Tazi J, Kornstädt U, Rossi F, Jeanteur P, Cathala G, Brunel C, Lührmann R
UA CNRS 1191, Génétique Moléculaire Université Montpellier II, France.
Nature. 1993 May 20;363(6426):283-6. doi: 10.1038/363283a0.
The U1 small nuclear ribonucleoprotein (snRNP) particle is one of the Sm class of snRNPs essential for splicing of precursor messenger RNA. Mammalian U1 snRNP contains a 165-nucleotide long RNA molecule and at least 11 proteins: the U1-specific 70K proteins A and C, and the common U snRNP proteins (B', B, D1, D2, D3, E, F and G). One of the functions of U1 snRNP is recognition of the 5' splice site, an event that requires both U1 RNA and U1 proteins. The 70K protein is the only heavily phosphorylated U1 protein in the cell. Isolated U1 snRNPs are associated with a kinase activity that selectively phosphorylates the 70K protein in vitro in a reaction requiring ATP. Here we investigate the role of phosphorylation of the 70K protein in the splicing of pre-mRNA. The 70K protein on U1 snRNPs was phosphorylated in vitro with either ATP, or with ATP-gamma S, which gave a thiophosphorylated product that was resistant to dephosphorylation by phosphatases. When HeLa nuclear splicing extracts that had been depleted of endogenous U1 snRNPs were complemented with U1 snRNPs possessing normal phosphorylated 70K protein, mature spliceosomes were generated and the splicing activity of the extracts was fully restored. By contrast, if thiophosphorylated U1 snRNPs were used instead, splicing was completely inhibited, although formation of the mature spliceosome was unaffected. Our data show that the state of phosphorylation of the U1-specific 70K protein is critical for its participation in a pre-catalytic step of the splicing reaction.
U1小核核糖核蛋白（snRNP）颗粒是Sm类snRNPs之一，对前体信使RNA的剪接至关重要。哺乳动物U1 snRNP包含一个165个核苷酸长的RNA分子和至少11种蛋白质：U1特异性的70K蛋白A和C，以及常见的U snRNP蛋白（B'、B、D1、D2、D3、E、F和G）。U1 snRNP的功能之一是识别5'剪接位点，这一过程需要U1 RNA和U1蛋白。70K蛋白是细胞中唯一高度磷酸化的U1蛋白。分离的U1 snRNPs与一种激酶活性相关，该激酶活性在需要ATP的反应中能在体外选择性地磷酸化70K蛋白。在此，我们研究70K蛋白磷酸化在前体mRNA剪接中的作用。U1 snRNPs上的70K蛋白在体外分别用ATP或ATP-γS进行磷酸化，ATP-γS产生的硫代磷酸化产物对磷酸酶的去磷酸化具有抗性。当耗尽内源性U1 snRNPs的HeLa细胞核剪接提取物用具有正常磷酸化70K蛋白的U1 snRNPs进行补充时，会产生成熟的剪接体，提取物的剪接活性也完全恢复。相比之下，如果使用硫代磷酸化的U1 snRNPs，则剪接被完全抑制，尽管成熟剪接体的形成不受影响。我们的数据表明，U1特异性70K蛋白的磷酸化状态对其参与剪接反应的催化前步骤至关重要。
