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 multiprotein complex, that catalyzes the coupled reaction of pre-mRNA cleavage and polyadenylation. In mammals, the cleavage and polyadenylation specificity factor (CPSF) constitutes the core of the 3' end processing machinery onto which the remaining factors, including cleavage stimulation factor (CstF) and poly(A) polymerase (PAP), assemble. These interactions are mediated by Fip1, a CPSF subunit characterized by high degree of intrinsic disorder. Here, we report two crystal structures revealing the interactions of human Fip1 (hFip1) with CPSF30 and CstF77. We demonstrate that CPSF contains two copies of hFip1, each binding to the zinc finger (ZF) domains 4 and 5 of CPSF30. Using polyadenylation assays we show that the two hFip1 copies are functionally redundant in recruiting one copy of PAP, thereby increasing the processivity of RNA polyadenylation. We further show that the interaction between hFip1 and CstF77 is mediated via a short motif in the N-terminal 'acidic' region of hFip1. In turn, CstF77 competitively inhibits CPSF-dependent PAP recruitment and 3' polyadenylation. Taken together, these results provide a structural basis for the multivalent scaffolding and regulatory functions of hFip1 in 3' end processing.

Natural variation in the plant polyadenylation complex

“…In mammals, CPSF160 coordinates the binding of two other CPSF subunits – CPSF30 and FY – to the polyadenylation signal and serves as a bridge between the PAS-binding module and the so-called cleavage module that consists of CPSF100 and CPSF73. FIP1 (the mammalian and yeast counterpart of FIPS5) recruits poly(A) polymerase to the PAC through interactions with both PAP and CPSF30 ( Kumar et al., 2021 ; Muckenfuss et al., 2022 ). Analogous interactions have been reported for FIPS5 ( Forbes et al., 2006 ; Hunt et al., 2008 ), as has a FIPS5-RNA interaction similar to that seen with the mammalian FIP1 ortholog ( Forbes et al., 2006 ).…”

“…CFIIm also plays roles in transcription termination ( Sadowski et al., 2003 ; Zhang et al., 2005 ; West and Proudfoot, 2008 ; Kamieniarz-Gdula et al., 2019 ). FIP1 is a scaffold that links PAP with other parts of the PAC ( Helmling et al., 2001 ; Kaufmann et al., 2004 ; Zhang et al., 2020 ; Muckenfuss et al., 2022 ). Symplekin and RBBP6 are additional scaffolds that coordinate the subcomplexes and enzymes in the course of the reaction ( Ghazy et al., 2009 ; Kennedy et al., 2009 ; Xiang et al., 2010 ; Ruepp et al., 2011 ; Di Giammartino et al., 2014 ; Zhang et al., 2020 ; Rodríguez-Molina et al., 2022 ; Schmidt et al., 2022 ).…”

Birth of a poly(A) tail: mechanisms and control of mRNA polyadenylation

“…Indeed, a non‐conserved region of yeast Fip1 appears to strongly bind a conserved cleft in Pap1 [ 37 ] (Fig. 1C ), but in humans multiple low‐affinity regions in hFip1 are required for its interaction with PAP [ 38 ]. The differential affinities of Pap1/PAP within CPAC complexes may underlie critical species‐specific regulatory mechanisms of polyadenylation, as discussed later in this review.…”

“…1B ). Specifically, crystal structures and solution nuclear magnetic resonance studies reveal extensive high‐affinity interaction of one Fip1 molecule with zinc finger 4 of Yth1/CPSF30, and another Fip1 molecule interacting with zinc finger 5 albeit with lower affinity [ 36 , 38 , 39 ]. This would, in principle, allow up to two polymerase molecules to be accommodated within the CPAC, which has been observed using native mass spectrometry analysis [ 3 ].…”

“…1B ). This interaction is mutually exclusive with the interaction between hFip1 and PAP [ 38 ] and may explain why PAP is not part of the pre‐cleavage complex [ 13 ]. Presumably, this state of the CPAC (without PAP) is competent for (and would even promote) cleavage, but recent studies show opposing views on the requirement for PAP in the cleavage activity of the CPAC [ 7 , 10 ].…”