Cytoplasmic LSM-1 protein regulates stress responses through the insulin/IGF-1 signaling pathway in Caenorhabditis elegans

Abstract

Genes coding for members of the Sm-like (LSm) protein family are conserved through evolution from prokaryotes to humans. These proteins have been described as forming homo- or heterocomplexes implicated in a broad range of RNA-related functions. To date, the nuclear LSm2-8 and the cytoplasmic LSm1-7 heteroheptamers are the best characterized complexes in eukaryotes. Through a comprehensive functional study of the LSm family members, we found that lsm-1 and lsm-3 are not essential for C. elegans viability, but their perturbation, by RNAi or mutations, produces defects in development, reproduction, and motility. We further investigated the function of lsm-1, which encodes the distinctive protein of the cytoplasmic complex. RNA-seq analysis of lsm-1 mutants suggests that they have impaired Insulin/IGF-1 signaling (IIS), which is conserved in metazoans and involved in the response to various types of stress through the action of the FOXO transcription factor DAF-16. Further analysis using a DAF-16::GFP reporter indicated that heat stress-induced translocation of DAF-16 to the nuclei is dependent on lsm-1. Consistent with this, we observed that lsm-1 mutants display heightened sensitivity to thermal stress and starvation, while overexpression of lsm-1 has the opposite effect. We also observed that under stress, cytoplasmic LSm proteins aggregate into granules in an LSM-1-dependent manner. Moreover, we found that lsm-1 and lsm-3 are required for other processes regulated by the IIS pathway, such as aging and pathogen resistance.

Keywords: Caenorhabditis elegans; LSM, daf-16; P bodies; stress granules; stress response.

FIGURE 1.

Characterization of lsm-1 and lsm-3 mutants. (A) Gene structures of lsm-1 and lsm-3. White boxes represent exons. Connecting lines represent introns. Black areas indicate the regions encoding the conserved Sm domain. Gray boxes represent regions deleted by lsm-1(tm3585) and lsm-3(tm5166) alleles; (bp) base pair scale. (B) Measure of animal motility using an automated locomotor tracking system (Simonetta and Golombek 2007). Locomotor activity plots showing the activity of L4 worms at 20°C (1 worm/well in a 96-well microplate, eight wells per genotype were analyzed). Each dot corresponds to the mean activity (measured in bins/hour) of 3 h intervals. (C) Graph representing the body length of young adults (grown for 48 h at 25°C from L1 stage). Mean and standard deviations are plotted (n ≥ 100 worms per genotype tested). (D) Brood sizes of wild type (N2) and the indicated mutant strains. Bars represent the standard deviation among individuals (n ≥ 20).

FIGURE 2.

lsm-1 promotes DAF-16 nuclear localization upon stress and influences stress responses. (A) lsm-1 is required for proper stress-induced DAF-16 nuclear translocation. Fluorescence images of DAF-16::GFP in wild-type (N2) and lsm-1 mutant animals grown at 25°C, and after a 40 min heat shock at 35°C. (B) Time-course analysis of DAF-16::GFP nuclear accumulation in response to heat stress. The histogram represents the average of percentages of worms with full nuclear DAF-16::GFP in three independent experiments (n ≥ 50 for each strain and time point). Error bars, SEM. (C) Representative graph of experiments (see Supplemental Fig. S4A) showing the significant reduced resistance to heat stress caused by mutations in lsm-1 and lsm-3 (P-value < 0.001). Young adult worms were incubated at 35°C and survival was scored every hour. (D) Representative graph of experiments (see Supplemental Fig. S4A) showing the significant resistance to heat stress caused by three distinct strains overexpressing lsm-1 (P-value < 0.001). daf-2(e1370) were used as positive control for heat stress resistance. Young adult worms were incubated at 35°C and survival was scored every 2 h. (E) lsm-1 and lsm-3 mutations reduce the survival of L1 larvae during starvation at 20°C. Ectopic expression of lsm-1 through the integrated reporter LSM-1::GFP displayed a significant survival to starvation compared with wild-type (N2) animals after 19 d (P-value < 0.001). Graph represents the mean percentage of survival and standard deviations from three experimental replicates (n ≥ 100 for each replicate and time point).

FIGURE 3.

Mutations in lsm-1 and lsm-3 cause hypersensitivity to pathogen infection and affect IIS-induced longevity. (A) Representative graph of survival experiments (see Supplemental Fig. S4B) of wild-type (N2) and mutant strains on Escherichia coli OP50, Staphylococcus aureus, and Enterococcus faecalis (n = 30). No significant differences were observed after 6 d on OP50 between lsm-1 and lsm-3 mutants compared with wild-type (N2) animals. Differences between lsm-1 and lsm-3 mutants were significant compared with wild-type (N2) after pathogen infection (0.01 < P-value < 0.001 in all cases). (B) Representative graphs showing lifespan analysis (see Supplemental Fig. S4C) of wild-type (N2) and different mutant and transgenic strains. daf-2(m577) and daf-16(mu86) mutants were used as control for extended and shortened lifespans, respectively.

FIGURE 4.

LSM proteins accumulate in cytoplasmic foci in specific stages and conditions. (A) LSM proteins accumulate in cytoplasmic granules during embryogenesis. Confocal images of embryos expressing LSM-1::GFP in the cytoplasm and LSM-4::GFP in the nucleus and the cytoplasm. Right panels show the same embryos visualized under differential interference contrast (DIC). (B) LSm accumulation in cytoplasmic granules in adult cells is induced by heat stress (1 h at 35°C) (additional images in Supplemental Fig. S5) and is dependent on lsm-1. Confocal images (Z hyperstack) of the head of young adult worms expressing LSM-1::GFP and LSM-4::GFP in control (20°C) and after heat-shock. (C) LSM-1 granules colocalize with TIAR-1 granules under heat-stress conditions (1 h at 35°C). Confocal images of the tail of L4 worms coexpressing LSM-1::GFP and RFP::TIAR-1.