The type 2C Ser/Thr phosphatase PP2Cγ is a pre-mRNA splicing factor
The type 2C Ser/Thr phosphatase PP2Cγ is a pre-mRNA splicing factor
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DOI：
10.1101/gad.13.1.87
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发表时间：
1999-01-01
影响因子：
10.5
通讯作者：
Krainer, AR
中科院分区：
文献类型：
--
作者：
Murray, MV;Kobayashi, R;Krainer, AR
为了确定人类前mrna剪接的活性，我们开发了一种将HeLa细胞核提取物分离成五个互补部分的方法。通过在其余四个部分存在的情况下分析剪接的重建，从其中一个部分中纯化了一种称为SCF1的活性。SCF1的一个组分被证明是PP2C γ，一种2C型丝氨酸/苏氨酸磷酸酶，其功能以前未知。先前的研究表明，剪接因子的去磷酸化可能对剪接体组装后的催化作用很重要，尽管所涉及的特定磷酸酶的身份尚不清楚。在这里，我们发现人类PP2C γ在体外剪接反应中与剪接体物理相关，但在剪接体组装的早期阶段首先需要有效形成A复合物。磷酸酶活性是PP2C γ剪接功能所必需的，因为活性位点突变体不支持剪接体组装。对PP2C γ的需求是高度特异性的，因为密切相关的磷酸酶PP2C α不能替代PP2C γ。与剪接的作用一致，PP2C γ在体内定位于细胞核。我们得出结论，剪接体的形成需要至少一个由PP2C γ催化的特异性去磷酸化事件。
To identify activities involved in human pre-mRNA splicing, we have developed a procedure to separate HeLa cell nuclear extract into five complementing fractions. An activity called SCF1 was purified from one of these fractions by assaying for reconstitution of splicing in the presence of the remaining four fractions. A component of SCF1 is shown to be PP2C gamma, a type 2C Ser/Thr phosphatase of previously unknown function. Previous work suggested that dephosphorylation of splicing factors may be important for catalysis after spliceosome assembly, although the identities of the specific phosphatases involved remain unclear. Here we show that human PP2C gamma is physically associated with the spliceosome in vitro throughout the splicing reaction, but is first required during the early stages of spliceosome assembly for efficient formation of the A complex. The phosphatase activity is required for the splicing function of PP2C gamma, as an active site mutant does not support spliceosome assembly. The requirement for PP2C gamma is highly specific, as the closely related phosphatase PP2C alpha cannot substitute for PP2C gamma. Consistent with a role in splicing, PP2C gamma localizes to the nucleus in vivo. We conclude that at least one specific dephosphorylation event catalyzed by PP2C gamma is required for formation of the spliceosome.