RNA-binding protein GLD-1/quaking genetically interacts with the mir-35 and the let-7 miRNA pathways in Caenorhabditis elegans

Abstract

Messenger RNA translation is regulated by RNA-binding proteins and small non-coding RNAs called microRNAs. Even though we know the majority of RNA-binding proteins and microRNAs that regulate messenger RNA expression, evidence of interactions between the two remain elusive. The role of the RNA-binding protein GLD-1 as a translational repressor is well studied during Caenorhabditis elegans germline development and maintenance. Possible functions of GLD-1 during somatic development and the mechanism of how GLD-1 acts as a translational repressor are not known. Its human homologue, quaking (QKI), is essential for embryonic development. Here, we report that the RNA-binding protein GLD-1 in C. elegans affects multiple microRNA pathways and interacts with proteins required for microRNA function. Using genome-wide RNAi screening, we found that nhl-2 and vig-1, two known modulators of miRNA function, genetically interact with GLD-1. gld-1 mutations enhance multiple phenotypes conferred by mir-35 and let-7 family mutants during somatic development. We used stable isotope labelling with amino acids in cell culture to globally analyse the changes in the proteome conferred by let-7 and gld-1 during animal development. We identified the histone mRNA-binding protein CDL-1 to be, in part, responsible for the phenotypes observed in let-7 and gld-1 mutants. The link between GLD-1 and miRNA-mediated gene regulation is further supported by its biochemical interaction with ALG-1, CGH-1 and PAB-1, proteins implicated in miRNA regulation. Overall, we have uncovered genetic and biochemical interactions between GLD-1 and miRNA pathways.

Keywords: Caenorhabditis elegans; SILAC; gld-1; let-7; miRNA.

Figure 1.

Whole genome RNAi screen identifies enhancers of gld-1(op236). (a) Schematic of the RNAi screen. (b) List of candidate genes with various levels of reproducibility indicated. Owing to variations in the effectiveness of RNAi, further validation was carried out by the analysis of mutants (genes marked with + or −) and five out of seven genes validated the RNAi results when tested with mutants (+). (c,d) Somatic defects in gld-1(op236); vig-1(ok2536). 29% of the surviving gld-1(op236); vig-1(ok2536) larvae develop an abnormal morphology, as indicated by arrowheads (*p < 0.01, Fisher's exact test). Left panel DIC image, right panel fluorescent image of the same animal that expresses col-19::GFP. As a control, wild-type animals are shown at the lower panel.

Figure 2.

gld-1 genetically interacts with mir-35 and let-7 family miRNAs. (a) Adult animals 24 h past L4 stage were allowed to lay eggs and quantitation of embryonic and larval lethality is depicted for mir-35–41(nDf50) and gld-1(op236); mir-35–41(nDf50) mutants at 20°C (left graph). gld-1(op236) animals are 100% viable. Number of eggs laid per worm is shown in the right graph. Each experiment was carried out in quadruplicate (n > 250), and the percentage of dead eggs and L1 worms was calculated (error bars = s.e.m.). (b) Lethality owing to adult-stage lethargus. Number of assayed worms is mentioned in parenthesis. m, maternal genotype; z, zygotic genotype. Synchronized L1 stage animals were grown to adult stage and assayed for lethality owing to internal hatching of embryos. Owing to the sterility of gld-1(null) animals, gld-1(null); let-7(mg279) phenotype is determined by slow movement and lack of pharyngeal activity during L4 to young adult transition. (c) Representative picture of a gld-1(op236); let-7(mg279) worm. The accumulation of late stage embryos is evident. Arrowhead indicates cuticle that failed to shed.

