Abstract
The RNA-binding proteins PTBP1 and PTBP2 control programs of alternative splicing during neuronal development. PTBP2 was found to maintain embryonic splicing patterns of many synaptic and cytoskeletal proteins during differentiation of neuronal progenitor cells (NPCs) into early neurons. However, the role of the earlier PTBP1 program in embryonic stem cells (ESCs) and NPCs was not clear. We show that PTBP1 controls a program of neuronal gene expression that includes the transcription factor Pbx1. We identify exons specifically regulated by PTBP1 and not PTBP2 as mouse ESCs differentiate into NPCs. We find that PTBP1 represses Pbx1 exon 7 and the expression of the neuronal Pbx1a isoform in ESCs. Using CRISPR-Cas9 to delete regulatory elements for exon 7, we induce Pbx1a expression in ESCs, finding that this activates transcription of neuronal genes. Thus, PTBP1 controls the activity of Pbx1 to suppress its neuronal transcriptional program prior to induction of NPC development.
Highlights
Alternative splicing is an important form of gene regulation during tissue development
Mouse embryonic stem cells (ESCs) (Day -2) were grown in aggregate culture for two days to form embryoid bodies (EBs; Day 0), which were treated with retinoic acid (RA) and a Sonic hedgehog (Shh) pathway agonist for 5 days to induce motor neurons (MNs) formation (Wichterle et al, 2002; Adams et al, 2015d)
Similar to observations in the brain, as ESCs differentiate into MNs, PTBP1 expression declines, while PTBP2 expression is induced (Figure 1B and C) (Boutz et al, 2007; Tang et al, 2011; Zheng et al, 2012)
Summary
Alternative splicing is an important form of gene regulation during tissue development. In the mammalian nervous system, large scale changes in splice site choice produce many new mRNAs encoding protein isoforms with different structures and functions that are specific to neurons (Li et al, 2007; Licatalosi and Darnell, 2006; Norris and Calarco, 2012a; Raj and Blencowe, 2015a; Yap and Makeyev, 2013; Zheng and Black, 2013) These splicing patterns are regulated in a temporal and cell-specific manner by the expression of specialized pre-mRNA binding proteins (RBPs) (Black, 2003; Braunschweig et al, 2013; Fu and Ares, 2014; Lee and Rio, 2015).
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