Research ArticleMOLECULAR BIOLOGY

N6-adenosine methylation of ribosomal RNA affects lipid oxidation and stress resistance

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Science Advances  22 Apr 2020:
Vol. 6, no. 17, eaaz4370
DOI: 10.1126/sciadv.aaz4370
  • Fig. 1 METL-5 N6-methylates adenosine 1717 on 18S rRNA in vivo.

    (A) UHPLC-MS/MS chromatography peaks can distinguish adenosine from N6-methylated adenosine (m6A) and cytidine from C3-methylated cytidine and C5-methylated cytidine based on retention time on the column. au, area units. (B) RNAi screen of 13 metl family members in C. elegans reveals that knockdown of C38D4.9/metl-5 causes a decrease in m6A levels on total RNA without any significant effects on m5C or m3C levels, as assessed by UHPLC-MS/MS. Each bar represents the mean ± SEM of two biological replicates performed in duplicate. *P < 0.05, as assessed by one-way analysis of variance (ANOVA). E.V., empty vector. (C) Schematic of metl-5 genomic DNA (gDNA), cDNA, and protein indicating the location of the catalytic domain and the mutations used in this study. aa, amino acid; Nt, N terminus; Ct, C terminus. (D) Two metl-5 mutant strains display decreases in m6A levels without any change in m3C or m5C levels, as assessed by UHPLC-MS/MS. Each bar represents the mean ± SEM of 4 to 12 biological replicates performed in duplicate. ****P < 0.0001, as assessed by one-way ANOVA. (E) Two metl-5 mutant strains display decreases in m6A levels on purified 18S rRNA without changes in m5C levels, as assessed by UHPLC-MS/MS. No detectable changes were observed in purified 28S or 5.8S and 5S in m6A or m5C. m3C was undetectable in all rRNA purifications. Each bar represents the mean ± SEM of two to four biological replicates performed in duplicate. ****P < 0.0001, as assessed by one-way ANOVA. (F) Directed RNA cleavage, followed by 32P labeling and thin-layer chromatography, demonstrates that adenosine 1717 on 18S rRNA is N6-adenosine methylated ~98% of the time in WT worms but is unmethylated in metl-5 mutant worms. The left blot represents the migration of unmethylated adenosines and N6-methylated adenosines, and the right blot represents the methylation of adenosine 1717 in 18S rRNA. The asterisk (*) indicates a nonspecific spot migrating above the m6A location.

  • Fig. 2 METL-5 N6-methylates adenosine 1717 on 18S rRNA in vitro.

    (A) Coomassie staining of SDS–polyacrylamide gel electrophoresis (SDS-PAGE) gels reveals that GST-tagged METL-5 WT and APPA catalytic mutant proteins are pure and migrate at the same location. (B) WT GST-tagged METL-5, but not the catalytically inactive mutant APPA, is able to methylate 18S rRNA purified from metl-5 mutant worms, as assessed by UHPLC-MS/MS of deuterated m6A. METL-5 was unable to methylate 18S rRNA purified from WT worms, which are already fully methylated. Deuterated S-adenosyl methionine (d3-SAM) was used as the methyl donor to ensure that methylation was added during methylation assays. Each bar represents the mean ± SEM of three independent experiments. **P < 0.005, as assessed by one-way ANOVA with Tukey’s multiple comparisons test. (C) WT GST-tagged METL-5, but not the catalytically inactive mutant APPA, is able to methylate both a short and a long oligo containing the sequence surrounding adenosine 1717 in 18S rRNA. This methylation is absent when the nucleoside representing adenosine 1717 is replaced with a guanosine despite the presence of additional adenosines. (Top) Each bar represents the mean ± SEM of three independent experiments, and (bottom) the oligo sequences are displayed with adenosine 1717 highlighted in red. Avg., average.

  • Fig. 3 metl-5 mutant worms are resistant to several stresses.

