Nanopore sequencing reveals full-length Tropomyosin 1 isoforms and their regulation by RNA-binding proteins during rat heart development.
Nanopore sequencing reveals full-length Tropomyosin 1 isoforms and their regulation by RNA-binding proteins during rat heart development.
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DOI：
10.1111/jcmm.16795
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发表时间：
2021-09
影响因子：
5.3
通讯作者：
Kuyumcu-Martinez MN
中科院分区：
文献类型：
--
作者：
Cao J;Routh AL;Kuyumcu-Martinez MN
选择性剪接（AS）通过从单个基因产生多个异构体而有助于蛋白质组的多样性。虽然短读RNA测序方法一直是确定基因AS模式的金标准，但它们难以定义使用不同外显子组合组装的全长mRNA亚型。原肌球蛋白1（TPM1）是一种肌动蛋白结合蛋白，在非肌肉细胞中的细胞骨架功能和肌肉细胞中的收缩所需。Tpm1经历AS调节以产生具有不同生理功能的肌肉与非肌肉TPM1蛋白同种型。目前尚不清楚AS产生哪些全长Tpm 1亚型，以及它们在心脏发育过程中如何受到调节。为了解决这些问题，我们利用纳米孔长读cDNA测序，而不进行基因特异性PCR扩增。在大鼠心脏中，我们鉴定了由具有特定外显子连接的不同外显子组成的全长Tpm 1亚型。我们发现，Tpm 1在胚胎心脏发育过程中经历AS转换，使得肌肉特异性外显子连接，在成人心脏中主要产生肌肉特异性Tpm 1亚型。我们发现，RNA结合蛋白RBFOX2控制大鼠Tpm 1外显子6a的AS，这对协同肌动蛋白结合很重要。此外，RBFOX2拮抗RNA结合蛋白PTBP 1调节外显子6a的Tpm1 AS。总之，我们定义了具有不同外显子组合的全长Tpm 1亚型，这些外显子组合在心脏发育过程中受到严格调控，并提供了对RNA结合蛋白调控Tpm 1 AS的见解。我们的研究结果表明，纳米孔测序是确定肌肉富集基因的全长AS变体的绝佳工具。
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.
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DOI：
10.1038/nrm.2017.27
发表时间：
2017-07
期刊：
Nature reviews. Molecular cell biology
影响因子：
--
作者：
Baralle FE;Giudice J
通讯作者：
Giudice J
影响因子：
14.9
作者：
Gooding C;Edge C;Lorenz M;Coelho MB;Winters M;Kaminski CF;Cherny D;Eperon IC;Smith CW
通讯作者：
Smith CW
影响因子：
5.8
作者：
Li, Heng
通讯作者：
Li, Heng
影响因子：
4
作者：
Gunning, Peter W.;Hardeman, Edna C.;Mulvihill, Daniel P.
通讯作者：
Mulvihill, Daniel P.
影响因子：
7.7
作者：
Cibi, Dasan Mary;Mia, Masum M.;Singh, Manvendra K.
通讯作者：
Singh, Manvendra K.