The cytoplasmic poly(A) polymerases GLD-2 and GLD-4 promote general gene expression via distinct mechanisms

Abstract

Post-transcriptional gene regulation mechanisms decide on cellular mRNA activities. Essential gatekeepers of post-transcriptional mRNA regulation are broadly conserved mRNA-modifying enzymes, such as cytoplasmic poly(A) polymerases (cytoPAPs). Although these non-canonical nucleotidyltransferases efficiently elongate mRNA poly(A) tails in artificial tethering assays, we still know little about their global impact on poly(A) metabolism and their individual molecular roles in promoting protein production in organisms. Here, we use the animal model Caenorhabditis elegans to investigate the global mechanisms of two germline-enriched cytoPAPs, GLD-2 and GLD-4, by combining polysome profiling with RNA sequencing. Our analyses suggest that GLD-2 activity mediates mRNA stability of many translationally repressed mRNAs. This correlates with a general shortening of long poly(A) tails in gld-2-compromised animals, suggesting that most if not all targets are stabilized via robust GLD-2-mediated polyadenylation. By contrast, only mild polyadenylation defects are found in gld-4-compromised animals and few mRNAs change in abundance. Interestingly, we detect a reduced number of polysomes in gld-4 mutants and GLD-4 protein co-sediments with polysomes, which together suggest that GLD-4 might stimulate or maintain translation directly. Our combined data show that distinct cytoPAPs employ different RNA-regulatory mechanisms to promote gene expression, offering new insights into translational activation of mRNAs.

Figure 1.

gld-2 but not gld-4 promotes mRNA abundance. (A and B) Domain structure of GLD-2 and GLD-4 proteins: dark blue–nucleotidyl transferase domain (NTD), light blue–poly(A) polymerase-associated domain. (C and D) Western blot analysis of protein levels in RNAi-treated adults. The asterisk marks a non-specific background band. Representative images are shown (n = 3). (E and F) mRNA abundance changes in RNAi-treated animals. All detectable 7649 genes are shown and the number of significant abundance changes is indicated. (G) The overlap of down-regulated genes between gld-2(RNAi) and gld-4(RNAi) is shown. (H) A GO-term analysis for gld-2(RNAi) and gld-4(RNAi) down-regulated genes was conducted and two representative categories are shown for each.

Figure 2.

GLD-2-associated mRNAs are less abundant. (A) Abundance changes of 538 previously described GLD-2-associated mRNAs (14) in our gld-2(RNAi) dataset. (B) RT-qPCR measurements of randomly selected down-regulated and unchanged genes, comparing mRNA abundance in gld-2(q497) mutants to wild-type. A plus sign indicates previously proposed GLD-2-associated mRNAs (14). Shown is the mean (±SD) of three independent experiments. Significance was calculated with a Student's t-test: ***, P < 0.001; **, P < 0.01; *, P < 0.05; n.s., not significant.

Figure 3.

gld-2 promotes bulk mRNA poly(A) tail extension. (A and B) Representative gels of at least three independent bulk poly(A) tail measurements. Equal amounts of radioactivity were loaded for each sample. WT, wild-type. (C and D) Line scans of bulk poly(A) profiles from (A).

Figure 4.

gld-2 promotes abundance of poorly translated germline mRNAs. (A) The relative gradient distribution of rpl-25.2 and gld-1 mRNA in animals treated with control RNAi is analyzed by RT-qPCR. Shown is the mean (±SEM) from 10 fractions in three independent experiments. (B–D) The gradient distribution of different groups of mRNAs was analyzed between polysome (P) and non-polysome (NP) fractions for control RNAi-treated animals. Shown are (B) all detected genes (dotted line–7649 mRNAs), (B, C and D) germline-enriched genes (solid black line–2243 mRNAs), (C) germline genes that are less abundant in gld-2(RNAi) (solid red line–444 mRNAs) and (D) germline genes that are less abundant in gld-4(RNAi) (solid green line–54 mRNAs). (E) P/NP distribution of the indicated groups of germline mRNAs. Significance was calculated with a Student's t-test: ***P < 0.001; n.s., not significant. (F) A large percentage of GLD-2-stabilized germline mRNAs are also GLD-1 and FBF-1 targets. The percent overlap between GLD-2-regulated mRNAs and putative GLD-1 and FBF-1 target mRNAs are given. mRNAs that are not decreased in gld-2(RNAi) are labeled as unchanged.

Figure 5.

gld-4 promotes general translation. (A andB) Translational efficiency changes of all 7649 detectable genes in (A) gld-2(RNAi) and (B) gld-4(RNAi). (C) Statistical analysis of translational efficiency changes. (D) GO-term analysis of NP-enriched mRNAs in gld-2(RNAi) and gld-4(RNAi). Student's t-test: ***, P < 0.001.

Figure 6.

GLD-4 co-sediments with polysomes and promotes polysome formation. (A and B) Typical absorbance profiles of a wild-type polysome gradient (A) without or (B) with prior EDTA treatment (n = 3). The positions of major ribonucleoprotein complexes are indicated. (C and D) Western blot analysis of fractionated material from (A) and (B). Equal exposure times for each antibody in (C) and (D); for GLD-2, also a longer exposure of the same blot is shown; asterisk marks an unspecific background band. (E and F) Representative absorbance profiles from extracts of RNAi-treated animals (n > 4). The numbers indicate the position of polyribosome peaks that are clearly detected in the control RNAi sample.