    (A) Two metl-5 mutant strains display a nonsignificant trend toward fewer progeny than WT worms grown at 20° or 25°C. Each bar represents the mean ± SEM of one to six independent experiments performed with three plates of 10 worms each. ns, not significant as assessed by two-way ANOVA and Tukey’s multiple comparisons test of one-way ANOVA. (B) Two metl-5 mutant strains display increased survival after L4 worms were placed at 37°C for the time indicated, then grown at 20°C, and assessed for survival after 24 hours. This graph represents the mean ± SEM of five independent experiments. Bar: *P < 0.05, as assessed by unpaired t test, and *P < 0.05 and **P < 0.005, as assessed by two-way ANOVA. (C) metl-5(gk747459) mutant worms display a 156% increase in average life span relative to WT worms when L4 worms were exposed to a 37°C heat shock for 5 hours and then grown at 20°C for the remainder of the assay. This graph represents one experiment of five and was performed with three plates of at least 30 worms per plate. Statistics and replicate experiments are presented in table S1. ****P < 0.0001, as assessed by log-rank Mantel-Cox survival analysis. (D) Two metl-5 mutant strains display increased survival after young adults were placed at 2°C for 6 hours before being returned to 20°C for the remainder of their life. This graph represents one experiment with three plates of 30 worms per plate, which was performed in triplicate. Statistics and replicate experiments are presented in table S1. **P < 0.005, as assessed by log-rank Mantel-Cox survival analysis. (E) Two metl-5 mutant strains display increased survival after young adults were exposed to 0.8 Joules and then grown at 20°C for the remainder of their life. This graph represents one experiment with three plates of 30 worms per plate, which was performed in triplicate. Statistics and replicate experiments are presented in table S1. ****P < 0.0001, as assessed by log-rank Mantel-Cox survival analysis. (F) metl-5 WT but not the catalytically inactive mutant APPA overexpression lines in metl-5(tm4561) mutant worms rescues 18S rRNA m6A methylation levels, as assessed by UHPLC-MS/MS. Each bar represents the mean ± SEM of two to three independent lines. ****P < 0.0001, as assessed by one-way ANOVA. Successful overexpression of metl-5 was validated by real-time polymerase chain reaction (RT-PCR), as shown in fig. S2J. (G) The heat shock resistance phenotype of three independent metl-5 WT but not the catalytically inactive mutant APPA overexpression lines in metl-5(tm4561) mutant worms reverts to the same levels as WT worms. Each bar represents the average ± SEM of three independent experiments performed with three plates of 20 to 50 worms each. The control represents three independent lines performed in three independent experiments in triplicate. Worms were treated with 37°C heat shock for 6 hours and then grown at 20°C and assessed for survival after 24 hours. *P < 0.05, **P < 0.005, and ***P = 0.0005, as assessed by one-way ANOVA relative to control-injected lines. (H) Increased heat shock survival of two metl-5 mutant strains was eliminated by inhibition of translation by cycloheximide treatment. This graph represents the mean ± SEM of three independent experiments of three plates containing 20 to 35 worms each. Bar: *P < 0.05 and **P < 0.005, as assessed by Tukey’s multiple comparisons test of one-way ANOVA analysis; strain: **P < 0.005 and ***P = 0.0005, as assessed by two-way ANOVA. (I) Increased survival of two metl-5 mutant strains exposed to 0.8 Joules was eliminated by inhibition of translation by cycloheximide treatment. This graph represents the mean ± SEM of three independent experiments of three plates containing 20 to 35 worms each. Bar: **P < 0.005, as assessed by one-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05 and **P < 0.005, as assessed by two-way ANOVA.

  • Fig. 4 metl-5 has no global effect on translation but regulates ribosome occupancy of cyp-29A3.

    (A) The profile of newly translated proteins changes in C. elegans in response to 3 hours at 37°C, but this is unaffected by metl-5(tm4561) mutation, as assessed by phosphor imaging of 35S incorporation. This blot is representative of three independent experiments. (B) Absolute 35S incorporation was unaffected by 3 hours at 37°C or metl-5(tm4561) mutation, as assessed by scintillation counting. Control (ctl) lanes represent worms that were treated with 35S-methionine for 1 min. Each bar represents the mean ± SEM of three independent experiments performed in duplicate. CPM, counts per minute. (C) Polysome profiles of metl-5(tm4561) mutant worms were indistinguishable from WT worms grown at 20°C or when profiles shifted to monosomes after 2 hours at 37°C. This graph is a representative experiment where UV absorbance at OD254 (optical density at 254 nm) is monitored continuously. (D) Volcano plot of translation efficiency as calculated by ribosome occupancy relative to mRNA transcript of WT and metl-5(tm4561) mutant worms grown at 20°C (top) or heat-shocked for 3 hours at 37°C (bottom) revealed no significant change in translation efficiency in mutant worms. (E) Ribosome sequencing clusters of WT and metl-5(tm4561) mutant worms grown at 20°C or heat-shocked for 3 hours at 37°C revealed global changes in transcripts bound by ribosomes in response to heat shock with no global change in response to metl-5 mutation. (F) Volcano plot of ribosome sequencing reveals that ribosomes are significantly differentially bound to the normalized transcripts of only cyp-29A3 and metl-5 after correction for multiple hypothesis testing. Ribosome sequencing was normalized by values within the replicate. (G) cyp-29A3 mRNA is at similar levels in WT and metl-5 mutant worms grown at 20°C and is increased to a similar extent in WT and metl-5 mutant worms in response to heat shock at 37°C for 5 hours. Each bar represents the mean ± SEM of two independent experiments performed in duplicate. (H) cyp-29A3 mRNA is higher in polysome fractions after heat shock, but this increase is blunted in the metl-5 mutant worms relative to WT worms, as assessed by polysome fractionation followed by qRT-PCR. cyp-29A3 expressions relative to the spiked-in internal standard of firefly luciferase were examined at each fraction of the polysome profile by qRT-PCR of WT and metl-5 mutant worms grown at 20°C (left) and 37°C for 5 hours (right). Relative expression is normalized to fraction #1 to demonstrate the relative change in mRNA presence. Fractions #1 and #2 represent the free RNA, fractions #4 to #6 represent the monosomes, and fractions #7 to #11 represent the polysomes.

