A CONSERVED ALTERNATIVE SPLICE IN THE VONRECKLINGHAUSEN NEUROFIBROMATOSIS (NF1) GENE PRODUCES 2 NEUROFIBROMIN ISOFORMS, BOTH OF WHICH HAVE GTPASE-ACTIVATING PROTEIN-ACTIVITY
A CONSERVED ALTERNATIVE SPLICE IN THE VONRECKLINGHAUSEN NEUROFIBROMATOSIS (NF1) GENE PRODUCES 2 NEUROFIBROMIN ISOFORMS, BOTH OF WHICH HAVE GTPASE-ACTIVATING PROTEIN-ACTIVITY
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DOI：
10.1128/mcb.13.1.487
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发表时间：
1993-01-01
影响因子：
5.3
通讯作者：
COLLINS, FS
中科院分区：
文献类型：
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作者：
ANDERSEN, LB;BALLESTER, R;COLLINS, FS
序列分析显示哺乳动物 ras GTP 酶激活蛋白 (GAP)、酵母 Ira1p 和 Ira2p（RAS-环 AMP 通路的抑制性调节因子）和神经纤维蛋白（NF1 基因编码的蛋白质）的催化区域之间存在显着同源性。酵母表达实验证实，神经纤维蛋白的 381 个氨基酸片段（称为 GAP 相关结构域 (GRD)）可以起到 GAP 的作用。使用 RNA 聚合酶链式反应和 NF1-GRD 侧翼的引物，我们已经确定了 NF1 基因该区域存在选择性剪接的证据。除了已经发表的序列（I 型）外，所有检查的组织中都存在携带 63 个核苷酸插入的替代 RNA（II 型），尽管 I 型和 II 型的相对量有所不同。该插入在物种间是保守的，但在 GAP、IRA1 或 IRA2 中不存在。尽管插入的基本氨基酸组成与核靶向序列具有相同的特征，但 GenBank 搜索未能识别插入序列与已知 DNA 或蛋白质序列之间的显着相似性。酵母表达研究表明，尽管这种插入部分破坏了神经纤维蛋白-IRA-GAP 同源性，但这两种形式的 NF1-GRD 都可以补充 IRA 功能的丧失。旨在比较两种选择性剪接形式的 NF1-GRD 的 GAP 活性的体内测定表明，两者都可以增加 GTP 结合 ras 向其 GDP 结合形式的转化，尽管 21 个氨基酸的插入削弱了这种效果。这种选择性剪接的高度保守性表明 I 型和 II 型亚型均介导神经纤维蛋白的重要生物学功能。
Sequence analysis has shown significant homology between the catalytic regions of the mammalian ras GTPase-activating protein (GAP), yeast Ira1p and Ira2p (inhibitory regulators of the RAS-cyclic AMP pathway), and neurofibromin, the protein encoded by the NF1 gene. Yeast expression experiments have confirmed that a 381-amino-acid segment of neurofibromin, dubbed the GAP-related domain (GRD), can function as a GAP. Using the RNA polymerase chain reaction with primers flanking the NF1-GRD, we have identified evidence for alternative splicing in this region of the NF1 gene. In addition to the already published sequence (type I), an alternative RNA carrying a 63-nucleotide insertion (type II) is present in all tissues examined, although the relative amounts of types I and II vary. The insertion is conserved across species but is not present in GAP, IRA1, or IRA2. GenBank searches have failed to identify significant similarity between the inserted sequence and known DNA or protein sequences, although the basic amino acid composition of the insertion shares features with nuclear targeting sequences. Expression studies in yeasts show that despite the partial disruption of the neurofibromin-IRA-GAP homology by this insertion, both forms of the NF1-GRD can complement loss of IRA function. In vivo assays designed to compare the GAP activity of the two alternatively spliced forms of the NF1-GRD show that both can increase the conversion of GTP-bound ras to its GDP-bound form, although the insertion of the 21 amino acids weakens this effect. The strong conservation of this alternative, splicing suggests that both type I and II isoforms mediate important biological functions of neurofibromin.