Nutrient control of growth and metabolism through mTORC1 regulation of mRNA splicing

Abstract

Cellular growth and organismal development are remarkably complex processes that require the nutrient-responsive kinase mechanistic target of rapamycin complex 1 (mTORC1). Anticipating that important mTORC1 functions remained to be identified, we employed genetic and bioinformatic screening in C. elegans to uncover mechanisms of mTORC1 action. Here, we show that during larval growth, nutrients induce an extensive reprogramming of gene expression and alternative mRNA splicing by acting through mTORC1. mTORC1 regulates mRNA splicing and the production of protein-coding mRNA isoforms largely independently of its target p70 S6 kinase (S6K) by increasing the activity of the serine/arginine-rich (SR) protein RSP-6 (SRSF3/7) and other splicing factors. mTORC1-mediated mRNA splicing regulation is critical for growth; mediates nutrient control of mechanisms that include energy, nucleotide, amino acid, and other metabolic pathways; and may be conserved in humans. Although mTORC1 inhibition delays aging, mTORC1-induced mRNA splicing promotes longevity, suggesting that when mTORC1 is inhibited, enhancement of this splicing might provide additional anti-aging benefits.

Keywords: C. elegans; SR proteins; development; gene expression; growth; human cell growth; longevity; mRNA splicing; mTORC1; metabolism; nutrient response.

Figure 1.. Identification of functional interactions involving mTORC1

(A) Genetic and bioinformatic screening to identify mechanisms that are regulated by mTORC1. See also Figure S1A–S1D, and Table S1. (B) Body size after daf-15 (Raptor) and let-363 (mTOR kinase) RNAi was performed in wild-type (WT) and raga-1(−/−) animals. EV, empty vector. (C) KEGG pathway analysis for 187 genetic screen hits that showed strong or moderate interactions with raga-1. Fisher’s exact test. (D) STRING analysis illustrating known or predicted protein-protein interactions among the 47 double-positive screen hits (Figure S1E). mRNA splicing and mTOR signaling genes are shown in red and blue, respectively. Confidence refers to the approximate probability of protein-protein association scored by known or predicted interactions. (E) Genetic interaction between raga-1 and rsp-3 or rsp-6. RNAi knockdown of rsp-3 (1:9 dilution) or rsp-6 reduced brood size in raga-1(−/−) but not WT animals. One-way ANOVA with Tukey’s correction, mean ± SEM, ***P < 0.001; n.s., not significant. Three biological replicates were performed.

Figure 2.. mTORC1 mediates nutrient regulation of gene expression.

(A) Scheme for RNA-seq analysis of the nutrient response. (B) Heatmap showing genes that were differentially expressed in response to feeding or mTORC1 pathway gene mutation in WT animals. All P-values for RNA-seq data have been calculated as false-discovery rate (FDR)-adjusted. P < 0.01 was used for the cutoff. Three biological replicates were performed. (C) Comparison of genes that were differentially up-regulated by feeding (WT Fed/WT Stv) and mTORC1 activity (WT Fed/raga-1 Fed). (D) GO terms enriched in genes that were up-regulated by both feeding and mTORC1 (WT Fed/raga-1 Fed). Fisher’s exact test. (E) Relative expression levels of mRNA splicing-related genes that exhibited strong genetic interactions with raga-1 (mTORC1) (Figure 1C; Table S1 and Table S2), normalized to WT levels. Mean ± SEM, ***P < 0.001, n.s., not significant.

Figure 3.. Nutrients broadly regulate alternative mRNA splicing through mTORC1.

(A) rMATS analysis of alternative splicing events. FDR-adjusted P-value < 0.05. To increase stringency the minimum delta PSI (Percent Spliced In) was set at 10% instead of the rMATS default of 5%. (B) Heatmap comparing 582 feeding-regulated splicing events detected in wild-type worms (WT Fed vs. WT Stv) to feeding effects detected in raga-1 and rsks-1 mutants. (C) Overlap between genes that were alternatively spliced in response to feeding (WT Fed/WT Stv) and mTORC1 activity (WT Fed/raga-1 Fed). (D) Processes that were enriched among the 133 genes for which alternative splicing was altered by altered by both feeding (WT Fed/WT Stv) and mTORC1 activity (WT Fed/raga-1 Fed). Fisher’s exact test. *P < 0.05. (E) rsp-6 isoform transcript structure. Isoform a encodes a functional protein. (F) RNA-seq and qPCR data indicate that raga-1 is required for feeding-induced alternative mRNA splicing events that increase levels of the functional rsp-6a isoform. Two-sided unpaired t-test, mean ± SEM, *P < 0.05, **P < 0.01. Three biological replicates were performed.

