Kinetic role for mammalian SF1/BBP in spliceosome assembly and function after polypyrimidine tract recognition by U2AF
Kinetic role for mammalian SF1/BBP in spliceosome assembly and function after polypyrimidine tract recognition by U2AF
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DOI：
10.1074/jbc.m001483200
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发表时间：
2000-12-01
影响因子：
4.8
通讯作者：
Valcárcel, J
中科院分区：
文献类型：
--
作者：
Guth, S;Valcárcel, J
高等真核生物内含子 3' 端之前有两个对前 mRNA 剪接重要的序列，即分支点 (BP) 和聚嘧啶 (Py) 区。这些信号的初始识别涉及剪接因子 SF1/哺乳动物分支点结合蛋白 (mBBP) 与 BP 以及 U2AF(65) 与副道的协同结合。在从纯化组分重构的反应中，这两个因素都是 U2 小核核糖核蛋白颗粒 (U2 snRNP) 募集至 BP 所必需的。相比之下，酿酒酵母中 ST1/BBP 的大量消耗不会显着损害剪接体组装或剪接。由于 BP 序列在哺乳动物中不太保守，这些差异可能反映出该系统中对 SF1/BBP 的更严格要求。我们在此报告，从 HeLa 细胞核提取物中大量去除 SF1/mBBP 只会导致其剪接体组装和剪接活性适度降低。其中一些效应反映了 U2 snRNP 结合动力学的差异。尽管 U2AF(65) 结合在耗尽的提取物中减少，但由 SF1/mBBP 耗尽引起的缺陷不能通过外源添加的 U2AF65 增加 Py 道的占用来完全恢复，这表明 SF1/mBBP 在 U2 snRNP 募集中的作用不同于促进 U2AF65 结合。
Two sequences important for pre-mRNA splicing precede the 3' end of introns in higher eukaryotes, the branch point (BP) and the polypyrimidine (Py) tract. Initial recognition of these signals involves cooperative binding of the splicing factor SF1/mammalian branch point binding protein (mBBP) to the BP and of U2AF(65) to the By tract. Both factors are required for recruitment of the U2 small nuclear ribonucleoprotein particle (U2 snRNP) to the BP in reactions reconstituted from purified components. In contrast, extensive depletion of ST1/BBP in Saccharomyces cerevisiae does not compromise spliceosome assembly or splicing significantly. As BP sequences are less conserved in mammals, these discrepancies could reflect more stringent requirements for SF1/BBP in this system. We report here that extensive depletion of SF1/mBBP from nuclear extracts of HeLa cells results in only modest reduction of their activity in spliceosome assembly and splicing. Some of these effects reflect differences in the kinetics of U2 snRNP binding. Although U2AF(65) binding was reduced in the depleted extracts, the defects caused by SF1/mBBP depletion could not be fully restored by an increase in occupancy of the Py tract by exogenously added U2AF65, arguing for a role of SF1/mBBP in U2 snRNP recruitment distinct from promoting U2AF65 binding.