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

<p> Titin ( TTN) is a major disease-causing gene in cardiac muscle. Titin ( TTN) contains 363 exons</p> <p>in human encoding various sizes of TTN protein due to alternative splicing regulated mainly by</p> <p>RNA binding motif 20 (RBM20). Three isoforms of TTN protein are produced by mutually exclusive</p> <p>exons 45 (Novex 1), 46 (Novex 2), and 48 (Novex 3). Alternatively splicing in Novex isoforms</p> <p>across species and whether Novex isoforms are associated with heart disease remains completely</p> <p>unknown. Cross-species exon comparison with the mVISTA online tool revealed that exon 45 is</p> <p>more highly conserved across all species than exons 46 and 48. Importantly, a conserved region</p> <p>between exons 47 and 48 across species was revealed for the first time. Reverse transcript polymerase</p> <p>chain reaction (RT-PCR) and DNA sequencing confirmed a new exon named as 480 in Novex 3.</p> <p>In addition, with primer pairs for Novex 1, a new truncated form preserving introns 44 and 45 was</p> <p>discovered. We discovered that Novex 2 is not expressed in the pig, mouse, and rat with Novex 2</p> <p>primer pairs. Unexpectedly, three truncated forms were identified. One TTN variant with intron 46</p> <p>retention is mainly expressed in the human and frog heart, another variant with co-expression of</p> <p>exons 45 and 46 exists predominantly in chicken and frog heart, and a third with retention of introns</p> <p>45 and 46 is mainly expressed in pig, mouse, rat, and chicken. Using Rbm20 knockout rat heart,</p> <p>we revealed that RBM20 is not a splicing regulator of Novex variants. Furthermore, the expression</p> <p>levels of Novex variants in human hearts with cardiomyopathies suggested that Novexes 2 and 3</p> <p>could be associated with dilated cardiomyopathy (DCM) and/or arrhythmogenic right ventricular</p> <p>cardiomyopathy (ARVC). Taken together, our study reveals that splicing diversity of Novex exons</p> <p>across species and Novex variants might play a role in cardiomyopathy.