Figure 4.. Partial rescue of mTORC1-dependent phenotypes by RSP-6 overexpression

(A) Schematic of transgenic RSP-6::GFP proteins. (B) Developmental stage of initially synchronized animals at 72 hours after treatment with RNAi against raga-1 (RAGA), ragc-1 (RAGC), rheb-1 (RHEB) or daf-15 (Raptor). In all such experiments RSP-6 OE is compared to rol-6 transgenic marker control. N > 25, chi-square-test. ***P < 0.001, n.s.= not significant. Three biological replicates were performed. (C) Representative images illustrating body length after 72 hours of treatment with RNAi against raga-1, ragc-1, rheb-1 or daf-15. Scale bars: 100μm. Three biological replicates were performed. (D) Quantification of (C). Two-way ANOVA with Tukey’s correction, *P <0.05, **P <0.01, ***P <0.001, n.s.= not significant. ###P < 0.001 compared to rol-6 control animals with EV. (E) Number of live progeny after treatment with RNAi against raga-1, ragc-1, rheb-1 or daf-15. Two-way ANOVA with Tukey’s correction *P < 0.05, **P < 0.01, ***P < 0.001, n.s.= not significant. Three biological replicates were performed. (F) Representative images illustrating body length after 72 hours of treatment with RNAi against raga-1. Scale bars: 100μm. Three biological replicates were performed. (G) Quantification of (F). Two-way ANOVA with Tukey’s correction, *P <0.05, **P <0.01, ***P <0.001, n.s.= not significant. ###P < 0.001 compared to rol-6 control animals with EV. (H) Number of live progeny after treatment with RNAi against raga-1. Two-way ANOVA with Tukey’s correction *P < 0.05, **P < 0.01, ***P < 0.001, n.s.= not significant. Three biological replicates were performed. (I) Relative expression of SR protein gene expression, detected by qPCR. Two-way ANOVA with Tukey’s correction, ***P < 0.001, #P < 0.05, ##P < 0.01, ###P < 0.001. Controls are rol-6 transgenic vector animals and EV RNAi. Three biological replicates were performed. (J) Semi-quantitative PCR analysis indicating that RSP-6 OE overcomes the effects of reduced mTORC1 activity on the rsp-4 coding transcript. Two-sided unpaired t-test, mean ± SEM, **P < 0.01, ***P < 0.001. Three biological replicates were performed.

Figure 5.. Feeding remodels metabolism through mTORC1 regulation of mRNA expression and splicing

(A) Scheme of metabolomics experiment. (B, C) Volcano plots of metabolites regulated by (B) feeding (Fed EV vs Stv EV) or (C) mTORC1 (Fed EV vs Fed raga-1 RNAi). Up- and down-regulated metabolites are indicated in red and blue, respectively, with gray showing metabolites with no significant difference. Total metabolites detected are indicated in parentheses. The horizontal dotted line indicates a P-value cutoff at 0.05. two-sided unpaired t-test. Three biological replicates were performed. (D) Venn diagram comparing feeding- and mTORC1-upregulated metabolites. (E) Heatmap showing relative levels of metabolites that were upregulated by both feeding and mTORC1 (raga-1-dependent). P-values are shown in the right panel, with a P-value cutoff of 0.05. two-sided unpaired t-test. Asterisks indicate metabolites that were previously detected as mTORC1-regulated in mammalian cell studies^–. (F) Metabolic pathway diagram illustrating regulation of metabolite levels, mRNA expression, and alternative mRNA isoform use by feeding and mTORC1. Asterisks indicate genes where mTORC1 and feeding increases expression of a functional protein-coding isoform. Red arrows indicate metabolic pathways where the direction of biosynthesis correlates with mTORC-1 (raga-1) increasing the levels of both the protein coding mRNA isoform and the metabolite immediately downstream. (G-I) Scatter plots comparing the effects of feeding with raga-1 or rsks-1 mutation on the expression of individual mRNA isoforms at genes in the indicated metabolic pathways. Only genes that encode two or more isoforms were included. X-axis; alteration of isoform expression by feeding (WT Fed/WT Stv), Y-axis; alteration of isoform expression by raga-1 or rsks-1 (WT Fed/raga-1 Fed or WT Fed/rsks-1 Fed). Spearman correlation coefficients (R) and associated P-values are shown in colors corresponding to the data plots. Points were excluded as outliers if their Z-score exceeded ±3 units. For evaluating the statistical significance of the difference in correlations, Fisher’s z-transformation was applied based on correlation coefficients. (J) Schematic of atic-1 isoforms. The bifunctional protein ATIC-1 (5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) formyltransferase/inosine monophosphate (IMP) cyclohydrolase) mediates IMP synthesis from AICAR, connecting the pentose phosphate and purine biosynthesis pathways (F). Isoform b encodes a functional protein. (K) Expression levels of atic-1 and mRNA isoforms are plotted from RNAseq data. two-sided unpaired t-test, mean ± SEM, *P < 0.05, **P < 0.01.

