Interaction between the U1 snRNP-A protein and the 160-kD subunit Of cleavage-polyadenylation specificity factor increases polyadenylation efficiency in vitro
Interaction between the U1 snRNP-A protein and the 160-kD subunit Of cleavage-polyadenylation specificity factor increases polyadenylation efficiency in vitro
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DOI：
10.1101/gad.10.3.325
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发表时间：
1996-02-01
影响因子：
10.5
通讯作者：
Alwine, JC
中科院分区：
文献类型：
--
作者：
Lutz, CS;Murthy, KGK;Alwine, JC
我们先前已经表明，U1 snRNP-A蛋白（U1 A）与SV 40晚期多聚腺苷酸化信号中的元件相互作用，并且这种关联增加了多聚腺苷酸化效率。据推测，这种相互作用的发生，以促进U1 snRNP和多聚腺苷酸化复合物的蛋白质的组分之间的蛋白质-蛋白质协会。我们现在已经使用GST融合蛋白实验，共免疫沉淀和远Western印迹分析，以证明U1 A和160 kD亚基的裂解-多聚腺苷酸化特异性因子（CPSF）之间的直接结合。此外，从CPSF纯化的各个阶段的馏分的蛋白质印迹分析表明，U1 A与CPSF共纯化到一个点，但可以在高度纯化的馏分中分离。这些数据表明，U1 A蛋白不是CPSF的组成部分，但可能能够相互作用并影响其活性。在这方面，将纯化的重组U1 A添加到含有CPSF、poly（A）聚合酶和预切割的RNA底物的聚腺苷酸化反应中导致聚腺苷酸化水平和poly（A）尾长的浓度依赖性增加。与U1 A引起的聚腺苷酸化效率的增加一致，重组U1 A稳定了CPSF与含有AAUAAA的底物RNA在电泳迁移率变化实验中的相互作用。这些发现表明，除了其在剪接中的功能，U1 A通过对多聚腺苷酸化的影响在RNA加工中发挥更全面的作用。
We have previously shown that the U1 snRNP-A protein (U1A) interacts with elements in the SV40 late polyadenylation signal and that this association increases polyadenylation efficiency. It was postulated that this interaction occurs to facilitate protein-protein association between components of the U1 snRNP and proteins of the polyadenylation complex. We have now used GST fusion protein experiments, coimmunoprecipitations and Far Western blot analyses to demonstrate direct binding between U1A and the 160-kD subunit of cleavage-polyadenylation specificity factor (CPSF). In addition, Western blot analyses of fractions from various stages of CPSF purification indicated that U1A copurified with CPSF to a point but could be separated in the highly purified fractions. These data suggest that U1A protein is not an integral component of CPSF but may be able to interact and affect its activity. In this regard, the addition of purified, recombinant U1A to polyadenylation reactions containing CPSF, poly(A) polymerase, and a precleaved RNA substrate resulted in concentration-dependent increases in both the level of polyadenylation and poly(A) tail length. In agreement with the increase in polyadenylation efficiency caused by U1A, recombinant U1A stabilized the interaction of CPSF with the AAUAAA-containing substrate RNA in electrophoretic mobility shift experiments. These findings suggest that, in addition to its function in splicing, U1A plays a more global role in RNA processing through effects on polyadenylation.