The Hinge Domain of the Cleavage Stimulation Factor Protein CstF-64 Is Essential for CstF-77 Interaction, Nuclear Localization, and Polyadenylation
The Hinge Domain of the Cleavage Stimulation Factor Protein CstF-64 Is Essential for CstF-77 Interaction, Nuclear Localization, and Polyadenylation
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DOI：
10.1074/jbc.m109.061705
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发表时间：
2010-01-01
影响因子：
4.8
通讯作者：
MacDonald, Clinton C.
中科院分区：
文献类型：
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作者：
Hockert, J. Andrew;Yeh, Hsiang-Jui;MacDonald, Clinton C.
因为多聚腺苷酸化对于细胞生长是必需的，所以多聚腺苷酸化蛋白功能的体内检查一直是困难的。在这里，我们描述了一种新的体内测定，允许对CstF-64进行结构-功能测定，CstF-64是一种与切割位点下游的前体mRNA结合的蛋白质，用于准确和有效的聚腺苷酸化。在该测定法（用于多聚腺苷酸化的茎环荧光素酶测定法，S）中，具有修饰的下游序列元件的荧光素酶前mRNA的表达取决于MS 2-CstF-64融合蛋白的共表达。我们在这里表明，SSTF准确地反映了CstF-64依赖的多聚腺苷酸化，证实了这种测定的有效性。使用SSCP，我们确定了参与RNA结合、与CstF-77（“铰链”结构域）相互作用以及与转录偶联的CstF-64结构域对于聚腺苷酸化是关键的。此外，我们表明铰链结构域是CstF-64与CstF-77相互作用和随后的核定位所必需的，这表明预先形成的CstF复合物的核输入是聚腺苷酸化中的重要步骤。
Because polyadenylation is essential for cell growth, in vivo examination of polyadenylation protein function has been difficult. Here we describe a new in vivo assay that allows structure-function assays on CstF-64, a protein that binds to pre-mRNAs downstream of the cleavage site for accurate and efficient polyadenylation. In this assay (the stem-loop luciferase assay for polyadenylation, SLAP), expression of a luciferase pre-mRNA with a modified downstream sequence element was made dependent upon co-expression of an MS2-CstF-64 fusion protein. We show here that SLAP accurately reflects CstF-64-dependent polyadenylation, confirming the validity of this assay. Using SLAP, we determined that CstF-64 domains involved in RNA binding, interaction with CstF-77 (the "Hinge" domain), and coupling to transcription are critical for polyadenylation. Further, we showed that the Hinge domain is necessary for CstF-64 interaction with CstF-77 and consequent nuclear localization, suggesting that nuclear import of a preformed CstF complex is an essential step in polyadenylation.