Both phosphorylation and dephosphorylation of ASF/SF2 are required for pre-mRNA splicing in vitro.
Both phosphorylation and dephosphorylation of ASF/SF2 are required for pre-mRNA splicing in vitro.
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发表时间：
1997-12
期刊：
影响因子：
4.5
通讯作者：
Wenhong Cao;S. Jamison;Mariano A. Garcia-Blanco
中科院分区：
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作者：
Wenhong Cao;S. Jamison;Mariano A. Garcia-Blanco
从前信使 RNA 中去除内含子的剪接反应需要剪接体在新生转录物上的组装、底物-酶复合物的正确折叠，以及最后的两个酯交换反应。剪接反应的这些阶段必须需要仔细协调。在这里，我们显示的数据表明 SR 蛋白的连续磷酸化和去磷酸化标志着剪接反应阶段之间的过渡。当我们在测量前剪接体向剪接体转化的反应中证明这些蛋白质的去磷酸化消除了它们的活性时，许多数据已经得出这样的想法：SR 蛋白质的磷酸化可以调节它们的活性（Roscigno RF，Garcia-Blanco MA，1995，RNA 1：692-706）。随后，Xiao和Manley(1997，Genes & Dev 11：334-344)表明，在用过量随机RNA攻击的反应中，磷酸化的ASF/SF2而非模拟磷酸化的ASF/SF2激活HIV tat前mRNA的剪接。在这里，我们确认并扩展了这两个发现。磷酸化的 ASF/SF2 有效地补充了 SR 蛋白缺陷的 HeLa S100 提取物，促进腺病毒 2 衍生的前信使 RNA 的剪接，而未磷酸化的 ASF/SF2 则不然。此外，我们发现，未磷酸化的 ASF/SF2 抑制 HeLa 核提取物中的剪接，而 ASF/SF2 的磷酸化则逆转了抑制并增强了剪接。我们还提供的数据表明，一旦剪接体组装，第一次酯交换反应就需要 ASF/SF2 的去磷酸化。硫代磷酸化的ASF/SF2不易去磷酸化，可以促进剪接体组装，但不能促进第一次酯交换反应。这些数据与其他观察结果一起首次表明前信使 RNA 体外剪接中需要 SR 蛋白去磷酸化。
The splicing reaction that removes introns from pre-messenger RNAs requires the assembly of the spliceosome on the nascent transcript, proper folding of the substrate-enzyme complex, and finally, two transesterification reactions. These stages in the splicing reaction must require careful orchestration. Here we show data that suggest that the sequential phosphorylation and dephosphorylation of SR proteins mark the transition between stages in the splicing reaction. Many data had already led to the idea that phosphorylation of SR proteins could modulate their activity, when we showed that dephosphorylation of these proteins abrogates their activity in a reaction measuring conversion of pre-spliceosomes to spliceosomes (Roscigno RF, Garcia-Blanco MA, 1995, RNA 1:692-706). Subsequently, Xiao and Manley (1997, Genes & Dev 11:334-344) showed that phosphorylated ASF/SF2, but not mock-phosphorylated ASF/SF2, activates the splicing of HIV tat pre-mRNA in reactions challenged with excess random RNA. Here we confirm and extend these two findings. Phosphorylated ASF/SF2 efficiently complemented an SR protein-deficient HeLa S100 extract in promoting the splicing of an adenovirus-2-derived pre-messenger RNA, whereas unphosphorylated ASF/ SF2 did not. Moreover, we show that, whereas unphosphorylated ASF/SF2 inhibited splicing in HeLa nuclear extracts, phosphorylation of the ASF/SF2 reversed the inhibition and enhanced splicing. We also present data that shows that dephosphorylation of ASF/SF2 is required for the first transesterification reaction once the spliceosome has assembled. Thiophosphorylated ASF/SF2, which cannot be readily dephosphorylated, can promote spliceosome assembly, but cannot promote the first transesterification reaction. These data, together with other observations, indicate for the first time a requirement for SR protein dephosphorylation in pre-messenger RNA splicing in vitro.