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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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TIF-IA function is required in muscle to maintain overall body growth and development.(A) Immunoblot indicates TIF-IA protein levels were reduced in 24 hr starved muscle compared to fed muscle, at 72 hr AEL. β tubulin levels indicate loading control. (B) qPCR indicates TIF-IA mRNA levels were reduced in 24 hr starved muscle compared to fed muscle, at 72 hr AEL. Data normalized to β tubulin. (*P = 0.009, Student's t-test). (C) qPCR indicates pre-rRNA levels were reduced in 24 hr starved muscle compared to fed muscle, at 72 hr AEL. Data normalized to β tubulin. (*P = 0.0059, Student's t-test). (D) qPCR indicates TIF-IA mRNA levels were reduced in dMef2>TIF-IA-IR muscle compared to control larval muscle (dMef2>+), at 72 hr AEL. Data normalized to β tubulin. (*P = 0.0022, Student's t-test). (E) qPCR indicates pre-rRNA levels were reduced in dMef2>TIF-IA-IR muscle compared to dMef2>+ (control) larval muscle, at 72 hr AEL. Data normalized to β tubulin. (*P = 0.015, Student's t-test). (F) Developmental timing from larval hatching to pupation of dMef2>+ and dMef2>TIF-IA IR animals, n = 134, n - number of larvae assessed per genotype, (mean time to pupation: dMef2>+, 6.1 days and dMef2>TIF-IA, 7.8 days, *P<0.05, Mann-Whitney U test). (G) Representative images of dMef2>+ (top) and dMef2>TIF-IA IR (bottom) larvae. Numbers at the bottom of the panel indicates hours AEL. (H) Representative images of dMef2>+ and dMef2>TIF-IA IR pupae, scale bar-200 µm. All error bars indicate SEM.
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pgen-1004750-g002: TIF-IA function is required in muscle to maintain overall body growth and development.(A) Immunoblot indicates TIF-IA protein levels were reduced in 24 hr starved muscle compared to fed muscle, at 72 hr AEL. β tubulin levels indicate loading control. (B) qPCR indicates TIF-IA mRNA levels were reduced in 24 hr starved muscle compared to fed muscle, at 72 hr AEL. Data normalized to β tubulin. (*P = 0.009, Student's t-test). (C) qPCR indicates pre-rRNA levels were reduced in 24 hr starved muscle compared to fed muscle, at 72 hr AEL. Data normalized to β tubulin. (*P = 0.0059, Student's t-test). (D) qPCR indicates TIF-IA mRNA levels were reduced in dMef2>TIF-IA-IR muscle compared to control larval muscle (dMef2>+), at 72 hr AEL. Data normalized to β tubulin. (*P = 0.0022, Student's t-test). (E) qPCR indicates pre-rRNA levels were reduced in dMef2>TIF-IA-IR muscle compared to dMef2>+ (control) larval muscle, at 72 hr AEL. Data normalized to β tubulin. (*P = 0.015, Student's t-test). (F) Developmental timing from larval hatching to pupation of dMef2>+ and dMef2>TIF-IA IR animals, n = 134, n - number of larvae assessed per genotype, (mean time to pupation: dMef2>+, 6.1 days and dMef2>TIF-IA, 7.8 days, *P<0.05, Mann-Whitney U test). (G) Representative images of dMef2>+ (top) and dMef2>TIF-IA IR (bottom) larvae. Numbers at the bottom of the panel indicates hours AEL. (H) Representative images of dMef2>+ and dMef2>TIF-IA IR pupae, scale bar-200 µm. All error bars indicate SEM.

