Thiophosphorylation of U1-70K protein inhibits pre-mRNA splicing
Thiophosphorylation of U1-70K protein inhibits pre-mRNA splicing
复制标题
U1-70K 蛋白的硫代磷酸化抑制前 mRNA 剪接
作者：
J. Tazi;U. Kornstädt;F. Rossi;P. Jeanteur;G. Cathala;C. Brunel;R. Lührmann
U1小核核糖核蛋白（snRNP）颗粒是前体信使RNA 1 -5剪接所必需的Sm类snRNP之一。哺乳动物U1 snRNP含有165个核苷酸长的RNA分子和至少11种蛋白质：U1特异性70 K蛋白A和C，以及常见的UsnRNP蛋白（B′、B、D1、D2、D3、E、F和G）。Ul snRNP的功能之一是识别5′剪接位点，这是需要Ul RNA和Ul蛋白的事件6 -10。70 K蛋白是细胞中唯一严重磷酸化的Ul蛋白11，12。分离的Ul snRNP与在需要ATP的反应中体外选择性磷酸化70 K蛋白的激酶活性相关。在这里，我们调查的70 K蛋白的磷酸化的作用，在前体mRNA的剪接。U1 snRNP上的70 K蛋白在体外用ATP或ATP-γS磷酸化，这产生了对磷酸酶的去磷酸化有抗性的硫代磷酸化产物。当用具有正常磷酸化70 K蛋白的Ul snRNP补充已经耗尽内源性Ul snRNP的HeLa核剪接提取物时，产生成熟的剪接体，并且提取物的剪接活性完全恢复。相反，如果使用硫代磷酸化的U1 snRNP，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，磷酸化状态的UL-特异性70 K蛋白是至关重要的，它参与了前催化步骤的剪接反应。
THE U1 small nuclear ribonucleoprotein (snRNP) particle is one of the Sm class of snRNPs essential for splicing of precursor messenger RNA1–5. Mammalian Ul snRNP contains a 165-nucleo-tide long RNA molecule and at least 11 proteins: the Ul-specific 70K proteins A and C, and the common U snRNP proteins (B′, B, Dl, D2, D3, E, F and G). One of the functions of Ul snRNP is recognition of the 5′ splice site, an event that requires both Ul RNA and Ul proteins6–10. The 70K protein is the only heavily phosphorylated Ul protein in the cell11,12. Isolated Ul 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 Ul snRNPs was phosphorylated in vitro with either ATP, or with ATP-γS, which gave a thiophosphorylated product that was resistant to dephos-phorylation by phosphatases. When HeLa nuclear splicing extracts that had been depleted of endogenous Ul snRNPs were complemented with Ul snRNPs possessing normal phosphorylated 70K protein, mature spliceosomes were generated and the splicing activity of the extracts was fully restored. By contrast, if thiophosphorylated Ul 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 Ul-specific 70K protein is critical for its participation in a pre-catalytic step of the splicing reaction.
酵母中的前 mRNA 剪接。
DOI：
10.1016/0168-9525(91)90276-v
发表时间：
1991
期刊：
Trends in genetics : TIG
影响因子：
--
作者：
Ruby,SW;Abelson,J
通讯作者：
Abelson,J
影响因子：
56.9
作者：
FU, XD;MANIATIS, T
通讯作者：
MANIATIS, T