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TIF-IA-dependent regulation of ribosome synthesis in drosophila muscle is required to maintain systemic insulin signaling and larval growth.

Ghosh A, Rideout EJ, Grewal SS - PLoS Genet. (2014)

Bottom Line: When we mimic this decrease in muscle ribosome synthesis using RNAi-mediated knockdown of TIF-IA, we observe delayed larval development and reduced body growth.This reduction in growth is caused by lowered systemic insulin signaling via two endocrine responses: reduced expression of Drosophila insulin-like peptides (dILPs) from the brain and increased expression of Imp-L2-a secreted factor that binds and inhibits dILP activity-from muscle.Finally, we show that activation of TOR specifically in muscle can increase overall body size and this effect requires TIF-IA function.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, and Clark H. Smith Brain Tumour Centre, Southern Alberta Cancer Research Institute, University of Calgary, Health Research Innovation Center, Calgary, Alberta, Canada.

ABSTRACT
The conserved TOR kinase signaling network links nutrient availability to cell, tissue and body growth in animals. One important growth-regulatory target of TOR signaling is ribosome biogenesis. Studies in yeast and mammalian cell culture have described how TOR controls rRNA synthesis-a limiting step in ribosome biogenesis-via the RNA Polymerase I transcription factor TIF-IA. However, the contribution of TOR-dependent ribosome synthesis to tissue and body growth in animals is less clear. Here we show in Drosophila larvae that ribosome synthesis in muscle is required non-autonomously to maintain normal body growth and development. We find that amino acid starvation and TOR inhibition lead to reduced levels of TIF-IA, and decreased rRNA synthesis in larval muscle. When we mimic this decrease in muscle ribosome synthesis using RNAi-mediated knockdown of TIF-IA, we observe delayed larval development and reduced body growth. This reduction in growth is caused by lowered systemic insulin signaling via two endocrine responses: reduced expression of Drosophila insulin-like peptides (dILPs) from the brain and increased expression of Imp-L2-a secreted factor that binds and inhibits dILP activity-from muscle. We also observed that maintaining TIF-IA levels in muscle could partially reverse the starvation-mediated suppression of systemic insulin signaling. Finally, we show that activation of TOR specifically in muscle can increase overall body size and this effect requires TIF-IA function. These data suggest that muscle ribosome synthesis functions as a nutrient-dependent checkpoint for overall body growth: in nutrient rich conditions, TOR is required to maintain levels of TIF-IA and ribosome synthesis to promote high levels of systemic insulin, but under conditions of starvation stress, reduced muscle ribosome synthesis triggers an endocrine response that limits systemic insulin signaling to restrict growth and maintain homeostasis.

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Nutrition-TOR signaling maintains TIF-IA mRNA and protein levels in larvae.(A) Immunoblot indicates TIF-IA protein levels were reduced in 24 hr starved larvae compared to fed larvae. (B) Immunoblot indicates TIF-IA protein levels were reduced in torΔP larvae compared to wild-type (WT) larvae, at 72 hr AEL. (C) Immunoblot indicates TIF-IA protein levels were unchanged between WT and s6k  (s6kL1) larvae, at 72 hr AEL. In all immunoblots, β tubulin levels indicate loading control. (D) qPCR indicates TIF-IA mRNA levels were reduced in 24 hr starved larvae compared to fed larvae. Data normalized to β tubulin. (*P = 3.46×10−6, Student's t-test). (E) qPCR indicates pre-rRNA levels were reduced in 24 hr starved larvae compared to fed larvae, at 72 hr AEL. Data normalized to β tubulin. (*P = 4.47×10−5, Student's t-test). (F) qPCR indicates TIF-IA mRNA levels were reduced in tor larvae compared to yw (control) larvae, at 72 hr AEL. Data normalized to actin. (*P = 0.01, Student's t-test). (G) qPCR indicates pre-rRNA levels were reduced in torΔP larvae compared to yw (control) larvae, at 72 hr AEL. Data normalized to actin. (*P = 0.04, Student's t-test). All error bars indicate SEM.
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pgen-1004750-g001: Nutrition-TOR signaling maintains TIF-IA mRNA and protein levels in larvae.(A) Immunoblot indicates TIF-IA protein levels were reduced in 24 hr starved larvae compared to fed larvae. (B) Immunoblot indicates TIF-IA protein levels were reduced in torΔP larvae compared to wild-type (WT) larvae, at 72 hr AEL. (C) Immunoblot indicates TIF-IA protein levels were unchanged between WT and s6k (s6kL1) larvae, at 72 hr AEL. In all immunoblots, β tubulin levels indicate loading control. (D) qPCR indicates TIF-IA mRNA levels were reduced in 24 hr starved larvae compared to fed larvae. Data normalized to β tubulin. (*P = 3.46×10−6, Student's t-test). (E) qPCR indicates pre-rRNA levels were reduced in 24 hr starved larvae compared to fed larvae, at 72 hr AEL. Data normalized to β tubulin. (*P = 4.47×10−5, Student's t-test). (F) qPCR indicates TIF-IA mRNA levels were reduced in tor larvae compared to yw (control) larvae, at 72 hr AEL. Data normalized to actin. (*P = 0.01, Student's t-test). (G) qPCR indicates pre-rRNA levels were reduced in torΔP larvae compared to yw (control) larvae, at 72 hr AEL. Data normalized to actin. (*P = 0.04, Student's t-test). All error bars indicate SEM.

