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  1. Evaluation Summary:

    The authors of this study characterize human Fip1, an important component of the 3' end processing machinery. They use X-ray crystallography to determine the molecular basis of the interactions between Fip1 and CPSF30 (at a 2:1 stoichiometry) and between Fip1 and CstF77 (at a 2:2 stoichiometry). Together with biochemical assays, they suggest that Fip1 may be central to regulating transitions with CPSF. The work will of relevance to colleagues interested in transcription and RNA processing.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)

  2. Reviewer #1 (Public Review):

    The goals of this paper were to provide structural and biochemical insight into the role that Fip1 plays as a protein interaction scaffold with other members of the cleavage and polyadenylation machinery. To that end, the authors solve two crystal structures as the featured basis for the study: FIp1 and CPSF30; Fip1 and Cst77. In both cases, subdomains of proteins were used for crystallography. These two structures are of high quality and are validated with rigorous and clear biochemistry. These points represent the technical strengths of the manuscript. One weakness of the study is the overall findings with those previously reported by the Tong laboratory. While the authors present some potentially newer information in terms of the number of PAP proteins capable of interacting with FIp1/CPSF30, there are experimental caveats that would need to be addressed first.

    The novelty of the Fip1/CstF77 structure represents the strongest aspect of the paper as well as the observation that CstF77 can inhibit polyadenylation. The in vitro data support this conclusion but additional experiments would strengthen this overall model.

  3. Reviewer #2 (Public Review):

    The work is carefully, rigorously, and comprehensively performed, and is well presented. The assays with recombinant multiprotein complexes are not straightforward but the figures are really clear. The crystal structure of Fip1-CPSF30 is similar to one reported in 2020 by the Tong lab. Nevertheless, there is a substantial new contribution to knowledge of this fundamental process. New hypotheses are generated that can be tested in the future.

    Intriguingly, the authors claim that only one PAP associates with the complex even though there are two PAP binding sites. In my view, the evidence for this is not conclusive - it is based on a SEC MALS trace in Fig 2D and I think it is difficult to exclude the possibility that there is a mixture of complexes containing one or two PAPs bound.

  4. Reviewer #3 (Public Review):

    The authors report the structure of CPSF30 bound to 2 molecules of FIP1, as well as the structure of FIP1 bound to CSTF77. Their data supports a model in which two molecules of FIP1, are present in the mPSF subcomplex of CPSF, although only one PAP may be bound to this complex. TheCstF77 binding to Fip1, which likely inhibits polyadenylation since it interferes with PAP binding, would occur as the complete complex assembles on the substrate, and result in the active cleavage complex not containing PAP or active in polyadenylation, only becoming active in polyadenylation after cleavage and loss of CstF from the complex.