We utilize PTBP2 binding and splicing maps to guide ASO disruption of PTBP2 binding to SYNGAP1, effectively redirecting splicing to increase gene expression. We perform extensive follow-up on one such gene, SYNGAP1, where variants lead to reduced expression (haploinsufficiency) and a phenotypically broad neurodevelopmental disorder 15, 16, 17. These negatively regulated genes represent potential targets for antisense splice switching for therapeutic upregulation. We identify genes that are alternatively spliced and repressed by PTBP2, several of which cause human disease when reduced in expression. We perform CLIP-seq analysis of PTBP2 binding in both human cortical tissue and human neurons derived from induced pluripotent stem cells (iPSC-neurons), and we combine this with splicing analysis following PTBP2 depletion in iPSC-neurons (Fig. Here we determine the direct targets of PTBP2-dependent AS in the human brain. However, neither PTBP2 binding nor splicing has been assessed in human neurons nor post-embryonic human brain tissue. Such an approach has identified axonogenesis-associated Ptbp2 targets in E18.5 mouse neocortex 12, 14. Yet to identify direct targets of PTBP2 splicing in the human brain, transcriptome-wide PTBP2 binding must be combined with differential splicing analysis upon PTBP2 manipulation. To identify binding sites of PTBPs, cross-linking immunoprecipitation followed by RNA sequencing (CLIP-seq) has been used in non-neuronal cells for PTBP1 13 and mouse embryonic cortical tissue for PTBP2 12. Analysis of differentially expressed transcripts upon PTBP2 ablation suggests preferential regulation of targets involved in pre- and post-synaptic assembly and synaptic transmission 11. PTBP2 is required for neuron development and survival and functions in part to suppress adult splicing patterns to control the temporal regulation of neuronal maturation 10, 11, 12. PTBP1 is broadly expressed across cell types but largely absent from neurons, while PTBP2 is predominantly neuronal (also referred to as “nPTB”). PTBP1 and PTBP2 are structurally similar and bind overlapping RNA targets yet differ by their cell type expression patterns. This ASO binds to the SMN2 pre-mRNA to disrupt a splice silencing RBP, in turn promoting SMN2 exon 7 inclusion and augmented SMN protein expression, improving disease trajectory 8, 9.ĪS of neuronal genes is controlled by the coordinated action of several RBPs, which include the polypyrimidine tract binding proteins (PTBP1 and PTBP2). Spinraza® is a prominent example of a splice-switching ASO that exerts therapeutic benefit in the central nervous system disorder spinal muscular atrophy (SMA). Since AS is controlled by trans-acting RNA-binding proteins (RBPs) that promote or repress splicing, steric-blocking ASOs that disrupt the interaction between these proteins and their target pre-mRNA can redirect AS for therapeutic benefit 6, 7. Depending on their chemistry, ASO binding can result in target gene degradation or modulation of RNA processing (for review, see ref. ASOs are short, single-stranded nucleic acid analogs that take advantage of Watson-Crick base pairing to target RNA molecules. Therapeutic targeting of AS with antisense oligonucleotides (ASOs) that redirect splicing has demonstrated clinical potential for treating neurological disorders 4. Concurrently, aberrant AS is implicated in multiple neurological disorders (for reviews, see refs. There is a particularly high frequency of AS in the brain, where it is required for all aspects of nervous system development and function. Our data comprehensively map PTBP2-dependent alternative splicing in human neurons and cerebral cortex, guiding development of novel therapeutic tools to benefit neurodevelopmental disorders.Īlternative splicing (AS) of precursor mRNA (pre-mRNA) is an essential mechanism for post-transcriptional gene diversification and regulation. In SYNGAP1 haploinsufficient iPSC-neurons generated from two patients, we show that PTBP2-targeting ASOs partially restore SYNGAP1 expression. We find that PTBP2 binding to SYNGAP1 mRNA promotes alternative splicing and nonsense-mediated decay, and that antisense oligonucleotides (ASOs) that disrupt PTBP binding redirect splicing and increase SYNGAP1 mRNA and protein expression. We map PTBP2 binding sites, characterize PTBP2-dependent alternative splicing events, and identify novel PTBP2 targets including SYNGAP1, a synaptic gene whose loss-of-function leads to a complex neurodevelopmental disorder. Here, we define the PTBP2 footprint in the human transcriptome using brain tissue and human induced pluripotent stem cell-derived neurons (iPSC-neurons). While PTBP1 is ubiquitously expressed, PTBP2 is predominantly neuronal. Alternative splicing of neuronal genes is controlled partly by the coordinated action of polypyrimidine tract binding proteins (PTBPs).
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