Structural basis for the activation of the C. elegans noncanonical cytoplasmic poly(A)-polymerase GLD-2 by GLD-3

Abstract

The Caenorhabditis elegans germ-line development defective (GLD)-2–GLD-3 complex up-regulates the expression of genes required for meiotic progression. GLD-2–GLD-3 acts by extending the short poly(A) tail of germ-line–specific mRNAs, switching them from a dormant state into a translationally active state. GLD-2 is a cytoplasmic noncanonical poly(A) polymerase that lacks the RNA-binding domain typical of the canonical nuclear poly(A)-polymerase Pap1. The activity of C. elegans GLD-2 in vivo and in vitro depends on its association with the multi-K homology (KH) domain-containing protein, GLD-3, a homolog of Bicaudal-C. We have identified a minimal polyadenylation complex that includes the conserved nucleotidyl-transferase core of GLD-2 and the N-terminal domain of GLD-3, and determined its structure at 2.3-Å resolution. The structure shows that the N-terminal domain of GLD-3 does not fold into the predicted KH domain but wraps around the catalytic domain of GLD-2. The picture that emerges from the structural and biochemical data are that GLD-3 activates GLD-2 both indirectly by stabilizing the enzyme and directly by contributing positively charged residues near the RNA-binding cleft. The RNA-binding cleft of GLD-2 has distinct structural features compared with the poly(A)-polymerases Pap1 and Trf4. Consistently, GLD-2 has distinct biochemical properties: It displays unusual specificity in vitro for single-stranded RNAs with at least one adenosine at the 3′ end. GLD-2 thus appears to have evolved specialized nucleotidyl-transferase properties that match the 3′ end features of dormant cytoplasmic mRNAs.

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

“…Finally, GLD-2 PAP -RNP-8 GB showed a preference for an RNA substrate with adenosines at the 3 ′ end (Fig. 1D), similarly to GLD-2 PAP -GLD-3 NT (Nakel et al 2015). We determined the structure of GLD-2 PAP-D -RNP-8 GB at a resolution of 2.5 Å and R free of 24.2% (Table 1).…”

“…Based on the substrate-bound structure of canonical PAP (Balbo and Bohm 2007) the cleft in GLD-2 is expected to contain the binding sites for RNA and ATP as well as the catalytic residues. There are two major differences of GLD-2 in the RNP-8 GB structure as compared to the previous GLD-3 NT structure (Nakel et al 2015). First, the catalytic domain is rotated toward the central domain and has a closer conformation of the active-site cleft ( Fig.…”

“…We previously characterized a nucleotidyl-transferase region of C. elegans GLD-2 suitable for biochemical and structural studies with the corresponding binding domain of GLD-3 (Nakel et al 2015). We will refer to these fragments as GLD-2 PAP for the wild-type enzyme, and GLD-2 PAP-D for the catalytically dead D668A mutant ( Fig.…”

“…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 ′ -32 P-labeled A 15 , U 15 , or U 10 A 1 oligomers (100 nM).…”

“…GLD-3 contains four noncanonical KH domains (Nakel et al 2010). It also contains an N-terminal region that wraps around GLD-2 and increases the RNA-binding properties and the stability of the poly(A)-polymerase, thereby promoting polyadenylation (Nakel et al 2015).…”