PTBP1 and PTBP2 Serve Both Specific and Redundant Functions in Neuronal Pre-mRNA Splicing

Abstract

Families of alternative splicing regulators often contain multiple paralogs presumed to fulfill different functions. Polypyrimidine tract binding proteins PTBP1 and PTBP2 reprogram developmental pre-mRNA splicing in neurons, but how their regulatory networks differ is not understood. To compare their targeting, we generated a knockin allele that conditionally expresses PTBP1. Bred to a Ptbp2 knockout, the transgene allowed us to compare the developmental and molecular phenotypes of mice expressing only PTBP1, only PTBP2, or neither protein in the brain. This knockin Ptbp1 rescued a forebrain-specific, but not a pan-neuronal, Ptbp2 knockout, demonstrating both redundant and distinct roles for the proteins. Many developmentally regulated exons exhibited different sensitivities to PTBP1 and PTBP2. Nevertheless, the two paralogs displayed similar RNA binding across the transcriptome, indicating that their differential targeting does not derive from their RNA interactions, but from possible different cofactor interactions.

Keywords: CLIP; GeneSplice; PTBP1; PTBP2; RNA binding proteins; RNA-seq; alternative splicing; brain development; neuronal differentiation; splicing reprogramming.

Figure 1. A Ptpb1 conditional knock-in mouse expresses inducible FLAG-PTBP1 from the Rosa26 locus

(A) Schematic of the targeting strategy. SA: splicing acceptor. PGK-Neo-tpA: PGK promoter-driven neomycin with PGK polyA site. NLS: nuclear localization signal. bPA: bovine growth hormone polyA site. F1, R2 and R3: genotyping primers. ► Loxp sites. Frt sites. (B) Genotyping shows distinct amplicons for the wildtype, Ptbp1-KI heterozygous and homozygous animals. (C) Southern blot of Kpn1-treated genomic DNA from the wildtype and Ptbp1-KI heterozygous animals. The wildtype allele is 37 kb and the mutant allele is 7.3kb. (D) Western blot shows the expression of Flag-PTBP1 protein using an anti-Flag antibody. Anti-U1-70k is used as a control. (E) Immunofluorescence staining shows the expression of FLAG-PTBP1 in the cortex of Emx1-Ptbp1 KI mice. Co-staining of FLAG-PTBP1 and neuronal marker NeuN in the neocortex (F) and hippocampus (G) of Emx1-Ptbp1 KI mice. Scale bars: 100 µm.

Figure 2. The FLAG-PTBP1 transgene partially rescues the slow growth and early mortality of Emx1-Ptbp2KO mice

(A) Postnatal day 10 animals of (1) Rosa26^{LSL-FLAG-Ptbp1/+} Ptbp2^loxp/loxp Emx1-Cre+ (Emx1-DM), (2) Ptbp2^loxp/loxp (considered as WT), and (3–4) Ptbp2^loxp/loxp Emx1-Cre+ (Emx1-Ptbp2KO). (B) Survival curves of wildtype-like (black), Emx1-DM (purple) and Emx1-Ptbp2KO (red) mice. The overall brain sizes (C) and structures shown by Nissl staining (D) were similar between Rosa26^{LSL-FLAG-Ptbp1/+} Ptbp2^loxp/loxp Emx1-Cre+ (Emx1-DM) mouse and wildtype-like mice at 18 months of age.

Figure 3. PTBP1 and PTBP2 regulate overlapping but distinct sets of alternative exons

RNA-Seq expression tracks show examples of exons that are (A) up-regulated or (B) repressed in the Emx1-Ptbp2KO (Ptbp2^loxp/loxp; Emx1-Cre+) neocortices and reversed in the Emx1-DM (Rosa26^{LSL-FLAG-Ptbp1/+}; Ptbp2^loxp/loxp; Emx1-Cre+) neocortices. Arrows point to the regulated exons. (C) A scatter plot of cassette exons shows splicing changes caused by FLAG-PTBP1 (y axis) and PTBP2 (x axis). Using a threshold of |ΔPSI| larger than 10, six exon groups in the colored sectors were identified by their responses to PTBP1 and PTBP2. (D) The averaged splicing changes induced by PTBP1 or PTBP2 were plotted for each exon group. Error bars represent standard deviation; with * P value < 1×10⁻¹³, using a paired Student’s t-test. (E) Conservation of each exon group, as well as non-PTBP-regulated alternative exons (AE) and constitutive exons.

Figure 4. PTBP1 and PTBP2 exhibit very similar RNA binding independent of regulation specificity

(A) A heat map shows the similarity between iCLIP libraries generated with anti- FLAG or PTBP2 antibodies (in yellow) from mouse neocortices of different genotypes (in green). The number of unique and significant iCLIP tags for each cluster was compared between iCLIP-Seq experiments to derive the R² values. (B) Logos plots of FLAG-PTBP1 or PTBP2 binding sites in each iCLIP experiment. Predicted crosslinked nucleotides are at the zero position. (C) Z scores of the triplets enriched within 30 nucleotides upstream or downstream of the crosslinking sites for each iCLIP experiment. A clustering dendrogram shows the similarity of the iCLIP libraries regarding Z scores of all possible motifs. (D) Bar graph showing within each group the percentage of exons containing FLAG-PTBP1 (blue) and PTBP2 (brown) significant binding sites in the flanking introns. n.s. indicates P value > 0.7. (E–H) Aligned genome browser tracks of RNA-Seq of Emx1-Ptbp2-cKO (red), Emx1-DM (Ptbp1-cKI, Ptbp2-cKO, dark blue) and Wt (orange), control iCLIP-Seq (black), PTBP1 iCLIP-seq (blue) and PTBP2 iCLIP-Seq (brown) for exons exhibiting different sensitivity to PTBP1 and PTBP2 but similar PTBP1 and PTBP2 binding. Arrows point to the regulated exons.

Figure 5. Developmental regulation of PTBP1 and PTBP2 target exons

(A–C) Heat maps show the hierarchical clustering of cassette exon groups based on their splicing levels at embryonic days 14 and 17 (E14, E17) and postnatal days 2 and 10 (P2, P10). Numbers on the dendrogram branches indicate individual exon clusters. Exon clusters exhibiting developmental-control were selected and their PSI values at E14, E17, P2 and P10 are plotted (D–J). * P value < 0.02., ** P value < 10⁻⁴, and *** P value < 10⁻⁹, determined using a Wilcoxon signed rank test.