Mentions: As well as controlling cell-autonomous growth, TOR activity in specific larval tissues is required for overall body growth in Drosophila. For example, reduced TOR signaling in larval muscle [28] and fat [25], [26] leads to reduced body growth. Given the importance of ribosome synthesis as an effector of TOR in the control of cell-autonomous growth, we examined whether TIF-IA-dependent ribosome synthesis could also exert non-autonomous effects on body growth. We first examined larval muscle. As with whole larvae, we found that protein starvation decreased both TIF-IA protein (Figure 2A) and mRNA (Figure 2B), and also pre-rRNA (Figure 2C) in larval muscle. To explore the consequence of this reduction in TIF-IA levels, we examined the effects of RNAi-mediated knockdown of TIF-IA in muscle, using a UAS-TIF-IA inverted repeat (IR) line. Ubiquitous expression of this TIF-IA IR line in larvae using the daughterless (da)-GAL4 driver (da>TIFIA-IR) phenocopied tif-ia mutants, and led to reduced TIF-IA protein levels (Figure S1B) and larval growth arrest (Figure S1A). Both of these effects were fully reversed by co-expression of a UAS-TIF-IA transgene (Figure S1A), confirming the specificity of the UAS-TIF-IA IR line. We then used the UAS-TIF-IA IR line to knock down TIF-IA specifically in muscle (using the dMef2-GAL4 driver – Figure S2). We found that RNAi-mediated knockdown of TIF-IA muscle mimicked the decrease in both TIF-IA mRNA (Figure 2D) and pre-rRNA (Figure 2E) levels following starvation. When we monitored larval growth and development, we observed that dMef2>TIF-IA IR larvae were smaller than age-matched control larvae (Figure 2G). Moreover, dMef2>TIF-IA IR larvae were significantly delayed in pupal development with respect to control (dMef2>+) larvae (Figure 2F), and only approximately 20% of dMef2>TIF-IA IR larvae formed pupae. These dMef2>TIF-IA IR pupae were malformed compared to control (dMef2>+) pupae (Figure 2H). We examined feeding by transferring dMef2>+ (control) and dMef2>TIF-IA IR larvae onto yeast paste colored with blue food dye. After 4 hours, we observed that both the control and TIF-IA IR larvae contained blue food in their guts (Figure S3), suggesting that knockdown of TIF-IA in larval muscle did not impair feeding. Together, these findings indicated that TIF-IA-dependent ribosome synthesis in muscle is required to maintain normal body growth and development.


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)

TIF-IA function is required in muscle to maintain overall body growth and development.(A) Immunoblot indicates TIF-IA protein levels were reduced in 24 hr starved muscle compared to fed muscle, at 72 hr AEL. β tubulin levels indicate loading control. (B) qPCR indicates TIF-IA mRNA levels were reduced in 24 hr starved muscle compared to fed muscle, at 72 hr AEL. Data normalized to β tubulin. (*P = 0.009, Student's t-test). (C) qPCR indicates pre-rRNA levels were reduced in 24 hr starved muscle compared to fed muscle, at 72 hr AEL. Data normalized to β tubulin. (*P = 0.0059, Student's t-test). (D) qPCR indicates TIF-IA mRNA levels were reduced in dMef2>TIF-IA-IR muscle compared to control larval muscle (dMef2>+), at 72 hr AEL. Data normalized to β tubulin. (*P = 0.0022, Student's t-test). (E) qPCR indicates pre-rRNA levels were reduced in dMef2>TIF-IA-IR muscle compared to dMef2>+ (control) larval muscle, at 72 hr AEL. Data normalized to β tubulin. (*P = 0.015, Student's t-test). (F) Developmental timing from larval hatching to pupation of dMef2>+ and dMef2>TIF-IA IR animals, n = 134, n - number of larvae assessed per genotype, (mean time to pupation: dMef2>+, 6.1 days and dMef2>TIF-IA, 7.8 days, *P<0.05, Mann-Whitney U test). (G) Representative images of dMef2>+ (top) and dMef2>TIF-IA IR (bottom) larvae. Numbers at the bottom of the panel indicates hours AEL. (H) Representative images of dMef2>+ and dMef2>TIF-IA IR pupae, scale bar-200 µm. All error bars indicate SEM.
