Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 Jul 25;9(7):e103365.
doi: 10.1371/journal.pone.0103365. eCollection 2014.

Diverse functions of mRNA metabolism factors in stress defense and aging of Caenorhabditis elegans

Aris Rousakis  1 , Anna Vlanti  2 , Fivos Borbolis  3 , Fani Roumelioti  3 , Marianna Kapetanou  4 , Popi Syntichaki  2
Affiliations

Diverse functions of mRNA metabolism factors in stress defense and aging of Caenorhabditis elegans

Aris Rousakis et al. PLoS One. .

Abstract

Processing bodies (PBs) and stress granules (SGs) are related, cytoplasmic RNA-protein complexes that contribute to post-transcriptional gene regulation in all eukaryotic cells. Both structures contain translationally repressed mRNAs and several proteins involved in silencing, stabilization or degradation of mRNAs, especially under environmental stress. Here, we monitored the dynamic formation of PBs and SGs, in somatic cells of adult worms, using fluorescently tagged protein markers of each complex. Both complexes were accumulated in response to various stress conditions, but distinct modes of SG formation were induced, depending on the insult. We also observed an age-dependent accumulation of PBs but not of SGs. We further showed that direct alterations in PB-related genes can influence aging and normal stress responses, beyond their developmental role. In addition, disruption of SG-related genes had diverse effects on development, fertility, lifespan and stress resistance of worms. Our work therefore underlines the important roles of mRNA metabolism factors in several vital cellular processes and provides insight into their diverse functions in a multicellular organism.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Aggregation of DCAP-1 protein in response to heat-shock.
(A) Representative confocal images of somatic tissues in 1-day adult worms expressing the transcriptional fusion Pdcap-1::gfp (BRF154) or the translational fusion dcap-1::gfp in N2 (BRF155 and BRF261) and germline-deficient glp-1(e2141) worms (BRF219), normally grown at 25°C (-HS) or transiently subjected to heat-shock (+HS, 35°C for 3 h). Arrows point to DCAP-1::GFP granules in body wall muscles that are highly induced upon stress. Asterisks denote the intestinal autofluorescence. Scale bar: 25 µm. (B) Quantification of data in (A). Values on Y axis show the number of granules per 2500 µm2 per worm (mean±SD, see Materials and Methods and Table S4). (C) Endogenous dcap-1(mRNA) levels in 1-day adults N2, grown at 25°C before (-HS) or after heat shock (+HS, 35°C for 3 h) were measured by quantitative RT-PCR and normalized to endogenous ama-1(mRNA) levels. Error bars represent the standard deviation of the means of five independent experiments (p-value = 0.3466, calculated by Student's t-test). (D) Western blot analysis of DCAP-1::GFP protein expression in 1-day adult BRF155 transgenic animals, before (-HS) or after heat shock (+HS), using anti-GFP or anti-Actin (as a loading control) antibodies. Band intensity of DCAP-1::GFP normalized to actin gives a value of 0.83, showing similar protein levels in the two conditions.
Figure 2
Figure 2. Accumulation of DCAP-1-containing granules under heat-shock is rapid, reversible and sensitive to cgh-1(RNAi).
(A) Representative confocal images of 1-day adult worms expressing dcap-1::gfp (BRF155 and BRF261 strains) normally grown at 25°C (-HS) or transiently subjected to heat-shock (+HS, 35°C for 3 h),following recovery for 3 h at 20°C (Recovery). (B) Quantification of data in (A). (C) Representative confocal images of 1-day adult worms expressing dcap-1::gfp (BRF155) fed from eggs with Control(RNAi) or cgh-1(RNAi) bacteria and grown at 25°C (-HS) or subjected to heat-shock at 35°C for 3 h (+HS). In all cases arrows point to DCAP-1::GFP granules in body wall muscles. (D). Quantification of data in (C). Values on Y axis show the number of granules per 2500 µm2 per worm (mean±SD, see Materials and Methods and Table S4). Scale bar: 25 µm.
