Tatei K, Takemura K, Tanaka H, Masaki T, Ohshima Y
J Biol Chem. 1987 Aug 25;262(24):11667-74.
A nuclear extract from HeLa cells was fractionated by DEAE-Sepharose chromatography. The obtained fractions were assayed for binding to an RNA transcript carrying a splice site sequence of 9-16 nucleotides by a filter binding technique. The U1 RNA-rich small nuclear ribonucleoprotein (snRNP) fractions, which showed binding activities for both 5' and 3' splice site RNAs, were studied for the sequence specificity of their binding. Results indicate that the U1-rich snRNP fraction can recognize both 5' and 3' splice site sequences. The U1 RNP, which was highly purified from the snRNP fractions, bound to at least some 5' splice site sequences, but not to a consensus 3' splice site sequence. Therefore, purified U1 RNP can directly recognize a 5' splice site, but not a 3' splice site. The binding activity for the 5' splice sites was lost either by digestion with micrococcal nuclease or by digestion of the 5' end of U1 RNA with RNase H and a complementary oligodeoxynucleotide, indicating the involvement of U1 RNA. Involvement of a protein moiety as well in this binding was suggested by the loss of binding activity upon heating at 60 degrees C. The binding activity to a 3' splice site sequence was not sensitive to digestion by micrococcal nuclease and was removed by protein A-coupled anti-Sm antibody. This activity was found in sucrose gradient fractions of about 8 S.
用DEAE-琼脂糖层析法对HeLa细胞的核提取物进行分级分离。通过滤膜结合技术，对获得的各组分进行检测，以确定其与携带9 - 16个核苷酸剪接位点序列的RNA转录本的结合情况。对富含U1 RNA的小核核糖核蛋白（snRNP）组分进行研究，该组分对5'和3'剪接位点RNA均显示出结合活性，以确定其结合的序列特异性。结果表明，富含U1的snRNP组分能够识别5'和3'剪接位点序列。从snRNP组分中高度纯化得到的U1 RNP与至少一些5'剪接位点序列结合，但不与共有3'剪接位点序列结合。因此，纯化的U1 RNP能够直接识别5'剪接位点，但不能识别3'剪接位点。用微球菌核酸酶消化或用RNase H和互补的寡聚脱氧核苷酸消化U1 RNA的5'末端后，5'剪接位点的结合活性丧失，这表明U1 RNA参与其中。在60℃加热后结合活性丧失，提示蛋白质部分也参与了这种结合。对3'剪接位点序列的结合活性对微球菌核酸酶消化不敏感，可被与蛋白A偶联的抗Sm抗体去除。这种活性存在于约8S的蔗糖梯度组分中。
