Nuclear ALG-2 Protein Interacts with Ca2+ Homeostasis Endoplasmic Reticulum Protein (CHERP) Ca2+-dependently and Participates in Regulation of Alternative Splicing of Inositol Trisphosphate Receptor Type 1 (IP3R1) Pre-mRNA*
Nuclear ALG-2 Protein Interacts with Ca2+ Homeostasis Endoplasmic Reticulum Protein (CHERP) Ca2+-dependently and Participates in Regulation of Alternative Splicing of Inositol Trisphosphate Receptor Type 1 (IP3R1) Pre-mRNA*
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DOI：
10.1074/jbc.m113.497479
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发表时间：
2013-09
期刊：
影响因子：
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通讯作者：
Kanae Sasaki-Osugi;C. Imoto;Terunao Takahara;H. Shibata;M. Maki
中科院分区：
文献类型：
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作者：
Kanae Sasaki-Osugi;C. Imoto;Terunao Takahara;H. Shibata;M. Maki
背景：ALG-2存在于细胞质和细胞核中，但其核功能尚不清楚。结果：ALG-2与SR超家族蛋白CHERP相互作用，并以Ca 2+依赖的方式在核斑点处积聚。结论：ALG-2和CHERP参与选择性剪接。意义：我们提出了ALG-2和CHERP在细胞核转录后加工中的新作用。细胞内Ca 2+信号通路对于从受精到细胞死亡的广泛细胞过程的控制是重要的。ALG-2是一种钙离子结合蛋白，含有5个连续重复的EF-手基序，并以钙离子依赖的方式与各种蛋白质相互作用。虽然ALG-2存在于细胞质和细胞核中，但对其核功能知之甚少。Ca 2+稳态内质网蛋白（CHERP）首先被鉴定为调节人类细胞内Ca 2+动员的内质网蛋白，但最近的蛋白质组学数据表明CHERP与剪接体之间的关联。在这里，我们报告，CHERP，含有Pro丰富的区域和磷酸化的Ser/Arg丰富的RS样结构域，是一种新的钙依赖性ALG-2的相互作用的目标在核中。免疫荧光显微镜分析显示CHERP定位于核质，在核斑点处具有显著的积累，核斑点是前体mRNA剪接因子的储存和修饰的位点。活细胞延时成像显示，细胞核ALG-2被招募到CHERP定位斑点后，Ca 2+动员。免疫共沉淀试验的结果显示，结合CHERP的磷酸化形式的RNA聚合酶II。HT 1080细胞中CHERP或ALG-2的敲低导致产生肌醇1，4，5-三磷酸受体1（IP 3R 1）前mRNA的可变剪接同种型，其除了缺少外显子40-42的主要同种型之外还包括外显子41和42。此外，通过使用针对CHERP的多克隆抗体的RNA免疫沉淀测定来检测CHERP和IP 3R 1 RNA之间的结合。这些结果表明，CHERP和ALG-2参与调节IP 3R 1前体mRNA的选择性剪接，并提供了新的见解剪接变体的转录后调节Ca 2+信号通路。
Background: ALG-2 is present both in the cytoplasm and nucleus, but little is known about its nuclear function. Results: ALG-2 interacts with the SR superfamily protein CHERP and accumulates at nuclear speckles in a Ca2+-dependent manner. Conclusion: ALG-2 and CHERP participate in alternative splicing. Significance: We propose a new role of ALG-2 and CHERP in post-transcriptional processing in the nucleus. The intracellular Ca2+ signaling pathway is important for the control of broad cellular processes from fertilization to cell death. ALG-2 is a Ca2+-binding protein that contains five serially repeated EF-hand motifs and interacts with various proteins in a Ca2+-dependent manner. Although ALG-2 is present both in the cytoplasm and in the nucleus, little is known about its nuclear function. Ca2+ homeostasis endoplasmic reticulum protein (CHERP) was first identified as an endoplasmic reticulum protein that regulates intracellular Ca2+ mobilization in human cells, but recent proteomics data suggest an association between CHERP and spliceosomes. Here, we report that CHERP, containing a Pro-rich region and a phosphorylated Ser/Arg-rich RS-like domain, is a novel Ca2+-dependent ALG-2-interactive target in the nucleus. Immunofluorescence microscopic analysis revealed localization of CHERP to the nucleoplasm with prominent accumulation at nuclear speckles, which are the sites of storage and modification for pre-mRNA splicing factors. Live cell time-lapse imaging showed that nuclear ALG-2 was recruited to the CHERP-localizing speckles upon Ca2+ mobilization. Results of co-immunoprecipitation assays revealed binding of CHERP to a phosphorylated form of RNA polymerase II. Knockdown of CHERP or ALG-2 in HT1080 cells resulted in generation of alternatively spliced isoforms of the inositol 1,4,5-trisphosphate receptor 1 (IP3R1) pre-mRNA that included exons 41 and 42 in addition to the major isoform lacking exons 40–42. Furthermore, binding between CHERP and IP3R1 RNA was detected by an RNA immunoprecipitation assay using a polyclonal antibody against CHERP. These results indicate that CHERP and ALG-2 participate in regulation of alternative splicing of IP3R1 pre-mRNA and provide new insights into post-transcriptional regulation of splicing variants in Ca2+ signaling pathways.