A developmentally regulated spliced variant of PTBP1 is upregulated in type 1 diabetic hearts

Abstract

Alternative splicing (AS) is dysregulated in Type 1 diabetic (T1D) hearts but mechanisms responsible are unclear. Here, we provide evidence that the RNA binding protein (RBP) PTBP1 is modulated in adult T1D hearts contributing to AS changes. We show that a spliced variant of PTBP1 that is highly expressed in normal newborn mouse hearts is aberrantly expressed in adult T1D mouse hearts. Comparing known PTBP1-target datasets to our T1D mouse transcriptome datasets, we discovered a group of genes with PTBP1 binding sites in their pre-mRNAs that are differentially spliced in T1D mouse hearts. We demonstrated that inducible expression of diabetes-induced PTBP1 spliced variant has less repressive splicing function. Notably, PTBP1 regulates AS of some of its targets antagonistically to RBFOX2. In sum, our results indicate that diabetic conditions disrupt developmental regulation of PTBP1 leading to differential AS of PTBP1 target genes. Identification of PTBP1 and PTBP1-regulated RNA networks can provide RNA-based therapies for the treatment of diabetes cardiac complications.

Keywords: Alternative splicing; Diabetic heart; PTBP1; RNA binding proteins.

Figure 1:. Regulation of PTBP1 spliced variant expression in the heart under diabetic conditions and during development.

A) Representative genome browser images of PTBP1 exon 8 exclusion in diabetic mouse hearts in comparison to the control mouse hearts. qRT-PCR analysis of PTBP1 exon 8 inclusion in B) control or streptozotocin induced diabetic (STZ:T1D), C) in control or non-obese diabetic (NOD:T1D) mouse hearts. qRT-PCR analysis of PTBP1 exon 8 inclusion D) during mouse heart development at different stages: embryonic day 18 (E18), newborn (NB), or adult (n≥3) and E) in H9c2 cells depleted of RBFOX2 or CELF1. (n≥3). Data represent means ± SD. Statistical significance was calculated using Student’s t-test for two groups and one-way ANOVA to compare three different groups. P-values are represented as **** < 0.0001, *** < 0.001 and ** < 0.01.

Figure 2:. PTBP1 binding sites are present within transcripts that are mis-spliced in Type 1 diabetic mouse hearts.

A) Number of transcripts differentially spliced in Type 1 diabetic mouse hearts that display PTBP1 binding sites. PTB binding sites were identified by extracting data from previously published CLIP-Seq data and overlapping these binding sites within transcripts (1318) mis-spliced in diabetic hearts. 58 of these transcripts exhibit PTBP1 binding sites. B) Distribution of cassette exon inclusion or exclusion of PTBP1 targets in diabetic hearts. C) Representative genome browser images of a known PTBP1 target Tpm2 exon 6b (E6B) in control vs STZ:T1D mice hearts. D) The KEGG pathway categories for PTBP1-regulated AS events in diabetic hearts using Enrichr.

Figure 3:. PTBP1 spliced variant regulates AS of transcripts differentially spliced in diabetic hearts.

A) Representative genome browser images of PTBP1 target Clip1 exon 9 in control vs STZ:T1D mice hearts. AS analysis of B) Clip1, Med23 and C) PTBP1 in 293 cells depleted of PTBP1/2 and stably expressing inducible WT (PTBP1+ex9) and diabetes-induced spliced variant (PTBP1Δex9) using qRT-PCR. D) Validation of endogenous PTBP1 depletion and ectopic expression of PTBP1+ex9 and PTBP1Δex9 by Western blot (WB) using an antibody against PTBP1. Total protein staining was used to confirm even protein loading. Data represent means ± SD. Statistical significance was calculated using one-way ANOVA to compare four different groups. P-values are represented as *** < 0.001, ** < 0.01, * < 0.05.

Figure 4:. PTBP1 regulates alternative splicing of Tmem184b mis-spliced in diabetes antagonistically to RBFOX2.

A) Diagram of PTBP1 (light gray boxes), and RBFOX2 binding sites (black box) within the Tmem184b pre-mRNA derived from CLIP-seq data and PTBP1 motif analysis. B) AS analysis of endogenous Tmem184b exon 9 in H9c2 cells transfected with scrambled (Control) or RBP specific siRNA pools (PTBP1/PTBP2siPool or RBFOX2siPool) (n=3) and transfected with GFP (Control), PTBP1GFP or RBFOX2GFP (n=3). Data represent means ± SD. Statistical significance was calculated using one-way ANOVA to compare three different groups. P-values are represented as *** < 0.001, ** < 0.01