Zhang Shuo Cheng, MacDonald Kimberley A, Baguma-Nibasheka Mark, Geldenhuys Laurette, Casson Alan G, Murphy Paul R
Department of Physiology and Biophysics, Dalhousie University, Halifax, Nova Scotia B3H 1X5, Canada.
BMC Mol Biol. 2008 Jan 23;9:10. doi: 10.1186/1471-2199-9-10.
GFG/NUDT is a nudix hydrolase originally identified as the product of the fibroblast growth factor-2 antisense (FGF-AS) gene. While the FGF-AS RNA has been implicated as an antisense regulator of FGF-2 expression, the expression and function of the encoded GFG protein is largely unknown. Alternative splicing of the primary FGF-AS mRNA transcript predicts multiple GFG isoforms in many species including rat. In the present study we focused on elucidating the expression and subcellular distribution of alternatively spliced rat GFG isoforms.
RT-PCR and immunohistochemistry revealed tissue-specific GFG mRNA isoform expression and subcellular distribution of GFG immunoreactivity in cytoplasm and nuclei of a wide range of normal rat tissues. FGF-2 and GFG immunoreactivity were co-localized in some, but not all, tissues examined. Computational analysis identified a mitochondrial targeting sequence (MTS) in the N-terminus of three previously described rGFG isoforms. Confocal laser scanning microscopy and subcellular fractionation analysis revealed that all rGFG isoforms bearing the MTS were specifically targeted to mitochondria whereas isoforms and deletion mutants lacking the MTS were localized in the cytoplasm and nucleus. Mutation and deletion analysis confirmed that the predicted MTS was necessary and sufficient for mitochondrial compartmentalization.
Previous findings strongly support a role for the FGF antisense RNA as a regulator of FGF2 expression. The present study demonstrates that the antisense RNA itself is translated, and that protein isoforms resulting form alternative RNA splicing are sorted to different subcellular compartments. FGF-2 and its antisense protein are co-expressed in many tissues and in some cases in the same cells. The strong conservation of sequence and genomic organization across animal species suggests important functional significance to the physical association of these transcript pairs.
GFG/NUDT是一种核苷二磷酸连接酶水解酶，最初被鉴定为成纤维细胞生长因子2反义（FGF-AS）基因的产物。虽然FGF-AS RNA被认为是FGF-2表达的反义调节因子，但编码的GFG蛋白的表达和功能在很大程度上尚不清楚。初级FGF-AS mRNA转录本的可变剪接预测在包括大鼠在内的许多物种中有多种GFG异构体。在本研究中，我们专注于阐明大鼠GFG可变剪接异构体的表达和亚细胞分布。
逆转录聚合酶链反应（RT-PCR）和免疫组织化学显示，在多种正常大鼠组织的细胞质和细胞核中，GFG mRNA异构体具有组织特异性表达以及GFG免疫反应性的亚细胞分布。FGF-2和GFG免疫反应性在一些但并非所有检测的组织中共定位。计算分析在三种先前描述的rGFG异构体的N端鉴定出一个线粒体靶向序列（MTS）。共聚焦激光扫描显微镜和亚细胞分级分离分析显示，所有带有MTS的rGFG异构体都特异性定位于线粒体，而缺乏MTS的异构体和缺失突变体则定位于细胞质和细胞核。突变和缺失分析证实，预测的MTS对于线粒体区室化是必要且充分的。
先前的研究结果有力地支持了FGF反义RNA作为FGF2表达调节因子的作用。本研究表明反义RNA本身被翻译，并且由可变RNA剪接产生的蛋白质异构体被分选到不同的亚细胞区室。FGF-2及其反义蛋白在许多组织中共同表达，在某些情况下在同一细胞中表达。跨动物物种的序列和基因组组织的高度保守表明这些转录本对的物理关联具有重要的功能意义。
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