Figure 3
Figure 3. Accumulation of PBs with age.
(A) Representative confocal images of 1-day and 5-day adults, grown at 25°C and expressing the transcriptional fusion Pdcap-1::gfp reporter (BRF154) or the translational fusions dcap-1::gfp (BRF155 and BRF261). Arrows point to age-induced DCAP-1::GFP granules. Scale bar: 25 µm. (B) Quantification of data in (A). Values on Y axis show the number of granules per head (mean±SD, see Materials and Methods and Table S4). (C) Endogenous dcap-1(mRNA) levels in 1-day and 7-day N2 adults grown at 25°C before (-HS) or after heat shock (+HS, 35°C for 3 h) were measured by quantitative RT-PCR and normalized to endogenous ama-1(mRNA) levels. Error bars represent the standard deviation of the means of six independent experiments (p-value = 0.9697, calculated by Student's t-test). (D) Western blot analysis of DCAP-1::GFP protein expression in 1-day and 5-day adult BRF155 transgenic animals, grown at 25°C, using anti-GFP or anti-β-Actin (as a loading control) antibodies. Band intensity of DCAP-1::GFP normalized to actin gives a value of 0.95, showing similar protein levels in the two conditions.
Figure 4
Figure 4. Disruption of mRNA degradation, but not of translation factors, triggers PBs formation that lack ife-2/eIF4E.
(A) Representative confocal images of 1-day adults expressing dcap-1::gfp in N2 (BRF155 and BRF261) fed with ife-2(RNAi), eIF2a(RNAi), rsks-1(RNAi), eIF2Bγ(RNAi) or xrn-1(RNAi) bacteria. (B) Quantification of data in (A). Values on Y axis show the number of granules per 2500 µm2 per worm (mean±SD, see Materials and Methods and Table S4). (C) Endogenous eIF2Bγ(mRNA) levels in 1-day adults BRF155 worms grown at 25°C and fed with eIF2Bγ(RNAi) from eggs, were measured by quantitative RT-PCR and normalized to ama-1(mRNA) levels. Error bars represent the standard deviation of the means of three independent experiments (***p-value<0.0001, in unpaired t-test). (D) Representative confocal images of 1-day adults expressing dcap-1::gfp in N2 (BRF155) or ife-2(ok306) (BRF220) background. (E) Representative confocal images of 1-day adults co-expressing dcap-1::rfp and ife-2::gfp (BRF313), and treated with xrn-1(RNAi) to monitor the lack of co-localization of the two proteins. In all cases, targeted RNAi was initiated from eggs at 25°C in parallel with Control(RNAi) to assess the localization of DCAP-1::GFP into PBs. Asterisks show the intestinal autofluorescence. Scale bar: 25 µm.
Figure 5
Figure 5. PB components are important for normal lifespan and stress response.
(A) Survival curves of N2, dcap-1 and dcap-2 mutants at 20°C. (B) Survival of adults dcap-1 and dcap-2 mutants in heat-shock (1-day adults at 35°C for 6 h), oxidative stress (1-day adults in 5 mM sodium arsenite for 48 h) and UV-irradiation (5-day adults at 0.2 J/cm2), compared to N2. (C) Survival curves of N2 worms treated post-developmentally with dcap-1/-2(RNAi), patr-1(RNAi) or xrn-1(RNAi) at 20°C. (D) Lifespan of daf-2, daf-2; dcap-1 and daf-2; dcap-2 mutants at 20°C and (E) survival of 1-day adults after heat-shock (35°C for 8 h). (F) Survival of 1-day adult glp-1 germline-deficient mutants that overexpress dcap-1::gfp after heat-shock (at 35°C for 6 h) or oxidative stress (5 mM sodium arsenite for 48 h). In lifespan assays the p-values were determined using the log-rank test (see Tables S5 and S6) and in stress assays the error bars show the SD in unpaired t-tests (see Materials and Methods). ** indicates very significant (p-value 0.001 to 0.01); *** indicates extremely significant (p<0.001).
Figure 6
Figure 6. Aggregation of TIAR-1 and TIAR-2 proteins in response to stress but not to age.
