Most eukaryotic pre-mRNAs must undergo 3′-end cleavage and polyadenylation prior to their export from the nucleus. A large number of proteins in several complexes participate in this 3′-end processing, including cleavage and polyadenylation specificity factor (CPSF) in mammals. The CPSF30 subunit contains five CCCH zinc fingers (ZFs), with ZF2–ZF3 being required for the recognition of the AAUAAA poly(A) signal. ZF4–ZF5 recruits the hFip1 subunit of CPSF, although the details of this interaction have not been characterized. Here we report the crystal structure of human CPSF30 ZF4–ZF5 in complex with residues 161–200 of hFip1 at 1.9 Å resolution, illuminating the molecular basis for their interaction. Unexpectedly, the structure reveals one hFip1 molecule binding to each ZF4 and ZF5, with a conserved mode of interaction. Our mutagenesis studies confirm that the CPSF30–hFip1 complex has 1:2 stoichiometry in vitro. Mutation of each binding site in CPSF30 still allows one copy of hFip1 to bind, while mutation of both sites abrogates binding. Our fluorescence polarization binding assays show that ZF4 has higher affinity for hFip1, with a K_d of 1.8 nM. We also demonstrate that two copies of the catalytic module of poly(A) polymerase (PAP) are recruited by the CPSF30–hFip1 complex in vitro, and both hFip1 binding sites in CPSF30 can support polyadenylation.

中文翻译：

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人CPSF30与hFip1相互作用的分子机制

大多数真核前mRNA在从细胞核中输出之前必须经过3'末端裂解和聚腺苷酸化。几种复合物中的大量蛋白质参与了该3'末端加工，包括哺乳动物中的裂解和聚腺苷酸特异性因子（CPSF）。CPSF30子单元包含五个CCCH锌指（ZF），需要ZF2-ZF3才能识别AAUAAA poly（A）信号。ZF4-ZF5募集了CPSF的hFip1亚基，尽管这种相互作用的细节尚未确定。在这里，我们报道了人类CPSF30 ZF4-ZF5的晶体结构，其复合物具有1.9Å分辨率的hFip1残基161-200，阐明了它们相互作用的分子基础。出乎意料的是，该结构揭示了一个hFip1分子与每个ZF4和ZF5结合，具有保守的相互作用方式。我们的诱变研究证实，CPSF30–hFip1复合物的体外化学计量比为1：2。CPSF30中每个结合位点的突变仍然允许hFip1的一个拷贝结合，而两个位点的突变都取消了结合。我们的荧光偏振结合测定表明ZF4对hFip1具有更高的亲和力，K _d为1.8 nM。我们还证明，CPSF30–hFip1复合物在体外募集了两个副本的poly（A）聚合酶（PAP）催化模块，并且CPSF30中的两个hFip1结合位点都可以支持聚腺苷酸化。