Mentions: In previous work, we showed that the nutrient/TOR pathway controls rRNA synthesis in developing larvae and that TOR signaling promotes TIF-IA recruitment to rDNA genes [6]. Here, we examined whether TOR signaling may function by controlling TIF-IA levels. Deprivation of dietary protein leads to reduced TOR signaling and decreased rRNA synthesis in larvae. We found that under protein starvation conditions (induced by transferring larvae to a sucrose-only diet), TIF-IA protein levels were reduced compared to fully fed controls (Figure 1A). We also found that TIF-IA protein levels were also reduced in tor mutant (torΔP) larvae compared to wild-type controls (Figure 1B). TOR can promote growth in part via its downstream effector kinase, ribosomal protein S6 kinase (S6K) [31]. However, we found that that TIF-IA protein levels were unaltered in s6k mutant (s6kL1) larvae compared to wild-type (Figure 1C). These results prompted us to examine TIF-IA mRNA levels. We found that both starved larvae and torΔP mutant larvae had reduced levels of both TIF-IA mRNA (Figure 1D, F) and pre-rRNA (Figure 1E, G) consistent with a reduction in synthesis of rRNA and hence ribosomes. Thus, during larval development nutrient/TOR signaling is required to maintain appropriate levels of TIF-IA mRNA and protein.


TIF-IA-dependent regulation of ribosome synthesis in drosophila muscle is required to maintain systemic insulin signaling and larval growth.

Ghosh A, Rideout EJ, Grewal SS - PLoS Genet. (2014)

Nutrition-TOR signaling maintains TIF-IA mRNA and protein levels in larvae.(A) Immunoblot indicates TIF-IA protein levels were reduced in 24 hr starved larvae compared to fed larvae. (B) Immunoblot indicates TIF-IA protein levels were reduced in torΔP larvae compared to wild-type (WT) larvae, at 72 hr AEL. (C) Immunoblot indicates TIF-IA protein levels were unchanged between WT and s6k  (s6kL1) larvae, at 72 hr AEL. In all immunoblots, β tubulin levels indicate loading control. (D) qPCR indicates TIF-IA mRNA levels were reduced in 24 hr starved larvae compared to fed larvae. Data normalized to β tubulin. (*P = 3.46×10−6, Student's t-test). (E) qPCR indicates pre-rRNA levels were reduced in 24 hr starved larvae compared to fed larvae, at 72 hr AEL. Data normalized to β tubulin. (*P = 4.47×10−5, Student's t-test). (F) qPCR indicates TIF-IA mRNA levels were reduced in tor larvae compared to yw (control) larvae, at 72 hr AEL. Data normalized to actin. (*P = 0.01, Student's t-test). (G) qPCR indicates pre-rRNA levels were reduced in torΔP larvae compared to yw (control) larvae, at 72 hr AEL. Data normalized to actin. (*P = 0.04, Student's t-test). All error bars indicate SEM.
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pgen-1004750-g001: Nutrition-TOR signaling maintains TIF-IA mRNA and protein levels in larvae.(A) Immunoblot indicates TIF-IA protein levels were reduced in 24 hr starved larvae compared to fed larvae. (B) Immunoblot indicates TIF-IA protein levels were reduced in torΔP larvae compared to wild-type (WT) larvae, at 72 hr AEL. (C) Immunoblot indicates TIF-IA protein levels were unchanged between WT and s6k (s6kL1) larvae, at 72 hr AEL. In all immunoblots, β tubulin levels indicate loading control. (D) qPCR indicates TIF-IA mRNA levels were reduced in 24 hr starved larvae compared to fed larvae. Data normalized to β tubulin. (*P = 3.46×10−6, Student's t-test). (E) qPCR indicates pre-rRNA levels were reduced in 24 hr starved larvae compared to fed larvae, at 72 hr AEL. Data normalized to β tubulin. (*P = 4.47×10−5, Student's t-test). (F) qPCR indicates TIF-IA mRNA levels were reduced in tor larvae compared to yw (control) larvae, at 72 hr AEL. Data normalized to actin. (*P = 0.01, Student's t-test). (G) qPCR indicates pre-rRNA levels were reduced in torΔP larvae compared to yw (control) larvae, at 72 hr AEL. Data normalized to actin. (*P = 0.04, Student's t-test). All error bars indicate SEM.
Mentions: In previous work, we showed that the nutrient/TOR pathway controls rRNA synthesis in developing larvae and that TOR signaling promotes TIF-IA recruitment to rDNA genes [6]. Here, we examined whether TOR signaling may function by controlling TIF-IA levels. Deprivation of dietary protein leads to reduced TOR signaling and decreased rRNA synthesis in larvae. We found that under protein starvation conditions (induced by transferring larvae to a sucrose-only diet), TIF-IA protein levels were reduced compared to fully fed controls (Figure 1A). We also found that TIF-IA protein levels were also reduced in tor mutant (torΔP) larvae compared to wild-type controls (Figure 1B). TOR can promote growth in part via its downstream effector kinase, ribosomal protein S6 kinase (S6K) [31]. However, we found that that TIF-IA protein levels were unaltered in s6k mutant (s6kL1) larvae compared to wild-type (Figure 1C). These results prompted us to examine TIF-IA mRNA levels. We found that both starved larvae and torΔP mutant larvae had reduced levels of both TIF-IA mRNA (Figure 1D, F) and pre-rRNA (Figure 1E, G) consistent with a reduction in synthesis of rRNA and hence ribosomes. Thus, during larval development nutrient/TOR signaling is required to maintain appropriate levels of TIF-IA mRNA and protein.

