Structural basis for the antagonistic roles of RNP-8 and GLD-3 in GLD-2 poly(A)-polymerase activity

Abstract

Cytoplasmic polyadenylation drives the translational activation of specific mRNAs in early metazoan development and is performed by distinct complexes that share the same catalytic poly(A)-polymerase subunit, GLD-2. The activity and specificity of GLD-2 depend on its binding partners. In Caenorhabditis elegans, GLD-2 promotes spermatogenesis when bound to GLD-3 and oogenesis when bound to RNP-8. GLD-3 and RNP-8 antagonize each other and compete for GLD-2 binding. Following up on our previous mechanistic studies of GLD-2-GLD-3, we report here the 2.5 Å resolution structure and biochemical characterization of a GLD-2-RNP-8 core complex. In the structure, RNP-8 embraces the poly(A)-polymerase, docking onto several conserved hydrophobic hotspots present on the GLD-2 surface. RNP-8 stabilizes GLD-2 and indirectly stimulates polyadenylation. RNP-8 has a different amino-acid sequence and structure as compared to GLD-3. Yet, it binds the same surfaces of GLD-2 by forming alternative interactions, rationalizing the remarkable versatility of GLD-2 complexes.

Keywords: C. elegans; cytoplasmic polyadenylation; germline development; nucleotidyl-transferase; translational regulation.

FIGURE 1.

Activity of a minimal GLD-2–RNP-8 complex. (A) Schematic representation of the domain structure of C. elegans GLD-2 and RNP-8. Globular domains are shown as rectangles and low-complexity sequences as lines. The catalytic (cat) and central domains of GLD-2 are colored in blue and pink, respectively. As in other nucleotidyltransferases, the central domain is composed of two noncontiguous polypeptide segments, which correspond in the structure to helix α1 and helices α4–α8 for the segments preceding and following the catalytic domain, respectively (see also Fig. 2). The RNA recognition motif (RRM) and the GLD-2-binding (GB) domains of RNP-8 are colored in gray and yellow, respectively. (B) Protein stability of GLD-2_PAP–RNP-8_GB, GLD-2_PAP–GLD-3_NT, and GLD-2_PAP as determined by thermofluor experiments. The normalized curves and the corresponding melting temperatures are shown on the left. The Coomassie-stained 4%–15% Bio-Rad TGX SDS-PAGE gel of the proteins used in the thermofluor and poly(A) polymerase assays (C,D) are shown on the right and in Supplemental Figure 1C. (C) Polyadenylation assay of C. elegans GLD-2_PAP or GLD-2_PAP-D in complex with either RNP-8_GB or GLD-3_NT (Nakel et al. 2015). (D) Polyadenylation assay of GLD-2_PAP–RNP-8_GB (0, 20, 100, and 500 nM) in the presence of 5′-³²P-labeled A₁₅, U₁₅, or U₁₀A₁ oligomers (100 nM).

FIGURE 2.

Structure of a GLD-2–RNP-8 core complex. (A) Ribbon diagram of the GLD-2_PAP-D–RNP-8_GB complex (PDB code 5JNB, left panel) and GLD-2_PAP-D–GLD-3_NT (PDB code 4ZRL, right panel) shown in the same orientation after optimal superposition of their central domains (in pink). The catalytic domains are in blue, RNP-8 in yellow, and GLD-3 in green. The N- and C-terminal residues of the proteins are labeled. Disordered loops are indicated with dashed lines. The five-stranded β-sheet (β1–β5) of the catalytic domain is indicated in both structures. In GLD-2_PAP-D–RNP-8_GB, an additional four-stranded β-sheet (β6–β9) is well ordered on top of the central domain. These secondary structure elements correspond to a conserved polypeptide segment between helices α7 and α8 and lay on top of helix α6. NRM, nucleotide recognition motif. (B) On the left is a zoom-in of the active site cleft of the GLD-2_PAP-D–RNP-8_GB complex shown after a 180° rotation around a vertical axis with respect to the view in panel A. The colors are the same as in panel A, with the peptide of a symmetry-related RNP-8 molecule (for explanations see main text) in stick representation and with carbon atoms in gray. On the right is the corresponding zoom-in view from the structure of canonical PAP bound to RNA and ATP (in stick representation, with carbon atoms in gray and black, respectively) (PDB code 2Q66, Balbo and Bohm 2007). For clarity, the zoom-in view lacks the RRM domain of canonical PAP (shown as a reference in the overall view in light gray). Magnesium and water molecules are shown as green and cyan spheres, respectively. A set of important residues in both GLD-2–RNP-8_GB and PAP are highlighted in stick representation and labeled. Note that the place of the two most 3′ end ribonucleotides is taken by RNP-8 Phe179 and His182 (instead of the nucleotide bases) and Asn181 (instead of the nucleotide ribose moieties). The place of ATP is taken by RNP-8 Thr177, Leu178, and Asp180 (instead of the adenosine) and by Asp180 and a sulfate ion from the crystallization buffer (instead of the phosphates). Remarkably, the GLD-2_PAP-D–RNP-8_GB structure also shows a similar arrangement of magnesium and water molecules in the active site as observed in the PAP-RNA-ATP structure (Balbo and Bohm 2007).

FIGURE 3.

Structural basis of mutually exclusive GLD-2–RNP-8 and GLD-2–GLD-3 complex formation. (A) Structural alignment from C. elegans RNP-8_GB and GLD-3_NT, with α-helical residues in cyan. Note that only the DDYV segment of RNP-8 and the ENFY segment of GLD-3 (both shown in bold letters) superpose well, with a C-α root mean square deviation of <0.1 Å. The dotted lines indicate the interactions of different GLD-3 and RNP-8 residues with the same side chains in the GLD-2 catalytic and central domains (in blue and pink above and below the sequences). (B) Zoom-in of RNP-8_GB interactions with patch 1, patch 2, and patch 3 surfaces of GLD-2 (right, middle, and left panels, respectively). In the middle panel, note that the RNP-8_GB helix is close to helix α4 of the GLD-2 central domain and that the GLD-2 loop containing Arg756 binds on top of the RNP-8_GB helix. (C) Corresponding zoom-in of GLD-3_NT interactions with the same surface patches of GLD-2 (PDB code 4ZRL, Nakel et al. 2015). In the left panel, two GLD-3 residues previously shown to contribute to RNA binding are indicated (Arg42, Lys43, Nakel et al. 2015). In the middle panel, note that the GLD-3_NT C-terminal helix is further apart from helix α4 of the GLD-2 central domain and that the GLD-2 loop containing Arg756 binds in between. (D) Superposition of RNP-8 and GLD-3 from the corresponding GLD-2–bound structures. The residues in the DDYV and ENFY segments are indicated. (E) Coomassie-stained 17% SDS/PAGE of His-pull-down experiments of coexpressed wild-type (wt) and mutant His-GLD-2_PAP with RNP-8_GB. Total lysate control is shown on the left; pulled-down protein eluate is shown on the right. The experiment includes the disrupting GLD-2 R547E mutant (which targets a central residue at the interaction interface) and as a control the GLD-2 N629A mutant (that still supports RNP-8 binding). (*) GST-6xHis-Tag contamination.