UCLA

Alternative splicing is an important form of gene regulation during tissue development. In the mammalian nervous system, complex splicing patterns produce new protein isoforms with different structures and functions specific to neurons. These splicing patterns are regulated in a temporal and cell-specific manner by the expression of specialized pre-mRNA binding proteins (RBPs). The polypyrimidine tract binding (PTB) proteins, PTBP1 and PTBP2, are RBPs that control extensive programs of alternative splicing during neuronal differentiation and maturation. However, the neuronal splicing programs regulated by PTBP1 and PTBP2 are not fully characterized, and the cellular function of their regulated isoforms remains largely unknown. Here we define the PTBP1 splicing program during in vitro neuronal differentiation and show that PTBP1 guides developmental gene expression programs by regulating an alternative exon in the homeodomain transcription factor Pbx1.

We use RNA-sequencing to identify thousands of alternative exons that are differentially spliced as mouse embryonic stem cells (ESC) differentiate into neuronal progenitor cells (NPC) and then into neurons, during the transition from PTBP1 expression to PTBP2. We then define the subset of these neuronal exons controlled by PTBP1 in ESCs and NPCs by RNA-sequencing analysis after PTBP1 depletion and PTBP1 crosslinking-immunoprecipitation (iCLIP-seq). Among these targets, we find that PTBP1 represses splicing of exon 7 in the Pbx1 transcript leading to expression of the Pbx1b isoform in ESCs and NPCs. To study the consequences of this Pbx1 splicing switch, we use the CRISPR-Cas9 technology to induce or eliminate exon 7 splicing in different cell types. We delete regulatory elements in Pbx1 intron 6 to force exon 7 splicing and Pbx1a expression in ESCs. We find that the early expression of Pbx1a enhances expression of a defined group of neurogenic genes, many of which were previously identified as Pbx1 targets. Cas9-targeted deletion of Pbx1 exon 7 forces expression of Pbx1b, with the loss of Pbx1a, in differentiated motor neurons. We find that these cells differentiate normally but exhibit altered expression of Pbx1 target genes. These data demonstrate that one component of the PTBP1 regulatory network is to control the activity of the transcription factor Pbx1 and thus to alter the transcriptional program of neuronal differentiation.