Ribosomal protein mutation suppresses gonadal leader cell migration defects in mig-17/ADAMTS mutants in Caenorhabditis elegans

Abstract

The migration of gonadal distal tip cells (DTCs) in Caenorhabditis elegans serves as an excellent model for studying the migration of epithelial tubes during organogenesis. Mutations in the mig-17/ADAMTS gene cause misdirected DTC migration during gonad formation, resulting in deformed gonad arms. An amino acid substitution in RPL-20, the ortholog of mammalian RPL18a/eL20, a component of the 60 S ribosomal large subunit, exhibited a slow-growth phenotype and strongly suppressed the mig-17 gonadal defects. Slow-growing mutations clk-1 and clk-2 also suppressed mig-17. Intestine-specific overexpression of mutant RPL-20 protein resulted in a slow-growth phenotype and suppressed the mig-17 gonadal defects, but these effects were much weaker when wild-type RPL-20 was overexpressed, suggesting that the mutant RPL-20 protein acquired a novel function. Analysis of ribosome profiles revealed reduced biogenesis of the 60 S subunit, leading to a reduction of 80 S ribosomes in the rpl-20 mutant. These results suggest that DTC migration defects in mig-17/ADAMTS mutants can be partly suppressed by growth retardation caused by the rpl-20 mutation. While defective ribosome biogenesis may contribute to the observed growth retardation, further investigation is needed to clarify the molecular basis of this phenomenon.

Keywords: ADAMTS; Growth retardation; Organogenesis; RPL18a/eL20; Ribosome protein mutation.

Fig. 1

Suppression of mig-17 and mig-18 DTC migration defects by rlp-20(tk73). (A) Gonad morphology (arrows) of wild type, mig-17(k174), and mig-17(k174); rpl-20(tk73) young-adult hermaphrodites. Posterior gonads are shown. Anterior to the left, dorsal to the top. Bar, 20 μm. (B) Quantitative analysis of mig-17 gonadal defects combined with rpl-20(tk73). N = 60 for each strain. P-values for Fisher’s exact test against mig-17(k174) for mig-17(k174) carrying strains are indicated: ***, P < 0.001; NS, not significant. (C) Transgenic rescue experiments of mig-17. mig-17 animals introduced with transgenic arrays containing PCR-amplified fragments of the rpl-20(tk73) gene were suppressed for their gonadal defects. #1 and #2 are independently established transgenic lines. N = 60 for each strain. P-values for Fisher’s exact test against mig-17(k174) are indicated: ***, P < 0.001. (D) Amino acid sequence homology between RPL-20 and human RPL18a. Identical amino acids are shown in black boxes. G82 mutated in rpl-20(tk73) is depicted with an arrow. (E-G) rpl-20(tk73) suppresses the DTC migration defects of mig-17 and mig-18 alleles (E), but not gon-1(RNAi) (F) or sqv-5(k175) and mig-22(k141) mutants (G). sqv-5(k175) and mig-22(k141) were marked with unc-13(e1091) and unc-32(e189), respectively. N = 60 for each strain. P-values for Fisher’s exact test against mig-17, mig-18, rpl-20(tk73) control RNAi, sqv-5, and mig-22 for the respective double mutants are indicated: ***, P < 0.001; **, P < 0.01; NS, not significant.

Fig. 2

Growth rate analysis of mig-17, rpl-20, mig-17; rpl-20, clk-1, and clk-2 mutants. (A-C) Percentages of larval stages were determined at 43 h (A) and 48 h (B) and 67 and 72 h (C) after hatching, as described in Methods. Color codes represent larval stage 2 (L2), larval stage 3 (L3), early larval stage 4 (EL4), mid-larval stage 4 (ML4), mid-late larval stage 4 (MLL4), late larval stage 4 (LL4), and adult stage (A), respectively. (D, E) Suppression of mig-17(k174) (D) and mig-17 and mig-18 alleles (E) by clk-1 and clk-2. N = 60 for each strain. P-values for Fisher’s exact test against mig-17 and unc-42 mig-17 for the respective double mutants are indicated: ***, P < 0.001; **, P < 0.01; *, P < 0.05.

Fig. 3

Tissue-specific expression of RPL-20. (A) Expression of mCherry-RPL-20(WT) and mCherry-RPL-20(tk73). Upper panels are Nomarski and confocal merged images for young adult animals and lower panels represent close-up images of the gonadal tip regions. Arrows and arrowheads indicate intestine and the distal tip cells, respectively. Bars, 50 μm. (B, C) Rescue experiments of the mig-17 gonadal defect by expressing rpl-20(tk73) (B) or rpl-20(WT) (C) genes under various tissue-specific promoters. N = 60 for each strain. P-values for Fisher’s exact test against mig-17(k174) for mig-17(k174) carrying strains are indicated: ***, P < 0.001; **, P < 0.01; NS, not significant. #1 and #2 are independently established transgenic lines. (D, E) Growth rate analysis of mig-17 animals carrying various plasmid constructs. Percentages of larval stages were determined at 43 h (D) and 48 h (E) after hatching, as described in Methods. #1 and #2 are independently established transgenic lines. Color codes represent larval stages, early L4 (EL4), mid-L4 (ML4), mid-late L4 (MLL4), late L4 (LL4), and adult (A), respectively. N ≥ 60 for each strain.

Fig. 4

Analysis of ribosomes. (A) Western blot analysis of ribosomes for wild type animals expressing mCherry-RPL-20(tk73) or mCherry-RPL-20(WT). Ribosomes were precipitated by dual sucrose cushion centrifugation and the samples of supernatants and precipitates were analyzed. Sup 1 and 2 and PPT 1 and 2 represent supernatant and precipitate samples from first and second centrifugation, respectively. Arrow indicates the bands corresponding to mCherry-RPL-20. The double bands may be generated due to post-translational modification of RPL-20. Original blot is presented in Supplementary Figure S3. (B) Polysome profiles of wild type and rpl-20(tk73) animals. The amounts of 60 S subunit and 80 S ribosome in rpl-20(tk73) were reduced relative to the wild type.