Figure 3.

gld-1 affects the let-7 regulated hypodermal development (a) Simplified diagram of the let-7 pathway leading to col-19 expression. (b) col-19::GFP expression in hypodermal hyp7 cells (error bars = s.e.m. of triplicate results). (c) Seam cell fusion defects assayed by the ajm-1::GFP junction marker upon either control RNAi or glp-1 RNAi (error bars = s.e.m. of quadruplicate results, n = 20 for each replicate). In glp-1 RNAi, only the animals without a germline were assayed. (d) Representative pictures of col-19::GFP expressing worms. Numbers indicate worms. Note the complete absence of signal in worm number 2 in the right panel. (e) Representative images of animals showing adult-stage alae and seam cell fusions. In wild-type worms, complete alae and complete seam cell fusion can be seen. Strong ectopic junctions (arrow heads), weak ectopic junctions (small, thin arrows) and lack of junctions (not shown) are observed in gld-1(op236), let-7(mg279) and gld-1(op236); let-7(mg279) worms (right hand panel). In the left-hand panels, partial alae or lack of alae are indicated by dashed lines and ectopic alae are indicated by small T-bars. (f) Schematic drawing of the seam cell fusion defects observable by AJM-1::GFP.

Figure 4.

gld-1(op236) induces vulva formation. (a) gld-1(op236) enhances the multi-vulva phenotype in the heterozygous let-60(n1046)/+ gain-of-function background. (b) gld-1(op236); let-60(ga89) shows increase in multi-vulva formation when switched from 20 to 25°C (n > 40 for each genotype, *p < 0.05, **p < 0.01 by Fisher's exact test).

Figure 5.

A let-7 sponge transgene generates a sensitive system to test miRNA function. (a) let-7 sponge (col-10::GFP::lin-41 3′UTR) causes mild bursting phenotype in let-7(mg279) mutants. Bursting dramatically increases in gld-1(op236); let-7(mg279); [let-7 sponge] animals (error bars = s.e.m.). Using lin-41 3′UTR with deleted let-7 binding sites ([Δlet-7sponge]) or [unc-54 3′UTR] in the sponge construct doesn't cause any phenotypes. (b) gld-1 expression under the control of the col-10 promoter causes lack of adult-stage alae. let-7 sponge partially rescues the alae defects in col-10::GLD-1 expressing animals. (c) col-10::GLD-1 expressing animals have a dumpy phenotype and short size. let-7 sponge partially rescues the dumpy phenotype and the short size of the animals are rescued to wild-type levels. The relative length of the animals is measured through time of flight by a COPAS biosorter (n > 2000). (d) Representative DIC images of animals expressing col-10::GLD-1, let-7 sponge and col-10::GLD-1; let-7 sponge.

Figure 6.

SILAC-based proteomics in let-7 and gld-1 mutants. (a) log₂ relative abundances of 2179 proteins in let-7(mg279); [let-7 sponge] (x-axis) and gld-1(op236); let-7(mg279); [let-7 sponge] (y-axis) animals compared with [let-7 sponge] animals alone. Solid black and grey lines indicate 1.2-fold and twofold thresholds, respectively. Dots represent 2179 proteins. Among them GLD-1 targets [55,56] are coloured blue, mirWIP database let-7 target predictions [57] are coloured red, and the possible GLD-1 and let-7 co-targets based on these lists are coloured purple. The remaining proteins are coloured in grey. CDL-1, DNJ-2 and B0303.3 are possible GLD-1 and let-7 targets that are upregulated more than 1.2-fold (arrows). (b) RNAi-mediated knockdown of 9 genes upregulated in the gld-1(op236); let-7(mg279); [let-7 sponge] animals (RNAi is done in the same strain). We picked six genes upregulated more than twofold and are GLD-1 or predicted let-7 targets (red and blue spots above the twofold line) and three genes upregulated more than 1.2-fold that are GLD-1 and predicted let-7 targets (purple spots above the 1.2-fold line). Empty vector RNAi and GFP RNAi are used as negative and positive controls respectively. B0303.3 RNAi results are omitted due to the early larval lethality in these animals (error bars = s.e.m. of triplicate, p-values calculated using Fisher's exact test).

Figure 7.

Protein interactors of GLD-1 and their effect on hypodermal development upon RNAi depletion. (a) List of protein interactors identified in both anti-GLD-1 antibody IPs in wild-type animals and anti-GFP IPs in gld-1::GFP expressing animals. Total peptides detected in Ab IPs and background peptides detected in mock IPs together with % coverage of the peptides are indicated. Original data is in electronic supplementary material, figure S6. (b) Quantification of bursting phenotype upon RNAi knockdown of indicated genes in let-7 sponge (sponge, grey) and let-7(mg279); let-7 sponge (green) genetic backgrounds (error bars = s.e.m., n > 50 for each replicate).