Crystal structure of human poly(A) polymerase gamma reveals a conserved catalytic core for canonical poly(A) polymerases

Abstract

In eukaryotes, the poly(A) tail added at the 3' end of an mRNA precursor is essential for the regulation of mRNA stability and the initiation of translation. Poly(A) polymerase (PAP) is the enzyme that catalyzes the poly(A) addition reaction. Multiple isoforms of PAP have been identified in vertebrates, which originate from gene duplication, alternative splicing or post-translational modifications. The complexity of PAP isoforms suggests that they might play different roles in the cell. Phylogenetic studies indicate that vertebrate PAPs are grouped into three clades termed α, β and γ, which originated from two gene duplication events. To date, all the available PAP structures are from the PAPα clade. Here, we present the crystal structure of the first representative of the PAPγ clade, human PAPγ bound to cordycepin triphosphate (3'dATP) and Ca(2+). The structure revealed that PAPγ closely resembles its PAPα ortholog. An analysis of residue conservation reveals a conserved catalytic binding pocket, whereas residues at the surface of the polymerase are more divergent.

Keywords: 3′ end processing; C-terminal domain; CTD; MCMC; Markov Chain Monte Carlo; N-terminal domain; NLS; NTD; PAP; PEG; RNA recognition motif; RRM; mRNA processing; neo-PAP; nuclear localization signals; poly(A) polymerase; poly(A) polymerase gamma; polyadenylation; polyethylene glycol.

Figure 1. Poly(A) polymerases are grouped into three clades

(A) Domain organization of human poly(A) polymerases α, β, and γ. CAT is the abbreviation for the catalytic domain; RRM, RNA recognition motif; NLS, nuclear localization signal; cdk-p, cyclin-dependent kinase phosphorylation sites; U1A, U1A-interaction motif; NTD, N-terminal domain; CTD, C-terminal domain. The domain and motif sizes are not proportional to the length of the amino acid sequences. A detailed sequence alignment is shown in Figure S1. (B) Phylogeny of vertebrate PAP sequences, constructed by MCMC simulation and the neighbor joining (NJ) algorithm (see METHODS for details). Statistical support for edges is described by MCMC posterior probabilities, followed by bootstrap support based on NJ calculations.

Figure 2. Structure of hPAPγ bound to 3′dATP and Ca²⁺

(A) Human PAPγ is shown in cartoon representation and the color scheme is the same as in Figure 1A. The catalytic domain comprises residues 59-172, the central domain residues 17-58 & 173-351, and the RRM, residues 352-501. 3′dATP is shown as a stick model and Ca²⁺ as a sphere (magenta). (B) A close-up view of 3′dATP in the binding pocket of hPAPγ. Residues interacting with 3′dATP and Ca²⁺ are shown and colored according to the domain they belong to. Hydrogen bonds are represented by red dashed lines. An Fo−Fc omit map contoured at 3 σ (calculated before building 3′dATP in the map) is shown as a green mesh and a 4 σ anomalous difference Fourier map (blue mesh) overlays on top of the Ca²⁺ ion.

Figure 3. Conservation of the PAP N-terminal domain

(A) The conservation of PAP NTD (central, catalytic, and RRM domain) was calculated with the ConSurf server and displayed with Pymol. Conserved residues are shown in shades of magenta and variable residues in shades of blue. The bound 3′dATP is shown in yellow. (B) RNA (orange) from the Pap1-RNA complex (PDB: 2Q66 ) and Fip1 fragment (residues 80-105, green) from the yeast Pap1-Fip1 complex (PDB: 3C66 ) were overlaid on top of the PAP NTD (bottom panel) to illustrate the binding surfaces for RNA and Fip1.

Figure 4. Superpositions of PAPs reveal domain movements

The structures were superimposed based on the central domain of hPAPγ (residues 17-58 & 173-351) to observe the movement of the catalytic domain and RRM. (A) Superposition of three mammalian PAP structures (bPAPα-3′dATP-Mn²⁺ (PDB: 1Q79 ), pink; bPAPα-3′dATP-Mg²⁺ (PDB: 1Q78 ), green; and hPAPγ-3′dATP-Ca²⁺ (PDB:4LT6), lightblue) (B) Superposition of three yeast complexes (yPap1-3′dATP-Mn²⁺ (PDB: 1FA0 ), purple; yPap1- Mg²⁺ (PDB: 2HHP ), dark green; and yPap1-ATP-Mg²⁺-RNA (PDB: 2Q66 ), cyan. ATP (yellow) and RNA (brown) from the yeast Pap1-RNA complex (PDB:2Q66 ) are modeled in the substrate binding cleft and shown in surface representation.