(A) The domain structure of TIAR proteins, designed using the Prosite MyDomains (http://prosite.expasy.org/mydomains/). The graphic was created using the Exon-Intron Graphic Maker (http://wormweb.org/exonintron). RRM: RNA-recognition motif, GLY_R: Glycine rich domain, GLN_R: Glutamine rich domain. (B) Representative confocal images of 1-day adults of the indicated transgenic strains grown at 25°C (Control 1-d ad), upon heat-shock (HS, 35°C for 3 h), oxidative stress (SA, 15 mM sodium arsenite for 3 h) or compared to 6-day adults under normal conditions. Arrows point to formed granules and quantification of the granule number is shown in the lower panel for each strain. Values on Y axis show the number of granules per head (BRF211), per 100 µM length of excretory cell (BRF255) or per 2500 µm2 per worm (mean±SD, see Materials and Methods and Table S4). Asterisks show the intestinal auto-fluorescence. Scale bar: 25 µm. (C–D) Expression levels of tiar-1 or tiar-2 genes in (C) the indicated transgenic strains expressing gfp::tiar-1 and gfp::tiar-2 by their own promoter (BRF211 and BRF255, respectively) or the muscle specific Pmyo-3::gfp::tiar-2 (BRF310) and (D) N2 worms after heat shock (35°C for 3 h). Quantification of each mRNA level, relative to ama-1(mRNA) levels, in 1-day adults and the mean ± SD of biological triplicates are shown (p>0.05 in Student's t-tests).
Figure 7
Figure 7. Rapid formation and dissociation of heat-induced SGs.
Representative confocal images of 1-day adults expressing gfp::tiar-1 and gfp::tiar-2 by their own promoter (BRF211 and BRF255, respectively) or the muscle specific Pmyo-3::gfp::tiar-2 (BRF310) under normal growth conditions (-HS) and upon heat-shock (+HS, 35°C for 45 min to 2.5 h), following recovery at 20°C for 2 h. Arrows point to formed granules and quantification of the granule number is shown in the lower panel for each strain. Values on Y axis show the number of granules per head (BRF211), per 100 µM length of excretory cell (BRF255) or per 2500 µm2 per worm (mean±SD, see Materials and Methods and Table S4). Asterisks show the intestinal auto-fluorescence. Scale bar: 25 µm.
Figure 8
Figure 8. Effects of tiar genes deletion on development, fertility, lifespan and stress survival.
(A) Developmental rate and brood size of tiar-1, tiar-2, tiar-3 and tiar-1;tiar-2 mutant worms, compared to N2. (B) Survival curves of the above mutants or of N2 worms treated post-developmentally with tiar-1(RNAi), tiar-2(RNAi) or tiar-3(RNAi). (C) Survival of adults of the above mutants in oxidative stress (1-day adults at 5 mM sodium arsenite for 48 h), heat-shock (1-day adults at 35°C for 6 h), UV-irradiation (5-day adults at 0.2 J/cm2) or osmotic stress (1-day adults at 400 mM NaCl for 24 h, compared to N2. (D) Survival of tiar-1 mutants expressing the gfp::tiar-1 rescuing transgene in oxidative stress (1-day adults at 5 mM sodium arsenite for 24 h) compared to N2 or tiar-1 animals carrying only the rol-6(su1006) roller marker. In lifespan assays the p-values were determined using the log-rank test (see Tables S5 and S6) and in stress assays the error bars show the SD in unpaired t-tests (see Materials and Methods). ns indicates not significant (p>0.05); * indicates significant (p-value 0.01 to 0.05); ** indicates very significant (p-value 0.001 to 0.01); *** indicates extremely significant (p<0.001).
See this image and copyright information in PMC

References

    1. Kenyon CJ (2010) The genetics of ageing. Nature 464: 504–512. - PubMed
    1. Spriggs KA, Bushell M, Willis AE (2010) Translational regulation of gene expression during conditions of cell stress. Mol Cell 40: 228–237. - PubMed
    1. Renoux AJ, Todd PK (2012) Neurodegeneration the RNA way. Prog Neurobiol 97: 173–189. - PMC - PubMed
    1. Wang W (2012) Regulatory RNA-binding proteins in senescence. Ageing Res Rev 11: 485–490. - PubMed
    1. Westmark CJ, Malter JS (2012) The regulation of AbetaPP expression by RNA-binding proteins. Ageing Res Rev 11: 450–459. - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources