Abstract
Cleavage and polyadenylation factor (CPF/CPSF) is a multiprotein complex essential for mRNA 3' end processing in eukaryotes. It contains an endonuclease that cleaves pre-mRNAs, and a polymerase that adds a poly(A) tail onto the cleaved 3' end. Several CPF subunits, including Fip1, contain intrinsically disordered regions (IDRs). IDRs within multiprotein complexes can be flexible, or can become ordered upon interaction with binding partners. Here, we show that yeast Fip1 anchors the poly(A) polymerase Pap1 onto CPF via an interaction with zinc finger 4 of another CPF subunit, Yth1. We also reconstitute a fully recombinant 850-kDa CPF. By incorporating selectively labeled Fip1 into recombinant CPF, we could study the dynamics of Fip1 within the megadalton complex using nuclear magnetic resonance (NMR) spectroscopy. This reveals that a Fip1 IDR that connects the Yth1- and Pap1-binding sites remains highly dynamic within CPF. Together, our data suggest that Fip1 dynamics within the 3' end processing machinery are required to coordinate cleavage and polyadenylation.
Highlights
Protein-coding genes in eukaryotes are transcribed by RNA polymerase II (Pol II) into precursor messenger RNAs
Cryo-electron microscopy structures of the polymerase modules from yeast and humans revealed an extensive network of interactions between Pfs2 and Cft1 (WDR33 and CPSF160 in humans), which function as a scaffold for assembly of the other subunits
To investigate how Fip1 and Pap1 interact with other CPF subunits, we first studied the five-subunit polymerase module purified from a baculovirus-mediated insect cell overexpression system as previously described (Casañal et al 2017)
Summary
Protein-coding genes in eukaryotes are transcribed by RNA polymerase II (Pol II) into precursor messenger RNAs (premRNAs). Eukaryotic 3′ end processing is carried out by a set of conserved multiprotein complexes that includes the cleavage and polyadenylation factor (CPF in yeast or CPSF in humans) and accessory cleavage factors (CF IA and CF IB in yeast or CF Im, CF IIm, and CstF in humans). Cryo-electron microscopy (cryoEM) structures of the polymerase modules from yeast and humans revealed an extensive network of interactions between Pfs and Cft (WDR33 and CPSF160 in humans), which function as a scaffold for assembly of the other subunits The structures provided a rationale for how WDR33 and CPSF30 (Yth in yeast) bind specific sequences in RNA (Clerici et al 2018; Sun et al 2018)
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