Multiple domains control the subcellular localization and activity of ETR-3, a regulator of nuclear and cytoplasmic RNA processing events
Multiple domains control the subcellular localization and activity of ETR-3, a regulator of nuclear and cytoplasmic RNA processing events
复制标题
DOI：
10.1242/jcs.01194
复制
发表时间：
2004-07-15
影响因子：
4
通讯作者：
Cooper, TA
中科院分区：
文献类型：
--
作者：
Ladd, AN;Cooper, TA
胚胎致死性视觉异常(ELAV)型RNA结合蛋白3(ETR-3；又称NAPOR、CUGBP2或BRNOL3)参与了核质RNA加工事件的调控，包括选择性剪接、RNA编辑、稳定性和翻译。在这里，我们报告了ETR-3蛋白包含多个区域，这些区域控制着它的亚细胞定位，并对其作为剪接调节因子的活性至关重要。我们从鸡心中克隆了ETR-3，并将其与绿色荧光蛋白(GFPcETR3vL)的C端进行了融合。GFPcETR3vL主要存在于细胞核中，在与心肌肌钙蛋白T微型基因的共转染实验中是选择性剪接的活性调节因子。ETR-3含有两个N-端RNA识别基序(RRMS)、一个210个氨基酸的分歧结构域和一个C-端RRM。我们证明，C末端包含一个与第三个RRM重叠的强烈的核定位信号，这可以使正常细胞质的丙酮酸激酶嵌合体进行核定位。进一步的缺失揭示了ETR-3的分歧结构域中的核定位和输出活动，以及前两个RRMS中对细胞质定位重要的区域。发散结构域的核输出活性对软霉素B敏感，表明向细胞质的输出是通过CRM1依赖的途径介导的。C末端和分叉结构域中的一个区域对ETR-3的剪接活性也是重要的。这是第一次对参与介导RNA加工调节因子CELF家族成员的亚细胞定位和剪接活性的蛋白质结构域进行了表征。
Embryonic lethal abnormal vision (ELAV) type RNA binding protein 3 (ETR-3; also called NAPOR, CUGBP2, or BRUNOL3) has been implicated in the regulation of nuclear and cytoplasmic RNA processing events, including alternative splicing, RNA editing, stability and translation. Here, we report that the ETR-3 protein contains multiple regions that control its subcellular localization and are important for its activity as a splicing regulator. We cloned ETR-3 from chicken heart and fused it to the C terminus of green fluorescent protein (GFPcETR3vL). GFPcETR3vL is found predominantly in the nucleus and is an active regulator of alternative splicing in cotransfection assays with a cardiac troponin T minigene. ETR-3 contains two N-terminal RNA recognition motifs (RRMs), a 210-amino acid divergent domain, and a C-terminal RRM. We demonstrate that the C terminus contains a strong nuclear localization signal overlapping the third RRM, which can confer nuclear localization on a normally cytoplasmic pyruvate kinase chimera. Additional deletions revealed nuclear localization and export activities in the divergent domain of ETR-3, as well as regions within the first two RRMs that are important for cytoplasmic localization. The nuclear export activity of the divergent domain is sensitive to leptomycin B, indicating that export to the cytoplasm is mediated via a CRM1-dependent pathway. The C terminus and a region within the divergent domain were also shown to be important for splicing activity of ETR-3. This is the first characterization of protein domains involved in mediating the subcellular localization and splicing activity of a member of the CELF family of RNA processing regulators.