Polyadenylation is the key aspect of GLD-2 function in C. elegans

Abstract

The role of many enzymes extends beyond their dedicated catalytic activity by fulfilling important cellular functions in a catalysis-independent fashion. In this aspect, little is known about 3'-end RNA-modifying enzymes that belong to the class of nucleotidyl transferases. Among these are noncanonical poly(A) polymerases, a group of evolutionarily conserved enzymes that are critical for gene expression regulation, by adding adenosines to the 3'-end of RNA targets. In this study, we investigate whether the functions of the cytoplasmic poly(A) polymerase (cytoPAP) GLD-2 in C. elegans germ cells exclusively depend on its catalytic activity. To this end, we analyzed a specific missense mutation affecting a conserved amino acid in the catalytic region of GLD-2 cytoPAP. Although this mutated protein is expressed to wild-type levels and incorporated into cytoPAP complexes, we found that it cannot elongate mRNA poly(A) tails efficiently or promote GLD-2 target mRNA abundance. Furthermore, germ cell defects in animals expressing this mutant protein strongly resemble those lacking the GLD-2 protein altogether, arguing that only the polyadenylation activity of GLD-2 is essential for gametogenesis. In summary, we propose that all known molecular and biological functions of GLD-2 depend on its enzymatic activity, demonstrating that polyadenylation is the key mechanism of GLD-2 functionality. Our findings highlight the enzymatic importance of noncanonical poly(A) polymerases and emphasize the pivotal role of poly(A) tail-centered cytoplasmic mRNA regulation in germ cell biology.

Keywords: germline development; poly(A) metabolism; poly(A) polymerase; translational regulation.

FIGURE 1.

The GLD-2(P652L) protein is robustly expressed and interacts with GLD-3 and RNP-8. (A) Cartoon of GLD-2 protein domain structure. Position and impact of q497, q540, and h292 mutations on GLD-2 protein are indicated. The black, thick bar indicates the region that was used to raise all antibodies against this protein. (B) A protein sequence alignment comprising the second half of the nucleotidyl transferase domain (NTD) from Caenorhabditis elegans (Ce), Drosophila melanogaster (Dm), Danio renio (Dr), Xenopus laevis (Xl), and Homo sapiens (Hs) GLD-2. On top of the alignment, the secondary structure elements of the C. elegans protein are shown (α, α-helices; β, β-sheet). The conserved proline in β-sheet number 4 is highlighted in red. (C) Western blot analysis of the indicated proteins in the different gld-2 mutants. (D) Immunoprecipitation of GLD-2(P652L)-containing complexes from adult animal extracts, tested for a coenrichment of both known GLD-2-interacting proteins. The star indicates a background band that is detected by the mouse anti-GLD-2 antibody.

FIGURE 2.

Impact of different GLD-2 mutant proteins on mRNA polyadenylation. (A) sPAT analysis of gld-1 and oma-2 mRNA in wild-type (wt) and different gld-2 backgrounds. The lane RH/dT is a wild-type sample that was treated with oligo(dT) and RNase H prior to the sPAT assay to indicate the size of a completely deadenylated mRNA. Line scans from data generated by the sPAT assay are given below. Two independent biological repeats were analyzed. (B) The distribution of bulk poly(A) tails was analyzed in wild-type and the given gld-2 backgrounds. (C) Line scans from bulk poly(A) measurements. Three independent biological repeats were analyzed. Regions of statistically significant differences indicated with a gray stripe and black bracket are calculated via the Student's t-test.

FIGURE 3.

Levels of GLD-2 target mRNAs are reduced in GLD-2(P652L)-expressing animals. (A) Abundance measurements of the indicated mRNAs via quantitative real-time PCR. Statistically significant differences are indicated and calculated via the Student's t-test. (***) P < 0.001, (**) P < 0.01, (*) P < 0.05; n.s., not significant. (B) Western blot analysis of the indicated proteins in fog-1 gld-2 double-mutant backgrounds. (C) mRNA abundance analysis during the development from L3 larvae to adults in given mutant backgrounds. L3, L3 larvae; L4, L4 larvae; earlyA, L4 + 12h; Adult, L4 + 24h.

FIGURE 4.

gld-2(q540) largely phenocopies the genetic null allele of gld-2. (A) Cartoon of the adult hermaphroditic syncytial germline tissue. Circles represent nuclei, T-structures are partial membranes. Important female germ cell stages are indicated; distal is top left, the position of sperm marks the most proximal end of the gonad. Color marked regions were analyzed in this work. (B) Analysis of the size of the proliferative region. Germ cell rows before meiotic entry were counted in the different genetic backgrounds as judged by DAPI staining. Significance was calculated by a two-tailed Student's t-test. (***) P ≤ 0.001. (C) Percentage of germlines that positively stain for diakinetic DNA in the proximal part, assessed by DAPI staining. (D–G) DAPI (purple in merge) staining of the indicated genetic backgrounds. NPC (green in merge), nuclear pore complex. Scale bar: (G) 15 µm, (blow-up) 5 µm.