Architecture of eukaryotic mRNA 3′-end processing machinery

Abstract

Newly transcribed eukaryotic precursor messenger RNAs (pre-mRNAs) are processed at their 3′ ends by the ~1-megadalton multiprotein cleavage and polyadenylation factor (CPF). CPF cleaves pre-mRNAs, adds a polyadenylate tail, and triggers transcription termination, but it is unclear how its various enzymes are coordinated and assembled. Here, we show that the nuclease, polymerase, and phosphatase activities of yeast CPF are organized into three modules. Using electron cryomicroscopy, we determined a 3.5-angstrom-resolution structure of the ~200-kilodalton polymerase module. This revealed four β propellers, in an assembly markedly similar to those of other protein complexes that bind nucleic acid. Combined with in vitro reconstitution experiments, our data show that the polymerase module brings together factors required for specific and efficient polyadenylation, to help coordinate mRNA 3′-end processing.

Structure–function relationships in the Nab2 polyadenosine‐RNA binding Zn finger protein family

“…Polyadenylation is the final processing step in the nuclear phase of the gene expression pathway. In S. cerevisiae , the 3′‐end processing machinery comprises cleavage factors IA and 1B and the cleavage and polyadenylation factor (CPF), a complex including the riboendonuclease, Ysh1/Brr5, the poly(A) polymerase, Pap1, and the Pap1 regulation factor, Fip1 . Rna15, associated with the scaffold protein, Rna14, and other CFIA subunits, Pcf11 and Clp1, positions the CPF to cleave the poly(A) site.…”

“…After the cleavage reaction, poly(A) polymerase, Pap1, synthesizes a poly(A) tail of ~60–80 adenosines in a template‐independent manner. Because isolated Pap1 is an inefficient distributive enzyme, Pap1 processivity requires stimulation/regulation by factors that bind Pap1 and tether it to the RNA . In mammals, the RRM‐containing nuclear poly(A) RNA binding protein, PABPN1, enhances the processivity of PAP and, in S. cerevisiae , the CPF component, Fip1, binds Pap1 directly and tethers it to CPF to stimulate/regulate Pap1 activity .…”

“…Key hydrophobic and basic residues that are important for binding to adenines (see also S. cerevisiae, the 3 0 -end processing machinery comprises cleavage factors IA and 1B and the cleavage and polyadenylation factor (CPF), a complex including the riboendonuclease, Ysh1/Brr5, the poly(A) polymerase, Pap1, and the Pap1 regulation factor, Fip1. 47 Rna15, associated with the scaffold protein, Rna14, and other CFIA subunits, Pcf11 and Clp1, positions the CPF to cleave the poly(A) site. After the cleavage reaction, poly(A) polymerase, Pap1, synthesizes a poly(A) tail of~60-80 adenosines in a template-independent manner.…”

Current approaches for the fitting and refinement of atomic models into cryo-EM maps usingCCP-EM

“…Several of the techniques available in Coot (Emsley et al , 2010) have successfully been used by structural biologists (see, for example, Casañal et al , 2017) for this purpose, notably Jiggle Fit for positioning and orienting the atomic model, Model Morphing to allow localized regions to be fitted into the map and the use of externally derived restraints that can be visualized as well as applied during refinement to aid stability and/or improve geometry. Recent efforts towards refinement parallelization have resulted in the ability to refine larger regions of the model concurrently.…”

Current developments in Coot for macromolecular model building of Electron Cryo‐microscopy and Crystallographic Data

“…Geman‐McClure restrains (limit 6 Å) are then applied (d) to perform chain refine (e) to obtain a final accurate fit (f). Map used for representation: EMD‐3908 . Fitted homologue: PDB 6f9n…”