Department of Neuroscience, and the Brown Institute for Brain Science, Brown University, Providence, RI 02912.
Department of Biology, Linfield College, McMinnville, OR 97128.
eNeuro. 2017 Oct 10;4(5). doi: 10.1523/ENEURO.0332-16.2017. eCollection 2017 Sep-Oct.
The majority of multiexon mammalian genes contain alternatively spliced exons that have unique expression patterns in different cell populations and that have important cell functions. The expression profiles of alternative exons are controlled by cell-specific splicing factors that can promote exon inclusion or exon skipping but with few exceptions we do not know which specific splicing factors control the expression of alternatively spliced exons of known biological function. Many ion channel genes undergo extensive alternative splicing including that encodes the voltage-gated Ca2.2 α1 subunit. Alternatively spliced exon 18a in RNA encodes 21 amino acids in the II-III loop of Ca2.2, and its expression differs across the nervous system and over development. Genome-wide, protein-RNA binding analyses coupled to high-throughput RNA sequencing show that RNA binding Fox (Rbfox) proteins associate with Ca2.2 () pre-mRNAs. Here, we link Rbfox2 to suppression of e18a. We show increased e18a inclusion in Ca2.2 mRNAs: (1) after siRNA knockdown of Rbfox2 in a neuronal cell line and (2) in RNA from sympathetic neurons of adult compared to early postnatal mice. By immunoprecipitation of Rbfox2-RNA complexes followed by qPCR, we demonstrate reduced Rbfox2 binding upstream of e18a in RNA from sympathetic neurons of adult compared to early postnatal mice. Ca2.2 currents in cell lines and in sympathetic neurons expressing only e18a-Ca2.2 are larger compared to currents from those expressing only Δ18a-Ca2.2. We conclude that Rbfox2 represses e18a inclusion during pre-mRNA splicing of Ca2.2, limiting the size of Ca2.2 currents early in development in certain neuronal populations.
大多数多外显子哺乳动物基因包含选择性剪接的外显子，这些外显子在不同的细胞群体中有独特的表达模式，具有重要的细胞功能。选择性外显子的表达谱受细胞特异性剪接因子控制，这些因子可以促进外显子包含或外显子跳过，但除了少数例外，我们不知道哪些特定的剪接因子控制已知生物学功能的选择性剪接外显子的表达。许多离子通道基因经历广泛的选择性剪接，包括编码电压门控 Ca2.2α1 亚基的基因。Ca2.2 RNA 中的选择性剪接外显子 18a 编码 Ca2.2 II-III 环中的 21 个氨基酸，其表达在神经系统和发育过程中存在差异。全基因组范围内，与高通量 RNA 测序相结合的蛋白质-RNA 结合分析表明，RNA 结合 Fox（Rbfox）蛋白与 Ca2.2（）前体 mRNA 结合。在这里，我们将 Rbfox2 与 e18a 的抑制联系起来。我们发现 Ca2.2 mRNA 中外显子 18a 的包含增加：（1）在神经元细胞系中用 siRNA 敲低 Rbfox2 后，以及（2）在成年而非新生小鼠的交感神经元的 RNA 中。通过 Rbfox2-RNA 复合物的免疫沉淀 followed by qPCR，我们证明了成年而非新生小鼠交感神经元的 RNA 中外显子 18a 上游的 Rbfox2 结合减少。与表达仅Δ18a-Ca2.2 的细胞系和交感神经元中的 Ca2.2 电流相比，表达仅 e18a-Ca2.2 的细胞系和交感神经元中的 Ca2.2 电流更大。我们得出结论，Rbfox2 在 Ca2.2 前体 mRNA 的剪接过程中抑制 e18a 的包含，从而限制了某些神经元群体在发育早期 Ca2.2 电流的大小。
