Department of Biochemistry, University of Zurich, Zurich, Switzerland.
Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland.
Elife. 2017 Dec 23;6:e33111. doi: 10.7554/eLife.33111.
3' polyadenylation is a key step in eukaryotic mRNA biogenesis. In mammalian cells, this process is dependent on the recognition of the hexanucleotide AAUAAA motif in the pre-mRNA polyadenylation signal by the cleavage and polyadenylation specificity factor (CPSF) complex. A core CPSF complex comprising CPSF160, WDR33, CPSF30 and Fip1 is sufficient for AAUAAA motif recognition, yet the molecular interactions underpinning its assembly and mechanism of PAS recognition are not understood. Based on cross-linking-coupled mass spectrometry, crystal structure of the CPSF160-WDR33 subcomplex and biochemical assays, we define the molecular architecture of the core human CPSF complex, identifying specific domains involved in inter-subunit interactions. In addition to zinc finger domains in CPSF30, we identify using quantitative RNA-binding assays an N-terminal lysine/arginine-rich motif in WDR33 as a critical determinant of specific AAUAAA motif recognition. Together, these results shed light on the function of CPSF in mediating PAS-dependent RNA cleavage and polyadenylation.
3' 多聚腺苷酸化是真核 mRNA 生物发生的关键步骤。在哺乳动物细胞中，这一过程依赖于切割和多聚腺苷酸化特异性因子 (CPSF) 复合物对 pre-mRNA 多聚腺苷酸化信号中六核苷酸 AAUAAA 基序的识别。由 CPSF160、WDR33、CPSF30 和 Fip1 组成的核心 CPSF 复合物足以识别 AAUAAA 基序，但构成其组装的分子相互作用及其 PAS 识别的机制尚不清楚。基于交联耦联质谱、CPSF160-WDR33 亚基复合物的晶体结构和生化测定，我们定义了核心人类 CPSF 复合物的分子结构，确定了参与亚基间相互作用的特定结构域。除了 CPSF30 中的锌指结构域外，我们还通过定量 RNA 结合测定确定了 WDR33 中的 N 端赖氨酸/精氨酸丰富基序是特异性识别 AAUAAA 基序的关键决定因素。这些结果共同阐明了 CPSF 在介导 PAS 依赖性 RNA 切割和多聚腺苷酸化中的作用。
