Fukumaki Y, Ghosh P K, Benz E J, Reddy V B, Lebowitz P, Forget B G, Weissman S M
Cell. 1982 Mar;28(3):585-93. doi: 10.1016/0092-8674(82)90213-6.
The reduced beta-globin synthesis characterizing the beta+ thalassemia phenotype has been shown to be caused by anomalous processing within the small intervening sequence (IVS1) of the beta-globin mRNA precursor. The beta-globin gene from such patients contains a single base substitution within IVS1, located 22 bp from the 3' junction between IVS1 and exon 2, creating an alternative splice site within IVS1 and resulting in retention of the 3'-terminal 19 bases of IVS1. We have identified this abnormally spliced mRNA in the reticulocyte RNA of two patients with beta+ thalassemia, by S1 nuclease mapping and primer-extension analysis. Moreover, a cloned beta+-thalassemic gene preferentially generated the anomalously spliced RNA when expressed in monkey kidney cells. The anomalously spliced RNA constituted approximately 80%--90%, and normal beta RNA approximately 10%--20%, of the total beta mRNA. In contrast, the small amount of beta mRNA present in reticulocytes from such patients consisted predominantly of normal beta mRNA. These results suggest that the reduced amount of normally functioning beta mRNA present in such patients results from preferential processing at the alternative splice site, with subsequent instability, reduced nuclear processing and/or inadequate cytoplasmic transport of the abnormal RNA species.
β⁺地中海贫血表型所特有的β-珠蛋白合成减少已被证明是由β-珠蛋白mRNA前体的小间隔序列（IVS1）内的异常加工所致。此类患者的β-珠蛋白基因在IVS1内有一个单碱基替换，位于IVS1与外显子2之间3'连接处的22个碱基对处，在IVS1内产生了一个替代剪接位点，并导致IVS1的3'末端19个碱基得以保留。我们通过S1核酸酶图谱分析和引物延伸分析，在两名β⁺地中海贫血患者的网织红细胞RNA中鉴定出了这种异常剪接的mRNA。此外，一个克隆的β⁺地中海贫血基因在猴肾细胞中表达时优先产生异常剪接的RNA。异常剪接的RNA约占总β mRNA的80% - 90%，正常β RNA约占10% - 20%。相比之下，此类患者网织红细胞中存在的少量β mRNA主要由正常β mRNA组成。这些结果表明，此类患者中正常功能的β mRNA数量减少是由于在替代剪接位点的优先加工，随后异常RNA种类不稳定、核加工减少和/或细胞质转运不足所致。
