Examining How Ribosomal Proteins Affect Growth And Body Size

Adequate amounts of nutrients are important for growth and development. Ingested nutrients are used for processes that meet the metabolic needs of the body allowing for growth and development. An important aspect of growth is ribosome biogenesis. The ribosome is the molecular machine that is responsible for protein synthesis, hence growth. However, what still remains unclear is the contribution of rRNA and ribosomal proteins to tissue and body growth in developing animals. The aim of this project was to study the role of certain ribosomal proteins in growth and development using Drosophila. Drosophila is a powerful genetic model system to study cell and organismal responses to growth cues. Over a 4-day period, Drosophila larvae can grow over 200 fold in mass in response to nutrient availability. For this project a class of Drosophila mutants known as Minutes were studied. These flies harbored mutations in different ribosomal proteins. These flies are lethal as homozygous mutants, however as heterozygotes with reduced levels of ribosome and protein synthesis, they have a characteristic slow rate of development.  The experiments conducted showed that mutations in certain ribosomal proteins result in no change or an increase in overall body size of Drosophila pupae in comparison to control. The results showed a link to delayed development to the pupal stage, suggesting an explanation to the increased body size. These results also showed a possible link of some ribosomal proteins functioning as tumor suppressors where suppressed expression results in increased overall body growth. These findings showed a striking contrast to the hypothesized decrease in overall growth from decreased ribosome synthesis, opening the door to numerous questions regarding the molecular mechanism of ribosomal proteins.

Chewing the Fat Regulating Autophagy in Drosophila

AbstractAutophagy is the major cellular process responsible for bulk cytoplasmic degradation. Two reports in this issue of Developmental Cell describe how both PI3 kinase and TOR signaling in Drosophila are critical for controlling autophagy in response to developmental and environmental cues

Nutritional control of gene expression in Drosophila larvae via TOR, Myc and a novel cis-regulatory element

<p>Abstract</p> <p>Background</p> <p>Nutrient availability is a key determinant of eukaryotic cell growth. In unicellular organisms many signaling and transcriptional networks link nutrient availability to the expression of metabolic genes required for growth. However, less is known about the corresponding mechanisms that operate in metazoans. We used gene expression profiling to explore this issue in developing <it>Drosophila </it>larvae.</p> <p>Results</p> <p>We found that starvation for dietary amino acids (AA's) leads to dynamic changes in transcript levels of many metabolic genes. The conserved insulin/PI3K and TOR signaling pathways mediate nutrition-dependent growth in <it>Drosophila </it>and other animals. We found that many AA starvation-responsive transcripts were also altered in TOR mutants. In contrast, although PI3K overexpression induced robust changes in the expression of many metabolic genes, these changes showed limited overlap with the AA starvation expression profile. We did however identify a strong overlap between genes regulated by the transcription factor, Myc, and AA starvation-responsive genes, particularly those involved in ribosome biogenesis, protein synthesis and mitochondrial function. The consensus Myc DNA binding site is enriched in promoters of these AA starvation genes, and we found that Myc overexpression could bypass dietary AA to induce expression of these genes. We also identified another sequence motif (Motif 1) enriched in the promoters of AA starvation-responsive genes. We showed that Motif 1 was both necessary and sufficient to mediate transcriptional responses to dietary AA in larvae.</p> <p>Conclusions</p> <p>Our data suggest that many of the transcriptional effects of amino acids are mediated via signaling through the TOR pathway in <it>Drosophila </it>larvae. We also find that these transcriptional effects are mediated through at least two mechanisms: via the transcription factor Myc, and via the Motif 1 cis-regulatory element. These studies begin to elucidate a nutrient-responsive signaling network that controls metabolic gene transcription in <it>Drosophila</it>.</p

Drosophila TIF-IA is required for ribosome synthesis and cell growth and is regulated by the TOR pathway