Figure 6.. Importance of mTORC1-mediated mRNA splicing regulation in metabolism and longevity.

(A and B) Volcano plot showing effects of RSP-6 OE on steady-state metabolite levels in (A) EV control and (B) raga-1 RNAi animals, compared to rol-6 transgene control. The horizontal dotted line indicates a P value cutoff of 0.05. Significantly up- and down-regulated metabolites are indicated by red and blue dots, respectively. Two-sided unpaired t-test. Three biological replicates were performed. (C) Heatmap showing levels of metabolites that were significantly decreased by raga-1 RNAi compared to EV, and rescued by RSP-6 OE in raga-1 RNAi animals (RSP-6 OE raga-1 RNAi/control raga-1 RNAi). P values are shown in the right panel; with a cutoff of 0.05. Two-sided unpaired t-test. (D) Comparison of metabolites that were upregulated under the indicated conditions. (E-H) qPCR analysis showing effects of raga-1 RNAi and RSP-6 OE on overall expression of selected genes in the glycolysis, pentose phosphate, and purine and pyrimidine metabolism pathways. Mean ± SEM, *P < 0.05, **P < 0.01. #P < 0.05, ##P < 0.01, compared to rol-6 transgenic control animals with EV RNAi. Two-sided unpaired t-test. Three biological replicates were performed. (I-K) Relative expression of individual mRNA isoforms at the indicated genes, assayed and illustrated as in (E-H). Mean ± SEM, **P < 0.01, ***P < 0.001. #P < 0.05, ##P < 0.01, compared to rol-6 transgenic control animals with EV RNAi. Two-sided unpaired t-test. Three biological replicates were performed. (L-N) Lifespan analysis of the indicated genotypes at 20°C, measured from day 1 of adulthood. Genetic mutants are shown in (L), with RNAi experiments shown in (M, N). A composite of 3 or 4 independent biological replicates is shown, with complete data presented in Table S6. Log-rank test, *P < 0.05, **P < 0.01, ***P < 0.001, n.s.= not significant.

Figure 7.. Conservation of mTORC1 gene regulation functions during human cell growth.

(A) Schematic of the regulatory pathways involved in mRNA splicing mediated by mTORC1, shown with inhibitors targeting its components. (B) Heatmap showing 9357 differentially expressed genes affected by Rapamycin (Rapa), Torin1, the S6K inhibitor PF4708671 (PF), the SRPK1 inhibitor SRPIN340 (SRPIN), or SRPK2 knockout (SRPK2 KO) in LAM cells. FDR < 0.05. Three biological replicates were performed per condition. (C) Effects of the indicated treatments on mRNA splicing events in LAM cells. SE; Skipped exon, RI; Retained intron, MX; Mutually exclusive exons, A3SS; Alternative 3’ splice sites, A5SS; Alternative 5’ splice sites. FDR < 0.05. (D) Processes that were enriched among the 2562 genes for which alternative mRNA transcript use was affected by mTORC1 activity (control/Torin1). (E) mTORC1 increases representation of protein-coding mRNA transcripts. Fold change of Torin1 effects is shown. The bar chart represents the number of protein-coding or non-coding (retained intron) transcripts that were affected. Two-sided chi-square test. ***P < 0.001. (F) Heatmap showing the effects of 4h feeding and Torin1 on gene expression in HEK 293E cells. FDR < 0.05. Three biological replicates were performed per condition. (G) GO terms for genes that are feeding-upregulated and mTORC1-dependent. (H) mTORC1-dependence of mRNA splicing protein gene expression. Two-sided unpaired t-test, mean ± SEM, **P < 0.01, ***P < 0.001. (I) mTORC1 dependence of feeding-induced alternative splicing events. FDR < 0.05. (J) GO terms for feeding-induced alternative splicing events that are mTORC1-dependent. (K) Effects of Torin1 on ATIC total expression and mRNA transcript levels. Two-sided unpaired t-test, mean ± SEM, *P < 0.05. (L) Model for nutrient-responsive mTORC1 regulation of gene expression and alternative mRNA splicing, and its effects on growth, metabolism, and longevity.