Most eukaryotic messenger RNAs (mRNAs) are processed at their 3′ end by the cleavage and polyadenylation specificity factor (CPF/CPSF). CPF mediates the endonucleolytic cleavage of the pre-mRNA and addition of a polyadenosine (poly(A)) tail, which together define the 3′ end of the mature transcript. The activation of CPF is highly regulated to maintain the fidelity of RNA processing. Here, using cryo-EM of yeast CPF, we show that the Mpe1 subunit directly contacts the polyadenylation signal sequence in nascent pre-mRNA. The region of Mpe1 that contacts RNA also promotes the activation of CPF endonuclease activity and controls polyadenylation. The Cft2 subunit of CPF antagonizes the RNA-stabilized configuration of Mpe1. In vivo, the depletion or mutation of Mpe1 leads to widespread defects in transcription termination by RNA polymerase II, resulting in transcription interference on neighboring genes. Together, our data suggest that Mpe1 plays a major role in accurate 3′ end processing, activating CPF, and ensuring timely transcription termination.

大多数真核信使 RNA (mRNA) 在其 3' 端被切割和聚腺苷酸化特异性因子 (CPF/CPSF) 加工。CPF 介导前体 mRNA 的核酸内切裂解和多聚腺苷 (poly(A)) 尾巴的添加，它们共同定义了成熟转录本的 3' 端。CPF 的激活受到高度调节，以维持 RNA 加工的保真度。在这里，我们使用酵母 CPF 的冷冻 EM，表明 Mpe1 亚基直接接触新生前体 mRNA 中的聚腺苷酸化信号序列。接触 RNA 的 Mpe1 区域也促进 CPF 核酸内切酶活性的激活并控制聚腺苷酸化。CPF 的 Cft2 亚基拮抗 Mpe1 的 RNA 稳定构型。体内, Mpe1 的缺失或突变导致 RNA 聚合酶 II 转录终止的广泛缺陷，从而导致对邻近基因的转录干扰。总之，我们的数据表明 Mpe1 在准确的 3' 末端处理、激活 CPF 和确保转录及时终止方面起着重要作用。