Synthesis of ribosomal RNA (rRNA) is a key step in ribosome biogenesis and is essential for cell growth. Few studies, however, have investigated rRNA synthesis regulation in vivo in multicellular organisms. Here, we present a genetic analysis of transcription initiation factor IA (TIF-IA), a conserved RNA polymerase I transcription factor. Drosophila melanogaster Tif-IA−/− mutants have reduced levels of rRNA synthesis and sustain a developmental arrest caused by a block in cellular growth. We find that the target of rapamycin (TOR) pathway regulates TIF-IA recruitment to rDNA. Furthermore, we show that the TOR pathway regulates rRNA synthesis in vivo and that TIF-IA overexpression can maintain rRNA transcription when TOR activity is reduced in developing larvae. We propose that TIF-IA acts in vivo as a downstream growth–regulatory target of the TOR pathway. Overexpression of TIF-IA also elevates levels of both 5S RNA and messenger RNAs encoding ribosomal proteins. Stimulation of rRNA synthesis by TIF-IA may therefore provide a feed-forward mechanism to coregulate the levels of other ribosome components

Rheb-TOR signaling promotes protein synthesis, but not glucose or amino acid import, in Drosophila

BACKGROUND: The Ras-related GTPase, Rheb, regulates the growth of animal cells. Genetic and biochemical tests place Rheb upstream of the target of rapamycin (TOR) protein kinase, and downstream of the tuberous sclerosis complex (TSC1/TSC2) and the insulin-signaling pathway. TOR activity is regulated by nutritional cues, suggesting that Rheb might either control, or respond to, nutrient availability. RESULTS: We show that Rheb and TOR do not promote the import of glucose, bulk amino acids, or arginine in Drosophila S2 cells, but that both gene products are important regulators of ribosome biogenesis, protein synthesis, and cell size. S2 cell size, protein synthesis, and glucose import were largely insensitive to manipulations of insulin signaling components, suggesting that cellular energy levels and TOR activity can be maintained through insulin/PI3K-independent mechanisms in S2 cell culture. In vivo in Drosophila larvae, however, we found that insulin signaling can regulate protein synthesis, and thus may affect TOR activity. CONCLUSION: Rheb-TOR signaling controls S2 cell growth by promoting ribosome production and protein synthesis, but apparently not by direct effects on the import of amino acids or glucose. The effect of insulin signaling upon TOR activity varies according to cellular type and context

Turingan et al raw data.xlsx

Raw data for Turingan et al 2024</p

An investigation of nutrient-dependent mRNA translation in Drosophila larvae

The larval period of the Drosophila life cycle is characterized by immense growth. In nutrient rich conditions, larvae increase in mass approximately two hundred-fold in five days. However, upon nutrient deprivation, growth is arrested. The prevailing view is that dietary amino acids drive this larval growth by activating the conserved insulin/PI3 kinase and Target of rapamycin (TOR) pathways and promoting anabolic metabolism. One key anabolic process is protein synthesis. However, few studies have attempted to measure mRNA translation during larval development or examine the signaling requirements for nutrient-dependent regulation. Our work addresses this issue. Using polysome analyses, we observed that starvation rapidly (within thirty minutes) decreased larval mRNA translation, with a maximal decrease at 6–18 hours. By analyzing individual genes, we observed that nutrient-deprivation led to a general reduction in mRNA translation, regardless of any starvation-mediated changes (increase or decrease) in total transcript levels. Although sugars and amino acids are key regulators of translation in animal cells and are the major macronutrients in the larval diet, we found that they alone were not sufficient to maintain mRNA translation in larvae. The insulin/PI3 kinase and TOR pathways are widely proposed as the main link between nutrients and mRNA translation in animal cells. However, we found that genetic activation of PI3K and TOR signaling, or regulation of two effectors – 4EBP and S6K – could not prevent the starvation-mediated translation inhibition. Similarly, we showed that the nutrient stress-activated eIF2α kinases, GCN2 and PERK, were not required for starvation-induced inhibition of translation in larvae. These findings indicate that nutrient control of mRNA translation in larvae is more complex than simply amino acid activation of insulin and TOR signaling