10.25376/hra.6083228.v1 Zhilong Chen Zhilong Chen Jiangping Song Jiangping Song Liang Chen Liang Chen Chaoqun Zhu Chaoqun Zhu Hanfang Cai Hanfang Cai Mingming SUn Mingming SUn Allysa Stern Allysa Stern Paul Mozdziak Paul Mozdziak Ying Ge Ying Ge Warrie Means Warrie Means Wei Guo Wei Guo Characterization of TTN Novex Splicing Variants across Species and the Role of RBM20 in Novex-Specific Exon Splicing Health Research Alliance 2018 TTN RBM20 Novex isoforms cardiomyopathy alternative splicing Cell Biology Molecular Biology Signal Transduction Physiology 2018-04-09 14:29:00 Journal contribution https://hra.figshare.com/articles/journal_contribution/Characterization_of_TTN_Novex_Splicing_Variants_across_Species_and_the_Role_of_RBM20_in_Novex-Specific_Exon_Splicing/6083228 <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.</p>