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. 2018 Mar 29;8(1):11.
doi: 10.1186/s13395-018-0156-z.

The complexity of titin splicing pattern in human adult skeletal muscles

Marco Savarese  1   2 , Per Harald Jonson  3 , Sanna Huovinen  4 , Lars Paulin  5 , Petri Auvinen  5 , Bjarne Udd  3   4   6 , Peter Hackman  3
Affiliations

The complexity of titin splicing pattern in human adult skeletal muscles

Marco Savarese et al. Skelet Muscle. .

Abstract

Background: Mutations in the titin gene (TTN) cause a large spectrum of diseases affecting skeletal and/or cardiac muscle. TTN includes 363 coding exons, a repeated region with a high degree of complexity, isoform-specific elements, and metatranscript-only exons thought to be expressed only during fetal development. Although three main classes of isoforms have been described so far, alternative splicing events (ASEs) in different tissues or in different developmental and physiological states have been reported.

Methods: To achieve a comprehensive view of titin ASEs in adult human skeletal muscles, we performed a RNA-Sequencing experiment on 42 human biopsies collected from 12 anatomically different skeletal muscles of 11 individuals without any skeletal-muscle disorders.

Results: We confirmed that the skeletal muscle N2A isoforms are highly prevalent, but we found an elevated number of alternative splicing events, some at a very high level. These include previously unknown exon skipping events and alternative 5' and 3' splice sites. Our data suggests the partial inclusion in the TTN transcript of some metatranscript-only exons and the partial exclusion of canonical N2A exons.

Conclusions: This study provides an extensive picture of the complex TTN splicing pattern in human adult skeletal muscle, which is crucial for a proper clinical interpretation of TTN variants.

Keywords: Alternative splicing events; Exon usage; RNA-sequencing; Splicing pattern; Titin; Titinopathies.

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Conflict of interest statement

Ethics approval and consent to participate

A written informed consent was signed by all the patients and the Tampere University Hospital (Tampere, Finland) Ethics Committee approved the study.

Consent for publication

Not applicable

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Isoform identification and titin alternative splicing events in human skeletal muscle. a The previously reported classes of isoforms differ from each other by the inclusion/exclusion of exons 48 (included only in Novex3 isoform) and 49 (included in all the other isoforms except the long skeletal muscle N2A-isoform). Our data suggests that N2A isoform is 20 times more expressed than Novex3. All the other isoforms have a very low expression. b We identified a low number of reads connecting exon 11 to its flanking exons. On the contrary, a high number of reads connect exon 10 to exon 12 and 13, thereby skipping exon 11. In line with the RNA-Seq results, a standard RT-PCR (forward primer on exon 9 and reverse primer on exon 13, red arrows) and agarose gel electrophoresis show a very low abundance of the transcript including exon 11. M1 = 100 bp ladder. c Several RT-PCRs and agarose gel electrophoresis show a variable expression of metatranscript-only exons, confirming the RNA-Seq results. In particular, no expression of exons 163 and 165 is detected; on the contrary, all the other RT-PCRs result in a detectable band corresponding to the expected size. M1 = 100 bp ladder; M2 = 1 kb ladder; d = PCR from a control DNA; c = RT-PCR from a control cDNA (obtained by a retrotranscription of RNA extracted from gracilis muscle). d Titin repeated region is composed of nine exons/blocks (here represented by different colors and named B1-B9) repeated three times. Within the repeated region, linear expression of consecutive exons has been detected. Moreover, a number of alternative splicing events has been identified. e We detected alternative splicing acceptors or donors leading to subtle changes in the produced protein. The splice-site strength for canonical splice sites (5′ss and 3′ss) as well as for alternative sites (alt 5′ss, alt 3′ss) has been calculated by Human Splice Finder (HSF)
Fig. 2
Fig. 2
Comparison of alternative splicing events among different tissues at different developmental stages. The analysis of publicly available total mRNA sequencing data from the ENCODE project shows that exon 11 is expressed only in cardiac muscles, whereas the expression of exon 148 is limited to skeletal muscles. Exons 213 and 217 show an increased expression in fetal skeletal (and, at least in part, cardiac) muscle compared to the adult expression. The reported values correspond to the inclusion values, based on the number of reads supporting each exon inclusion or exclusion in TTN transcripts
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