Siegall C B, Hla T T, Kumar A
George Washington University, Department of Genetics, Washington D.C. 20037.
Biochem Biophys Res Commun. 1988 Aug 15;154(3):1010-7. doi: 10.1016/0006-291x(88)90240-9.
Functional reconstitution of U1 small nuclear ribonucleoprotein particle (U1 snRNP) was performed using in vitro transcribed U1 snRNA. Hela cell nuclear extract was depleted of its constituent snRNPs by centrifugation at 100,000 X g. The supernatant was devoid of snRNAs and lacked cleavage activity in splicing reactions using in vitro transcribed beta-globin pre-mRNA as substrate. The resulting pellet which contained the snRNAs, retained 5' splice site cleavage activity in a similar splicing reaction. Supplementation of the inactive supernatant fraction with in vitro transcribed U1 snRNA, partially restored 5' splice site cleavage activity thereby demonstrating the specific requirement of U1 snRNP in the initial stage of pre-mRNA splicing.
使用体外转录的U1小核核糖核蛋白颗粒（U1 snRNP）进行功能重建。通过在100,000×g下离心，从HeLa细胞核提取物中去除其组成的snRNP。上清液不含snRNA，并且在以体外转录的β-珠蛋白前体mRNA为底物的剪接反应中缺乏切割活性。含有snRNA的所得沉淀在类似的剪接反应中保留了5'剪接位点切割活性。用体外转录的U1 snRNA补充无活性的上清液部分，部分恢复了5'剪接位点切割活性，从而证明了U1 snRNP在mRNA前体剪接初始阶段的特定需求。
