We show that the splicing regulator PTBP2 controls a genetic program essential for neuronal maturation. Depletion of PTBP2 in developing mouse cortex leads to degeneration of these tissues over the first three postnatal weeks, a time when the normal cortex expands and develops mature circuits. Cultured Ptbp2−/− neurons exhibit the same initial viability as wild type, with proper neurite outgrowth and marker expression. However, these mutant cells subsequently fail to mature and die after a week in culture. Transcriptome-wide analyses identify many exons that share a pattern of mis-regulation in the mutant brains, where isoforms normally found in adults are precociously expressed in the developing embryo. These transcripts encode proteins affecting neurite growth, pre- and post-synaptic assembly, and synaptic transmission. Our results define a new genetic regulatory program, where PTBP2 acts to temporarily repress expression of adult protein isoforms until the final maturation of the neuron.
Li et al. Supplemental File 1: Table 1. Exons validated by RT/PCR.
Table S1 for Li et al. A variety of exons showing differences in splicing between wild type and Ptbp2 nestin-KO brain at embryonic day 18 were subjected to further analysis by RT/PCR. These included exons identified by MJAY array, RNAseq, and exons previously reported to be PTBP targets. Gene names, exon coordinates, and percent spliced in values (PSI) are listed. Genome coordinates are for mm9.
Supplemental File 1 Table S1 RT-PCR validated E18 exons.docx
Li et al. Supplemental File 2: Table 2. Splicing Changes Identified by Exon Junction Microarray.
RNA from WT and Ptbp2-NestinKO E18 pups was analyzed on Affymetrix MJAY arrays (see Methods). Splicing events yielding Sepscores greater than 0.345 are listed.
Supplemental File 2 Table_S2_MJAY Array_top_Splicing_events_by_SepScore.xls
Li et al. Supplemental File 3: Table 3. Splicing Changes Identified by RNAseq analysis in Ptbp2-NesKO mice.
RNAseq reads from E18 WT and NesKO mice were subjected to Splicetrap analysis (see Methods). Events with a change in PSI of greater than 10% are shown with genome coordinates, read numbers, and coverage statistics.
Supplemental File 3 Table S3_nestin_nptbko_SpliceTrap_psi_over10percent_with_pvalues.xls
Li et al. Supplemental File 4: Table 4. Gene Ontology Analysis of Functional or Process Enrichment for Genes Showing Splicing Changes in Ptbp2-NesKO mice.
Enrichments are relative to all genes expressed in wildtype brain at E18.
Supplemental File 4 Table_S4_GO_analysis_nestin_splicetrap_exons.xls
Li et al. Supplemental File 5: Table 5. Expression changes in Ptbp2-NesKO mice.
Genes showing overall expression changes were identified by Cuffdiff analysis.
Supplemental File 5 Table_S5_cuffdiff_NesKOvWT_expression_change_V3.xls
Li et al. Supplemental File 6: Table 6. Gene Ontology analysis of genes up-regulated in Ptbp2-NesKO mice.
Genes showing a two-fold or greater increase in expression in the KO relative to WT were analyzed for functional or process enrichment relative to all genes expressed in wildtype brain.
Supplemental File 6 Table_S6_gene_ontology_2_fold_upregulated transcripts_v2.xls
Li et al. Supplemental File 7: Table 7. Gene Ontology analysis of genes down-regulated in Ptbp2-NesKO mice.
Genes showing two-fold or greater reduced expression in the KO relative to WT were analyzed for functional or process enrichment relative to all genes expressed in wildtype brain.
Supplemental File 7 Table_S7_gene_ontology_2_fold_change_down_regulated_transcripts_v2.xls
Li et al. Supplemental File 8: Table 8. Splicing Changes Identified by RNAseq analysis in Ptbp2-EmxKO mice.
RNAseq reads from P1 WT and EmxKO mice were subjected to Splicetrap analysis (see Methods). Events with a change in PSI of greater than 10% are shown with genome coordinates, read numbers, and coverage statistics.
Supplemental File 8 Table_S8_Emx_P1_SpliceTrap_psi_over10percent_with_pvalues.xls
