Fip1 is a multivalent interaction scaffold for processing factors in human mRNA 3’ end biogenesis

Abstract

3’ end formation of most eukaryotic mRNAs is dependent on the assembly of a ~1.5 megadalton 3 multiprotein complex, that catalyzes the coupled reaction of pre-mRNA cleavage and polyadenylation. 4 In mammals, the cleavage and polyadenylation specificity factor (CPSF) constitutes the core of the 5 3’ end processing machinery onto which the remaining factors, including cleavage stimulation factor 6 (CstF) and poly(A) polymerase (PAP), assemble. These interactions are mediated by Fip1, a CPSF 7 subunit characterized by high degree of intrinsic disorder. Here, we report two crystal structures 8 revealing the interactions of human Fip1 (hFip1) with CPSF30 and CstF77. We demonstrate that CPSF 9 contains two copies of hFip1, each binding to the zinc finger (ZF) domains 4 and 5 of CPSF30. Using 10 polyadenylation assays we show that the two hFip1 copies are functionally redundant in recruiting one 11 copy of PAP, thereby increasing the processivity of RNA polyadenylation. We further show that the 12 interaction between hFip1 and CstF77 is mediated via a short motif in the N-terminal “acidic” region of 13 hFip1.