(A) Equilibrium binding of CPSF160FL-WDR33M1-K410-CPSF30FL -Fip1FL to an Atto532-labelled, 16-nucleotide RNA containing the PAS hexanucleotide (AAUAAA), measured by fluorescence polarization. The polarization amplitude is normalized to 1. Error bars indicate standard error of means (SEM) for five consecutive measurements of a single representative sample. Each binding experiment was performed in triplicate and the mean KDvalues ± SEM are reported in Table 2. (B) Inter-subunit cross-link map of the CPSF160FL-WDR33M1-G474-CPSF30FL-Fip1FL complex. Observed inter-molecular cross-links between CPSF160FL, WDR33M1-G474 and CPSF30FL (upper panel) and between FipFL and the other three CPSF subunits CPSF160FL, WDR33M1-G474 and CPSF30FL (lower panel) are represented as dashed lines. A list of the cross-linked peptides identified by mass spectrometry is reported in Figure 1—source data 1. Proteins are indicated as schematic diagrams (not to scale) with featured domains highlighted in color. (CD, Fip1 conserved domain; ZF1-5, CPSF30 zinc finger domains 1 to 5; ZK, CPSF30 zinc knuckle domain; CTD, CPSF160 C-terminal domain; bP1-3, CPSF160 beta-propeller domains 1 to 3; NTD, WDR33 N-terminal domain. CPSF160: Uniprot Q10570; WDR33: Uniprot Q9C0J8-1; CPSF30: Uniprot O95639-3; Fip1: Uniprot Q6UN15-4).