Brain and muscle express a unique alternative transcript of αII spectrin
Brain and muscle express a unique alternative transcript of αII spectrin
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DOI：
10.1021/bi991458k
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发表时间：
1999-11-30
期刊：
影响因子：
2.9
通讯作者：
Morrow, JS
中科院分区：
文献类型：
--
作者：
Cianci, CD;Zhang, ZS;Morrow, JS
α和β血影蛋白的前mRNA转录物的选择性剪接已经成为该基因家族多样性的重要产生者，但这种多样性的功能后果和程度仍然未知。我们已经从人胎脑中克隆并鉴定了全长α II血影蛋白cDNA（GenBank U83867和U26396）。基于预测的氨基酸序列，已经鉴定出11个氨基酸取代，推测代表多态性，其将该α II血影蛋白与人肺成纤维细胞α II血影蛋白区分开。此外，人胎脑血影蛋白显示一种新的5个氨基酸插入重复15，产生的选择性mRNA剪接，并区分这种血影蛋白从肺成纤维细胞α II血影蛋白。这一发现，连同两个先前确定的区域的选择性mRNA剪接α II血影蛋白表明，多达八种不同的剪接形式的成熟蛋白质可能存在，如果所有的组合（在插入I，2，和3）的选择性mRNA剪接被利用。为了评估这种可能性，选择外显子使用的组织分布进行了调查，半定量PCR与内含子跳跃引物集。检查的组织来自小鼠，包括心脏、肾脏、肺、肝脏、胸腺、脾脏、脑、卵巢、睾丸和骨骼肌以及小鼠胚胎组织。在所有组织中鉴定了具有和不具有插入物1（代表α II血影蛋白重复序列10内的60 bp插入物）的转录物。与此相反，转录插入2，新的15 bp插入报道，只在大脑，心脏，骨骼肌和胚胎组织中表达。在检查的所有组织中，仅扩增插入片段3（重复序列21中的18 bp插入片段）阳性的转录物，即使在人工制备的对照样品中可以容易地检测到30%水平的插入片段3阴性转录物的条件下也是如此。插入片段1和插入片段2的所有组合在单独的转录物中一起鉴定，验证了α II血影蛋白的至少四种不同的同种型。雅阁目前血影蛋白的命名惯例，这些被命名为α II Sigma 1至α II Sigma 4。动态分子模拟的第15个重复单位纳入插入2预测，剪接序列形成一个环之间的螺旋A和B，并建议，这个插入可能构成一个新的蛋白质相互作用位点。在α II σ 3和α II σ 4血影蛋白中存在该序列表明该分子的第15个重复序列具有专门的和迄今为止未预料到的功能。
Alternative splicing of pre-mRNA transcripts of alpha and beta spectrin has emerged as an important generator of diversity in this gene family, yet the functional consequences and extent of this diversity remains unknown. We have cloned and characterized full-length alpha II spectrin cDNA from human fetal brain (GenBank U83867 and U26396). On the basis of the predicted amino acid sequence, 11 amino acid substitutions, presumably representing polymorphisms, have been identified that distinguish this alpha II spectrin from human lung fibroblast aII spectrin. In addition, human fetal brain spectrin displays a novel five amino acid insertion in repeat 15 that arises from alternative mRNA splicing and that distinguishes this spectrin from lung fibroblast aII spectrin. This discovery, together with two previously identified regions of alternative mRNA splicing in (alpha II spectrin suggest that as many as eight different splice forms of the mature protein might exist if all combinations (at inserts I, 2, and 3) of alternative mRNA splicing are utilized. To assess this possibility, the tissue distribution of alternative exon usage was investigated by semiquantitative PCR with intron-jumping primer sets. Tissues examined were from mouse and included heart, kidney, lung, liver, thymus, spleen, brain, ovary, testis, and skeletal muscle, as well as mouse embryonic tissue. Transcripts both with and without insert 1, representing a 60 bp insertion within (alpha II spectrin repeat 10, were identified in all tissues. In contrast, transcripts with insert 2, the novel 15 bp insertion reported here, were only expressed in brain, heart, skeletal muscle, and embryonic tissue. In all tissues examined only transcripts positive for insert 3, an 18 bp insertion in repeat 21, were amplified, even under conditions in which a 30% level of insert 3 negative transcript could be easily detected in artificially prepared control samples. All combinations of insert 1 and insert 2 were identified together in individual transcripts, verifying at least four distinct isoforms of alpha II spectrin. These have been named alpha II Sigma 1 through alpha II Sigma 4, in accord with current spectrin naming conventions. Dynamic molecular modeling of the 15th repeat unit incorporating insert 2 predicts that the spliced sequence forms a loop between helices A and B, and suggests that this insert might constitute a novel protein interaction site. The presence of this sequence in alpha II Sigma 3 and alpha II Sigma 4 spectrin suggests a specialized and heretofore unanticipated function for the 15th repeat of this molecule.