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pgen-1004750-g002: TIF-IA function is required in muscle to maintain overall body growth and development.(A) Immunoblot indicates TIF-IA protein levels were reduced in 24 hr starved muscle compared to fed muscle, at 72 hr AEL. β tubulin levels indicate loading control. (B) qPCR indicates TIF-IA mRNA levels were reduced in 24 hr starved muscle compared to fed muscle, at 72 hr AEL. Data normalized to β tubulin. (*P = 0.009, Student's t-test). (C) qPCR indicates pre-rRNA levels were reduced in 24 hr starved muscle compared to fed muscle, at 72 hr AEL. Data normalized to β tubulin. (*P = 0.0059, Student's t-test). (D) qPCR indicates TIF-IA mRNA levels were reduced in dMef2>TIF-IA-IR muscle compared to control larval muscle (dMef2>+), at 72 hr AEL. Data normalized to β tubulin. (*P = 0.0022, Student's t-test). (E) qPCR indicates pre-rRNA levels were reduced in dMef2>TIF-IA-IR muscle compared to dMef2>+ (control) larval muscle, at 72 hr AEL. Data normalized to β tubulin. (*P = 0.015, Student's t-test). (F) Developmental timing from larval hatching to pupation of dMef2>+ and dMef2>TIF-IA IR animals, n = 134, n - number of larvae assessed per genotype, (mean time to pupation: dMef2>+, 6.1 days and dMef2>TIF-IA, 7.8 days, *P<0.05, Mann-Whitney U test). (G) Representative images of dMef2>+ (top) and dMef2>TIF-IA IR (bottom) larvae. Numbers at the bottom of the panel indicates hours AEL. (H) Representative images of dMef2>+ and dMef2>TIF-IA IR pupae, scale bar-200 µm. All error bars indicate SEM.
Mentions: As well as controlling cell-autonomous growth, TOR activity in specific larval tissues is required for overall body growth in Drosophila. For example, reduced TOR signaling in larval muscle [28] and fat [25], [26] leads to reduced body growth. Given the importance of ribosome synthesis as an effector of TOR in the control of cell-autonomous growth, we examined whether TIF-IA-dependent ribosome synthesis could also exert non-autonomous effects on body growth. We first examined larval muscle. As with whole larvae, we found that protein starvation decreased both TIF-IA protein (Figure 2A) and mRNA (Figure 2B), and also pre-rRNA (Figure 2C) in larval muscle. To explore the consequence of this reduction in TIF-IA levels, we examined the effects of RNAi-mediated knockdown of TIF-IA in muscle, using a UAS-TIF-IA inverted repeat (IR) line. Ubiquitous expression of this TIF-IA IR line in larvae using the daughterless (da)-GAL4 driver (da>TIFIA-IR) phenocopied tif-ia mutants, and led to reduced TIF-IA protein levels (Figure S1B) and larval growth arrest (Figure S1A). Both of these effects were fully reversed by co-expression of a UAS-TIF-IA transgene (Figure S1A), confirming the specificity of the UAS-TIF-IA IR line. We then used the UAS-TIF-IA IR line to knock down TIF-IA specifically in muscle (using the dMef2-GAL4 driver – Figure S2). We found that RNAi-mediated knockdown of TIF-IA muscle mimicked the decrease in both TIF-IA mRNA (Figure 2D) and pre-rRNA (Figure 2E) levels following starvation. When we monitored larval growth and development, we observed that dMef2>TIF-IA IR larvae were smaller than age-matched control larvae (Figure 2G). Moreover, dMef2>TIF-IA IR larvae were significantly delayed in pupal development with respect to control (dMef2>+) larvae (Figure 2F), and only approximately 20% of dMef2>TIF-IA IR larvae formed pupae. These dMef2>TIF-IA IR pupae were malformed compared to control (dMef2>+) pupae (Figure 2H). We examined feeding by transferring dMef2>+ (control) and dMef2>TIF-IA IR larvae onto yeast paste colored with blue food dye. After 4 hours, we observed that both the control and TIF-IA IR larvae contained blue food in their guts (Figure S3), suggesting that knockdown of TIF-IA in larval muscle did not impair feeding. Together, these findings indicated that TIF-IA-dependent ribosome synthesis in muscle is required to maintain normal body growth and development.

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