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

Titin ( TTN) is a major disease-causing gene in cardiac muscle. Titin ( TTN) contains 363 exons

in human encoding various sizes of TTN protein due to alternative splicing regulated mainly by

RNA binding motif 20 (RBM20). Three isoforms of TTN protein are produced by mutually exclusive

exons 45 (Novex 1), 46 (Novex 2), and 48 (Novex 3). Alternatively splicing in Novex isoforms

across species and whether Novex isoforms are associated with heart disease remains completely

unknown. Cross-species exon comparison with the mVISTA online tool revealed that exon 45 is

more highly conserved across all species than exons 46 and 48. Importantly, a conserved region

between exons 47 and 48 across species was revealed for the first time. Reverse transcript polymerase

chain reaction (RT-PCR) and DNA sequencing confirmed a new exon named as 480 in Novex 3.

In addition, with primer pairs for Novex 1, a new truncated form preserving introns 44 and 45 was

discovered. We discovered that Novex 2 is not expressed in the pig, mouse, and rat with Novex 2

primer pairs. Unexpectedly, three truncated forms were identified. One TTN variant with intron 46

retention is mainly expressed in the human and frog heart, another variant with co-expression of

exons 45 and 46 exists predominantly in chicken and frog heart, and a third with retention of introns

45 and 46 is mainly expressed in pig, mouse, rat, and chicken. Using Rbm20 knockout rat heart,

we revealed that RBM20 is not a splicing regulator of Novex variants. Furthermore, the expression

levels of Novex variants in human hearts with cardiomyopathies suggested that Novexes 2 and 3

could be associated with dilated cardiomyopathy (DCM) and/or arrhythmogenic right ventricular

cardiomyopathy (ARVC). Taken together, our study reveals that splicing diversity of Novex exons

across species and Novex variants might play a role in cardiomyopathy.