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An investigation of nutrient-dependent mRNA translation in Drosophila larvae.

Nagarajan S, Grewal SS - Biol Open (2014)

Bottom Line: 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.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.

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, HRIC, 3330 Hospital Drive NW, Calgary, AB T2N 4N1, Canada.

No MeSH data available.


Related in: MedlinePlus

Translational control of genes whose total mRNA levels are decreased by starvation.(A) Total mRNA levels of each of the six genes were measured by qRT-PCR in fed vs. 18 hr starved larvae. Data are presented as mean ± SEM. (B–G) qRT-PCR analysis of each of the six selected genes. Each data point in the figure shows the mean (± SEM) % of total mRNA in each of the twelve fractions. Grey bars, fed larvae; green bars, starved larvae.
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f04: Translational control of genes whose total mRNA levels are decreased by starvation.(A) Total mRNA levels of each of the six genes were measured by qRT-PCR in fed vs. 18 hr starved larvae. Data are presented as mean ± SEM. (B–G) qRT-PCR analysis of each of the six selected genes. Each data point in the figure shows the mean (± SEM) % of total mRNA in each of the twelve fractions. Grey bars, fed larvae; green bars, starved larvae.

Mentions: To examine these starvation effects further, we analyzed translation of specific mRNAs by measuring their association with polysomes. We previously used microarray analyses to explore genome-wide transcript changes in response to starvation in larvae (Li et al., 2010). We used this data set to select 18 mRNAs to test for translational changes – six of these mRNAs showed little or no change in total levels upon starvation, six showed a marked increase, and six showed a decrease. The analyses for the three sets of genes are shown in Figs 3–5. We first confirmed the starvation-mediated changes in total mRNA levels using qRT-PCR analysis, and in general found good agreement with our microarray analyses (Fig. 3A, Fig. 4A, Fig. 5A). Although by no means an exhaustive set of mRNAs, the selection of these 18 genes allowed us to a) examine how translation of individual mRNAs responds to starvation, and b) identify any potential correlation between changes in total transcript levels versus specific changes in translation. To perform the translation analyses, we selected an 18 hr starvation time point and following sucrose density centrifugation, we divided the gradient contents into 12 equal fractions and performed qRT-PCR to measure mRNA levels in each fraction (Fig. 3B). Two general themes emerged from this analysis. First, in fed animals, for 16 out of 18 genes, most mRNA was found in fractions 7–9. This corresponds to a polysome containing 5–8 ribosomes. The remaining two genes (4EBP and CG7224 – Fig. 5C,F) are both small genes, which may limit the numbers of ribosomes that can associate with their mRNAs. These polysome data suggest that translation is generally at a high level in feeding larvae. Second, we observed that upon starvation, for almost all genes the peak of mRNAs shifted to fractions 5–7, which contains polysomes with 2–5 ribosomes. These effects were seen regardless of whether total mRNA levels for the genes were unchanged (Fig. 3), downregulated (Fig. 4) or upregulated (Fig. 5). Hence, even upon starvation almost all mRNAs are still polysomal, albeit with a shift in polysome association consistent with reduced translation. With the exception of two RP mRNAs, we saw little or no increase in mRNAs in fractions 1–5 upon starvation. These fractions contain untranslated mRNAs (e.g. mRNAs associated with mRNP complexes or sequestered in P-bodies). Together these findings suggest that translation of all mRNAs was reduced, but not abolished, upon starvation, regardless of the change in total mRNA levels.


An investigation of nutrient-dependent mRNA translation in Drosophila larvae.

Nagarajan S, Grewal SS - Biol Open (2014)

Translational control of genes whose total mRNA levels are decreased by starvation.(A) Total mRNA levels of each of the six genes were measured by qRT-PCR in fed vs. 18 hr starved larvae. Data are presented as mean ± SEM. (B–G) qRT-PCR analysis of each of the six selected genes. Each data point in the figure shows the mean (± SEM) % of total mRNA in each of the twelve fractions. Grey bars, fed larvae; green bars, starved larvae.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4232759&req=5

f04: Translational control of genes whose total mRNA levels are decreased by starvation.(A) Total mRNA levels of each of the six genes were measured by qRT-PCR in fed vs. 18 hr starved larvae. Data are presented as mean ± SEM. (B–G) qRT-PCR analysis of each of the six selected genes. Each data point in the figure shows the mean (± SEM) % of total mRNA in each of the twelve fractions. Grey bars, fed larvae; green bars, starved larvae.
Mentions: To examine these starvation effects further, we analyzed translation of specific mRNAs by measuring their association with polysomes. We previously used microarray analyses to explore genome-wide transcript changes in response to starvation in larvae (Li et al., 2010). We used this data set to select 18 mRNAs to test for translational changes – six of these mRNAs showed little or no change in total levels upon starvation, six showed a marked increase, and six showed a decrease. The analyses for the three sets of genes are shown in Figs 3–5. We first confirmed the starvation-mediated changes in total mRNA levels using qRT-PCR analysis, and in general found good agreement with our microarray analyses (Fig. 3A, Fig. 4A, Fig. 5A). Although by no means an exhaustive set of mRNAs, the selection of these 18 genes allowed us to a) examine how translation of individual mRNAs responds to starvation, and b) identify any potential correlation between changes in total transcript levels versus specific changes in translation. To perform the translation analyses, we selected an 18 hr starvation time point and following sucrose density centrifugation, we divided the gradient contents into 12 equal fractions and performed qRT-PCR to measure mRNA levels in each fraction (Fig. 3B). Two general themes emerged from this analysis. First, in fed animals, for 16 out of 18 genes, most mRNA was found in fractions 7–9. This corresponds to a polysome containing 5–8 ribosomes. The remaining two genes (4EBP and CG7224 – Fig. 5C,F) are both small genes, which may limit the numbers of ribosomes that can associate with their mRNAs. These polysome data suggest that translation is generally at a high level in feeding larvae. Second, we observed that upon starvation, for almost all genes the peak of mRNAs shifted to fractions 5–7, which contains polysomes with 2–5 ribosomes. These effects were seen regardless of whether total mRNA levels for the genes were unchanged (Fig. 3), downregulated (Fig. 4) or upregulated (Fig. 5). Hence, even upon starvation almost all mRNAs are still polysomal, albeit with a shift in polysome association consistent with reduced translation. With the exception of two RP mRNAs, we saw little or no increase in mRNAs in fractions 1–5 upon starvation. These fractions contain untranslated mRNAs (e.g. mRNAs associated with mRNP complexes or sequestered in P-bodies). Together these findings suggest that translation of all mRNAs was reduced, but not abolished, upon starvation, regardless of the change in total mRNA levels.

Bottom Line: 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.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.

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, HRIC, 3330 Hospital Drive NW, Calgary, AB T2N 4N1, Canada.

No MeSH data available.


Related in: MedlinePlus