Nanopore sequencing reveals full-length Tropomyosin 1 isoforms and their regulation by RNA-binding proteins during rat heart development

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Abstract

Alternative splicing (AS) contributes to the diversity of the proteome by producing multiple isoforms from a single gene. Although short-read RNA-sequencing methods have been the gold standard for determining AS patterns of genes, they have a difficulty in defining full-length mRNA isoforms assembled using different exon combinations. Tropomyosin 1 (TPM1) is an actin-binding protein required for cytoskeletal functions in non-muscle cells and for contraction in muscle cells. Tpm1 undergoes AS regulation to generate muscle versus non-muscle TPM1 protein isoforms with distinct physiological functions. It is unclear which full-length Tpm1 isoforms are produced via AS and how they are regulated during heart development. To address these, we utilized nanopore long-read cDNA sequencing without gene-specific PCR amplification. In rat hearts, we identified full-length Tpm1 isoforms composed of distinct exons with specific exon linkages. We showed that Tpm1 undergoes AS transitions during embryonic heart development such that muscle-specific exons are connected generating predominantly muscle-specific Tpm1 isoforms in adult hearts. We found that the RNA-binding protein RBFOX2 controls AS of rat Tpm1 exon 6a, which is important for cooperative actin binding. Furthermore, RBFOX2 regulates Tpm1 AS of exon 6a antagonistically to the RNA-binding protein PTBP1. In sum, we defined full-length Tpm1 isoforms with different exon combinations that are tightly regulated during cardiac development and provided insights into the regulation of Tpm1 AS by RNA-binding proteins. Our results demonstrate that nanopore sequencing is an excellent tool to determine full-length AS variants of muscle-enriched genes.

Original languageEnglish (US)
Pages (from-to)8352-8362
Number of pages11
JournalJournal of Cellular and Molecular Medicine
Volume25
Issue number17
DOIs
StatePublished - Sep 2021

Keywords

  • RNA-binding proteins
  • alternative splicing
  • heart development
  • long-read DNA sequencing
  • tropomyosin

ASJC Scopus subject areas

  • Molecular Medicine
  • Cell Biology

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