Characterization of TTN Novex Splicing Variants across Species and the Role of RBM20 in Novex-Specific Exon Splicing

Figures

• 

  Figure 1. Schematic splicing pattern of titin (TTN) Novexes 1, 2, and 3 and conserved sequence analysis of other species to human sequence using mVISTA software. (A) Schematic splicing pattern of TTN Novexes 1, 2, and 3 with comparison to N2B isoform. (B) Sequence comparison of exons 44–48 in the Novex-1, -2, and-3 mutually exclusive splicing regions between species. Blue box indicates a highly conserved region between exon 47 and 48. Percentage on the right side represents sequence homology by comparing to human sequence. Large percentage of the red region indicates a high sequence homology to human sequence. E: Exon.

• 

  Figure 2. Splicing analysis of TTN Novex 1 between species. (A) PCR products with primers spanning exons 42–45 (1) and Exons 45–49 (2); expression between exons 42–45 is consistent across species, while different expression pattern is observed between exons 45 and 49. (B) Schematic diagram of the reported Novex 1 splicing pattern in all species but mouse. (C) A newly identified truncated splicing form with Intron 44 retention in pig, mouse, and chicken heart. M: DNA marker; E: Exon; black boxes: Exons; white boxes: Intron retention; black lines: Introns.

• 

  Figure 3. Splicing analysis of TTN Novex 2 between species. (A) PCR products with primer pairs spanning exon 42 and 46 (1) and exon 46 and 49 (2); human, chicken, and frog express Exons 42–46 and Exons 46–49 with a lower level; no products could be detected in pig, mouse, or rat. (B) Schematic diagram of the reported Novex 2 splicing pattern in human, chicken, and frog heart. (C) Schematic diagram of PCR results; a truncated form with Intron 46 retention was found in human and frog heart. (D) Schematic diagram of PCR results; a new truncated form with co-expression of exons 45 and 46 and Intron 46 retention was observed in chicken and frog hearts. (E) PCR products with primer spanning exon 45 and Intron 46 between species; DNA band was observed in pig, mouse, and rat heart. (F) Schematic diagram of a newly identified truncated form with co-expression of Exons 45 and 46 and Introns 45 and 46 retention in pig, mouse, rat, and chicken. M: DNA marker; E: Exon; black boxes: Exons; white boxes: Intron retention; black lines: Introns.

• 

  Figure 4. Species difference in splicing of TTN Novex 3 between species. (A) PCR products with primer pair spanning exons 44 and 48; the lower band was expressed across all species with a lower level in frog. An additional band was amplified in all species with a lower level in rat; in frog heart, an even larger product appears. (B) Schematic diagram of splicing pattern of the reported Novex 3. (C) Schematic diagram of a newly identified isoform of Novex 3 with an additional exon 48′ insertion in all species with a lower expression in rats between exon 47 and 48. (D) Sequencing of exon 48′ and alignment of new Novex 3 isoform between species; the size of exon 48′ is either 85 or 100 bp due to alternatively used 15 bp sequence highlighted in black box, and this sequence is conserved between species. (E) Schematic diagram of another new splicing isoform of Novex 3 with co-expression of exons 45 and 48 and additional exon 48′ insertion. E: Exon, M: DNA marker.

• 

  Figure 5. RBM20 regulation of TTN Novex alternatively used exons. (A) PCR products with primers spanning exons 44–45 (Novex 1), Exons 44–46 (Novex 2), and exons 44–48 (Novex 3) in rat fetal heart of WT and KO; no splicing difference was found between WT and KO. (B) PCR products with the same primers in adult rat heart of WT and KO; no difference between WT and KO was found. GAPDH: PCR internal control. M: 1 Kb DNA marker; WT: Wild type; KO: Knockout.

• 

  Figure 6. Splicing pattern and expression level of Novex isoforms in human cardiomyopathies. (A) PCR products with primers spanning exons 44–45 (Novex 1), exons 44–46 (Novex 2), and exons 44–48 (Novex 3) in normal and diseased hearts; no splicing pattern difference was found between normal and diseased hearts, but expression levels of Novex isoforms was observed. (B) Quantification of DNA band intensity of Novex 1 in HCM, DCM, and ARVC. (C) Quantification of DNA band intensity of Novex 2 in HCM, DCM, and ARVC. (D) Quantification of DNA band intensity of Novex 3 in HCM, DCM, and ARVC; Gapdh: internal control; HCM: hypertrophy cardiomyopathy; DCM: Dilated cardiomyopathy; ARVC: Arrhythmogenic right ventricular cardiomyopathy. Mean ± (Standard Error of the Mean) SEM, ** p < 0.001; #### p < 0.00001.