Animal Science, University of Wyoming, Laramie, WY 82071, USA.
Animal Science, University of Wyoming, Laramie, WY 82071, USA; Department of Cardiology, Xi Jing Hospital, Fourth Military Medical University, Xi'an 710032, China.
Biochim Biophys Acta Mol Basis Dis. 2017 Sep;1863(9):2363-2371. doi: 10.1016/j.bbadis.2017.06.023. Epub 2017 Jul 1.
Titin, a giant sarcomeric protein, is largely responsible for the diastolic properties of the heart. It has two major isoforms, N2B and N2BA due to pre-mRNA splicing regulated mainly by a splicing factor RNA binding motif 20 (RBM20). Mis-splicing of titin pre-mRNA in response to external stimuli may lead to altered ratio of N2B to N2BA, and thus, impaired cardiac contractile function. However, little is known about titin alternative splicing in response to external stimuli. Here, we reported the detailed mechanisms of titin alternative splicing in response to insulin. Insulin treatment in cultured neonatal rat cardiomyocytes (NRCMs) activated the PI3K-Akt-mTOR kinase axis, leading to increased N2B expression in the presence of RBM20, but not in NRCMs in the absence of RBM20. By inhibiting this kinase axis with inhibitors, decreased N2B isoform was observed in NRCMs and also in diabetic rat model treated with streptozotocin, but not in NRCMs and diabetic rats in the absence of RBM20. In addition to the alteration of titin isoform ratios in response to insulin, we found that RBM20 expression was increased in NRCMs with insulin treatment, suggesting that RBM20 levels were also regulated by insulin-induced kinase axis. Further, knockdown of p70S6K1 with siRNA reduced both RBM20 and N2B levels, while knockdown of 4E-BP1 elevated expression levels of RBM20 and N2B. These findings reveal a major signal transduction pathway for insulin-induced titin alternative splicing, and place RBM20 in a central position in the pathway, which is consistent with the reputed role of RBM20 in titin alternative splicing. Findings from this study shed light on gene therapeutic strategies at the molecular level by correction of pre-mRNA mis-splicing.
肌联蛋白是一种巨大的肌节蛋白，主要负责心脏的舒张特性。它有两个主要的同工型，N2B 和 N2BA，这主要是由于前体 mRNA 的剪接受到 RNA 结合基序 20（RBM20）的调控。肌联蛋白前体 mRNA 对外界刺激的错误剪接可能导致 N2B 与 N2BA 的比例发生改变，从而导致心脏收缩功能受损。然而，人们对肌联蛋白对外界刺激的选择性剪接知之甚少。在这里，我们报道了肌联蛋白对外界刺激选择性剪接的详细机制。胰岛素处理培养的新生大鼠心肌细胞（NRCMs）激活了 PI3K-Akt-mTOR 激酶轴，导致 RBM20 存在的情况下 N2B 表达增加，但在 NRCMs 中不存在 RBM20 的情况下则没有。通过用抑制剂抑制这个激酶轴，我们观察到 NRCMs 和用链脲佐菌素处理的糖尿病大鼠模型中的 N2B 同工型减少，但在没有 RBM20 的 NRCMs 和糖尿病大鼠中则没有。除了肌联蛋白同工型比例的改变对胰岛素的反应，我们还发现胰岛素处理后的 NRCMs 中 RBM20 的表达增加，这表明 RBM20 的水平也受到胰岛素诱导的激酶轴的调节。此外，用 siRNA 敲低 p70S6K1 会降低 RBM20 和 N2B 的水平，而敲低 4E-BP1 则会提高 RBM20 和 N2B 的表达水平。这些发现揭示了胰岛素诱导的肌联蛋白选择性剪接的主要信号转导途径，并将 RBM20 置于该途径的中心位置，这与 RBM20 在肌联蛋白选择性剪接中的公认作用一致。这项研究的结果为通过纠正前体 mRNA 的错误剪接，从分子水平上为基因治疗策略提供了启示。
