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

Abstract

Alternative splicing (AS) increases the variety 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 linkages. We showed that Tpm1 undergoes AS transitions during embryonic heart development such that muscle-specific exons are connected together generating predominantly muscle specific Tpm1 isoforms in adult hearts. Nanopore sequencing combined with polyA sequencing revealed that the RNA binding protein RBFOX2 controls AS of muscle specific Tpm1 isoforms and expression of TPM1 protein via terminal exon splicing impacting its polyadenylation. Furthermore, RBFOX2 regulates Tpm1 AS antagonistically to PTBP1. In sum, we defined full length Tpm1 isoforms with different exon combinations that are tightly regulated during cardiac development and provided insights into regulation of muscle specific isoforms of Tpm1 by RNA binding proteins. Our results demonstrate that nanopore sequencing is an excellent tool to determine full-length AS variants of muscle enriched genes.

Droplet-based Single-cell Total RNA-seq Reveals Differential Non-Coding Expression and Splicing Patterns during Mouse Development

“…In addition, Tropomyosin 1 (Tpm1) stood out with three DISNs, a CE and a pair of mutually exclusive exons, that had some of the highest confidence levels detected (Tpm1_29, Tpm1_22 and Tpm1_25 corresponding to exon 9a, 6a and 6b respectively). These splicing events have been shown to be part of a coordinated transition between a smooth muscle and striated muscle program, orchestrated by the antagonistic splicing regulation between PTBP1 and RBFOX2 (Cao et al, 2021). This transition was captured along a differentiation trajectory encompassing the early caudal epiblast (ECE), the FHF and PHT (Figures 7C and S5D) which also highlighted a switch for the N-(Tpm1_14, exon 1b) and C-(Tpm1_32, 9b) termini that has been shown to modulate the protein's interaction with actin and troponin, hereby assisting muscle contraction (Cao et al, 2021;Hammell and Hitchcock-DeGregori, 1996).…”

“…These splicing events have been shown to be part of a coordinated transition between a smooth muscle and striated muscle program, orchestrated by the antagonistic splicing regulation between PTBP1 and RBFOX2 (Cao et al, 2021). This transition was captured along a differentiation trajectory encompassing the early caudal epiblast (ECE), the FHF and PHT (Figures 7C and S5D) which also highlighted a switch for the N-(Tpm1_14, exon 1b) and C-(Tpm1_32, 9b) termini that has been shown to modulate the protein's interaction with actin and troponin, hereby assisting muscle contraction (Cao et al, 2021;Hammell and Hitchcock-DeGregori, 1996). Since Tpm1 has many cell type specific isoforms (Gooding et al, 2013), we further visualized single-cell ψ values for the aforementioned splicing nodes on the UMAP, which showed cell type specific patterning across the atlas (Figures 7D and S5E-F), underlining the potential of the VASA-seq method to capture AS at the single-cell level.…”