Department of Structural Cell Biology, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany.
Department of Structural Cell Biology, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany
RNA. 2024 Jun 17;30(7):795-806. doi: 10.1261/rna.079915.123.
3' end processing of most eukaryotic precursor-mRNAs (pre-mRNAs) is a crucial cotranscriptional process that generally involves the cleavage and polyadenylation of the precursor transcripts. Within the human 3' end processing machinery, the four-subunit mammalian polyadenylation specificity factor (mPSF) recognizes the polyadenylation signal (PAS) in the pre-mRNA and recruits the poly(A) polymerase α (PAPOA) to it. To shed light on the molecular mechanisms of PAPOA recruitment to mPSF, we used a combination of cryogenic-electron microscopy (cryo-EM) single-particle analysis, computational structure prediction, and in vitro biochemistry to reveal an intricate interaction network. A short linear motif in the mPSF subunit FIP1 interacts with the structured core of human PAPOA, with a binding mode that is evolutionarily conserved from yeast to human. In higher eukaryotes, however, PAPOA contains a conserved C-terminal motif that can interact intramolecularly with the same residues of the PAPOA structured core used to bind FIP1. Interestingly, using biochemical assay and cryo-EM structural analysis, we found that the PAPOA C-terminal motif can also directly interact with mPSF at the subunit CPSF160. These results show that PAPOA recruitment to mPSF is mediated by two distinct intermolecular connections and further suggest the presence of mutually exclusive interactions in the regulation of 3' end processing.
真核生物前体 mRNA（pre-mRNA）的 3' 末端加工是一个关键的共转录过程，通常涉及前体转录本的切割和多聚腺苷酸化。在人类 3' 末端加工机制中，四亚基哺乳动物多聚腺苷酸化特异性因子（mPSF）识别 pre-mRNA 中的多聚腺苷酸化信号（PAS），并将多聚腺苷酸化酶 α（PAPOA）募集到其上。为了阐明 PAPOA 募集到 mPSF 的分子机制，我们使用低温电子显微镜（cryo-EM）单颗粒分析、计算结构预测和体外生物化学相结合的方法，揭示了一个复杂的相互作用网络。mPSF 亚基 FIP1 中的一个短线性基序与人类 PAPOA 的结构核心相互作用，其结合模式在从酵母到人等高等生物中是保守的。然而，在高等真核生物中，PAPOA 含有一个保守的 C 末端基序，该基序可以与 PAPOA 结构核心的相同残基进行分子内相互作用，用于与 FIP1 结合。有趣的是，通过生化测定和 cryo-EM 结构分析，我们发现 PAPOA C 末端基序也可以直接与 mPSF 的亚基 CPSF160 相互作用。这些结果表明，PAPOA 募集到 mPSF 是由两个不同的分子间连接介导的，并进一步表明 3' 末端加工的调节中存在相互排斥的相互作用。