Bottom Line: When we mimic this decrease in muscle ribosome synthesis using RNAi-mediated knockdown of TIF-IA, we observe delayed larval development and reduced body growth.This reduction in growth is caused by lowered systemic insulin signaling via two endocrine responses: reduced expression of Drosophila insulin-like peptides (dILPs) from the brain and increased expression of Imp-L2-a secreted factor that binds and inhibits dILP activity-from muscle.Finally, we show that activation of TOR specifically in muscle can increase overall body size and this effect requires TIF-IA function.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, and Clark H. Smith Brain Tumour Centre, Southern Alberta Cancer Research Institute, University of Calgary, Health Research Innovation Center, Calgary, Alberta, Canada.

ABSTRACT
The conserved TOR kinase signaling network links nutrient availability to cell, tissue and body growth in animals. One important growth-regulatory target of TOR signaling is ribosome biogenesis. Studies in yeast and mammalian cell culture have described how TOR controls rRNA synthesis-a limiting step in ribosome biogenesis-via the RNA Polymerase I transcription factor TIF-IA. However, the contribution of TOR-dependent ribosome synthesis to tissue and body growth in animals is less clear. Here we show in Drosophila larvae that ribosome synthesis in muscle is required non-autonomously to maintain normal body growth and development. We find that amino acid starvation and TOR inhibition lead to reduced levels of TIF-IA, and decreased rRNA synthesis in larval muscle. When we mimic this decrease in muscle ribosome synthesis using RNAi-mediated knockdown of TIF-IA, we observe delayed larval development and reduced body growth. This reduction in growth is caused by lowered systemic insulin signaling via two endocrine responses: reduced expression of Drosophila insulin-like peptides (dILPs) from the brain and increased expression of Imp-L2-a secreted factor that binds and inhibits dILP activity-from muscle. We also observed that maintaining TIF-IA levels in muscle could partially reverse the starvation-mediated suppression of systemic insulin signaling. Finally, we show that activation of TOR specifically in muscle can increase overall body size and this effect requires TIF-IA function. These data suggest that muscle ribosome synthesis functions as a nutrient-dependent checkpoint for overall body growth: in nutrient rich conditions, TOR is required to maintain levels of TIF-IA and ribosome synthesis to promote high levels of systemic insulin, but under conditions of starvation stress, reduced muscle ribosome synthesis triggers an endocrine response that limits systemic insulin signaling to restrict growth and maintain homeostasis.

Show MeSH
Related in: MedlinePlus