The mammalian pre-mRNA 3′-end-processing machinery consists of cleavage and polyadenylation specificity factor (CPSF), cleavage stimulation factor (CstF), and other proteins, but the overall architecture of this machinery remains unclear. CPSF contains two functionally distinct modules: a cleavage factor (mCF) and a polyadenylation specificity factor (mPSF). Here, we have produced recombinant human CPSF and CstF and examined these factors by electron microscopy (EM). We find that mPSF is the organizational core of the machinery, while the conformations of mCF and CstF and the position of mCF relative to mPSF are highly variable. We have identified by cryo-EM a segment in CPSF100 that tethers mCF to mPSF, and we have named it the PSF interaction motif (PIM). Mutations in the PIM can abolish CPSF formation, indicating that it is a crucial contact in CPSF. We have also obtained reconstructions of mCF and CstF77 by cryo-EM, assembled around the mPSF core.

哺乳动物前 mRNA 3' 末端加工机制由切割和多聚腺苷酸化特异性因子 (CPSF)、切割刺激因子 (CstF) 和其他蛋白质组成，但该机制的整体架构仍不清楚。 CPSF 包含两个功能不同的模块：切割因子 (mCF) 和聚腺苷酸化特异性因子 (mPSF)。在这里，我们生产了重组人 CPSF 和 CstF，并通过电子显微镜 (EM) 检查了这些因子。我们发现mPSF是该机制的组织核心，而mCF和CstF的构象以及mCF相对于mPSF的位置变化很大。我们通过冷冻电镜鉴定了 CPSF100 中将 mCF 与 mPSF 连接的片段，并将其命名为 PSF 相互作用基序 (PIM)。 PIM 的突变可以消除 CPSF 的形成，表明它是 CPSF 中的关键接触点。我们还通过冷冻电镜获得了 mCF 和 CstF77 的重建，并围绕 mPSF 核心组装。