a switch in the AS of transcription factors (TFs) required to establish specific cell lineages, and favours the splicing of a differentiated cell-specific isoform of DNMT3B. Our results showed that the differential expression of the core spliceosomal factor U2AF1, between stem cells and the precursors of the three germ layers regulates a cell-type-specific alternative splicing programme and a transcriptional network involved in cell-fate determination via the modulation of gene expression and alternative splicing of transcription regulators.

Keywords: Cell-fate determination; RNA; Spliceosome; Splicing; Stem cells; Transcription.

Figure 1.

U2AF1 cell specific expression and the impact of U2AF1 down-regulation on pluripotency genes.

Figure 2.

Developmentally regulated genes and transcription factors are enriched for among the U2AF1 target genes.

Figure 3.

U2AF1 down-regulation governs a specific AS program and impacts open reading frame of transcription factors.

Figure 4.

U2AF1 down-regulation preferentially modulates TFs’ AS and favors the DNMT3B isoform specific to differentiated cell-types(A, B) Sashimi plots showing RNA-Seq reads of two genes involved in cell-fate regulation, and how the alternative splicing events of these genes localize to their functional domains, leading to the creation of a new protein isoform or to the disruption of the ORF. (C) Sashimi plots showing the RNA- seq reads of DNMT3B localized to its functional domains. In addition, these plots show how the down- regulation of U2AF1 in hiPSCs favors the splicing of the DNMT3B. (D) Schematic representation of DNMT3B functional domains impacted by U2AF1 down-regulation.

Figure 5.

U2AF1 controls a cell-specific AS program.

Figure 6.

Model of a level-dependent action of the core spliceosomal factor U2AF1 during cell fate regulation. Modulation of U2AF1 expression levels trigger gene expression changes via both transcriptional regulation and AS changes.