  • Fig. 5 cyp-29A3 and EPA derivatives mediate heat stress resistance of metl-5 mutant worms.

    (A) cyp-29A3 mutant worms display increased stress resistance relative to WT worms after 5 hours of 37°C heat shock. Each bar represents the mean ± SEM of two independent experiments of three plates containing 20 to 35 worms each. **P < 0.01, as assessed by unpaired t test. (B) The heat shock resistance phenotype of three independent cyp-29A3 driven by the ubiquitous promoter of eft-3 but not control lines in metl-5(tm4561) mutant worms reverts to the same levels as WT worms. Each bar represents the average ± SEM of three independent experiments performed with three plates of 20 to 50 worms each. The control represents three independent lines performed in three independent experiments in triplicate. Worms were treated with 37°C heat shock for 6 hours, then grown at 20°C, and assessed for survival after 24 hours. *P < 0.05 and **P < 0.005, as assessed by one-way ANOVA relative to control-injected lines. Note that the WT, metl-5, and control-injected strains are the same as in Fig. 3G, as these experiments were performed together. Successful overexpression of cyp-29A3 was validated by qRT-PCR, as shown in fig. S6B. (C) metl-5 and cyp-29A3 mutant worms have lower lipid concentrations. as assessed by ORO fluorescence. Each column represents the mean ± SD of quantification of 19 to 30 worms each. ****P < 0.0001, as assessed by one-way ANOVA with Dunnett’s multiple comparisons test. (D) metl-5(gk747459) and cyp-29A3(gk827495) mutant worms display lower levels of eicosanoid EETs 17,18-EpETE, 17,18-DiHETE, and 20-HEPE than WT worms, as assessed by GC-MS. metl-5(gk747459) mutant worms have equivalent levels of PUFAs AA and EPA compared to WT worms, but cyp-29A3 mutant worms display lower levels of these PUFAs. Each bar represents the mean ± SEM of three independent experiments of ~6000 larval stage L4 worms each. *P < 0.05 and **P < 0.01, as assessed by Dunnett’s multiple comparisons test of one-way ANOVA. (E) Dietary supplementation with eicosanoid EETs 17,18-EpETE, 17,18-DiHETE, and 20-HEPE reverts the increased stress resistance of metl-5(gk747459) mutant worms, but supplementation with AA or EPA does not cause a significant reversion of the increased stress resistance of metl-5(gk747459) mutant worms after 5 hours of 37°C heat shock. Each column represents the mean ± SEM of three to five independent experiments of three plates containing 20 to 54 worms each. *P < 0.05 and **P < 0.01, as assessed by multiple t tests and two-way ANOVA. (F) Dietary supplementation with eicosanoid EETs 17,18-EpETE, 17,18-DiHETE, and 20-HEPE reverts the increased stress resistance of cyp-29A3(gk827495) mutant worms, but supplementation with AA or EPA does not cause a significant reversion of the increased stress resistance of cyp-29A3(gk827495) mutant worms after 5 hours of 37°C heat shock. Each column represents the mean ± SEM of three independent experiments of three plates containing 20 to 54 worms each. *P < 0.05 and **P < 0.01, as assessed by multiple t tests and two-way ANOVA. (G) The model depicts how METL-5 can methylate the 18S rRNA of the 40S ribosomal subunit, which regulates binding to cyp-29A3 transcripts, which, in turn, controls the oxidation of EPA to eicosanoid EETs, which regulate stress resistance in C. elegans.

Supplementary Materials

  • Supplementary Materials

    N6-adenosine methylation of ribosomal RNA affects lipid oxidation and stress resistance

    Noa Liberman, Zach K. O’Brown, Andrew Scott Earl, Konstantinos Boulias, Maxim V. Gerashchenko, Simon Yuan Wang, Colette Fritsche, Paul-Enguerrand Fady, Anna Dong, Vadim N. Gladyshev, Eric Lieberman Greer

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