Association of polyadenylation cleavage factor I with U1 snRNP
Association of polyadenylation cleavage factor I with U1 snRNP
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DOI：
10.1261/rna.5104603
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发表时间：
2003-11-01
期刊：
影响因子：
4.5
通讯作者：
Alwine, JC
中科院分区：
文献类型：
--
作者：
Awasthi, S;Alwine, JC
剪接和多聚腺苷酸化因子相互作用以控制多聚腺苷酸化以及剪接和多聚腺苷酸化的偶联。我们的文件之间的相互作用U1 snRNP和哺乳动物的多聚腺苷酸化裂解因子I（CF Im），需要在多聚腺苷酸化位点的前mRNA的裂解的几个多聚腺苷酸化因子之一。蔗糖密度梯度离心证明CF Im分离成两个部分，一个轻的部分，其中包含已知的CF Im亚基（72，68，59，和25 kD），和一个重的部分，富含snRNP，其中主要包含68-和25-kD的CF Im亚基。使用特异性抗体，我们发现，重馏分含有U1 snRNP/CF Im共沉淀复合物。这些复合物对RNA酶处理不敏感，表明共沉淀不是由于RNA束缚。体外结合实验表明，68-和25-kD亚基结合和共迁移与U1 snRNP。此外，25-kD CF Im亚基特异性结合U1 snRNP的70 K蛋白（U1 70 K）。这种结合可以解释CF Im/U1 snRNP相互作用。在这些研究中，我们发现mAb 2.73（mAb 2.73），一种已建立的U1 70 K抗体，有效地从细胞提取物中沉淀大量的CF Im。由于mAb 2.73已用于与U1 snRNP和U1 70 K蛋白相关的许多先前研究中，因此在基于mAb 2.73的使用评价过去和未来数据时必须考虑CF Im的沉淀。
Splicing and polyadenylation factors interact for the control of polyadenylation and the coupling of splicing and polyadenylation. We document an interaction between the U1 snRNP and mammalian polyadenylation cleavage factor I (CF Im), one of several polyadenylation factors needed for the cleavage of the pre-mRNA at the polyadenylation site. Sucrose density gradient centrifugation demonstrated that CF Im separated into two fractions, a light fraction which contained the known CF Im subunits (72, 68, 59, and 25 kD), and a heavy fraction, rich in snRNPs, which contained predominately the 68- and 25-kD CF Im subunits. Using specific antibodies we found that the heavy fraction contains U1 snRNP/CF Im coprecipitable complexes. These complexes were insensitive to RNase treatment, suggesting that the coprecipitation is not due to RNA tethering. In vitro binding experiments show that both the 68- and 25-kD subunits bind to and comigrate with U1 snRNP. In addition, the 25-kD CF Im subunit binds specifically to the 70K protein of U1 snRNP (U1 70K). This binding may account for the CF Im/U1 snRNP interaction. During these studies we found that mAb 2.73 (mAb 2.73), an established U1 70K antibody, efficiently precipitates the bulk of the CF Im from cellular extracts. Because mAb 2.73 has been used in a number of previous studies related to the U1 snRNP and the U1 70K protein, the precipitation of CF Im must be considered in evaluating past and future data based on the use of mAb 2.73.