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