scholarly journals Natural Genetic Variation Modifies Gene Expression Dynamics at the Protein Level During Pheromone Response in Saccharomyces cerevisiae

2016 ◽  
Author(s):  
Daniel A. Pollard ◽  
Ciara K. Asamoto ◽  
Homa Rahnamoun ◽  
Austin S. Abendroth ◽  
Suzanne R. Lee ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Synthesis ◽  
Genetic Variation ◽  
Steady State ◽  
Protein Expression ◽  
Expression Patterns ◽  
Synthesis Rate ◽  
Protein Synthesis Rate ◽  
Mrna Levels ◽  
Expression Divergence

ABSTRACTHeritable variation in gene expression patterns plays a fundamental role in trait variation and evolution, making understanding the mechanisms by which genetic variation acts on gene expression patterns a major goal for biology. Both theoretical and empirical work have largely focused on variation in steady-state mRNA levels and mRNA synthesis rates, particularly of protein-coding genes. Yet in order for this variation to affect higher order traits it must lead to differences at the protein level. Variation in protein-specific processes including protein synthesis rates and protein decay rates could amplify, mask, or even reverse effects transmitted from the transcript level, but the extent to which this happens is unclear. Moreover, mechanisms that underlie protein expression variation under dynamic conditions have not been examined. To address this challenge, we analyzed how mRNA and protein expression dynamics covary between two strains ofSaccharomyces cerevisiaeduring mating pheromone response. Although divergentsteady-statemRNA expression levels explained divergentsteady-stateprotein levels for four out of five genes in our study, the same was true for only one out of five genes for expressiondynamics. By integrating decay rate and allele-specific protein expression analyses, we resolved that expression divergence for Fig1p was caused by genetic variation acting intranson protein synthesis rate, expression divergence for Ina1p was caused bycis-by-transepistatic effects on transcript level and protein synthesis rate, and expression divergence for Fus3p and Tos6p were caused by divergence in protein synthesis rates. Our study demonstrates that steady-state analysis of gene expression is insufficient to understand the impact of genetic variation on gene expression variation. An integrated and dynamic approach to gene expression analysis - comparing mRNA levels, protein levels, protein decay rates, and allele-specific protein expression - allows for a detailed analysis of the genetic mechanisms underlying protein expression divergences.

scholarly journals Differential regulation of mRNA fate by the human Ccr4-Not complex is driven by coding sequence composition and mRNA localization

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Sarah L. Gillen ◽  
Chiara Giacomelli ◽  
Kelly Hodge ◽  
Sara Zanivan ◽  
Martin Bushell ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Synthesis ◽  
Mrna Stability ◽  
Mrna Localization ◽  
Synthesis Rate ◽  
Protein Synthesis Rate ◽  
Translational Efficiency ◽  
Mrna Levels ◽  
Control Of Gene Expression ◽  
Mrna Fate

Abstract Background Regulation of protein output at the level of translation allows for a rapid adaptation to dynamic changes to the cell’s requirements. This precise control of gene expression is achieved by complex and interlinked biochemical processes that modulate both the protein synthesis rate and stability of each individual mRNA. A major factor coordinating this regulation is the Ccr4-Not complex. Despite playing a role in most stages of the mRNA life cycle, no attempt has been made to take a global integrated view of how the Ccr4-Not complex affects gene expression. Results This study has taken a comprehensive approach to investigate post-transcriptional regulation mediated by the Ccr4-Not complex assessing steady-state mRNA levels, ribosome position, mRNA stability, and protein production transcriptome-wide. Depletion of the scaffold protein CNOT1 results in a global upregulation of mRNA stability and the preferential stabilization of mRNAs enriched for G/C-ending codons. We also uncover that mRNAs targeted to the ER for their translation have reduced translational efficiency when CNOT1 is depleted, specifically downstream of the signal sequence cleavage site. In contrast, translationally upregulated mRNAs are normally localized in p-bodies, contain disorder-promoting amino acids, and encode nuclear localized proteins. Finally, we identify ribosome pause sites that are resolved or induced by the depletion of CNOT1. Conclusions We define the key mRNA features that determine how the human Ccr4-Not complex differentially regulates mRNA fate and protein synthesis through a mechanism linked to codon composition, amino acid usage, and mRNA localization.

scholarly journals Differential regulation of mRNA fate by the human Ccr4-Not complex is driven by CDS composition and mRNA localisation

2021 ◽  
Author(s):  
Sarah L Gillen ◽  
Kelly Hodge ◽  
Sara Zanivan ◽  
Martin Bushell ◽  
Ania Wilczynska
Keyword(s):  
Gene Expression ◽  
Protein Synthesis ◽  
Mrna Stability ◽  
Ribosome Profiling ◽  
Synthesis Rate ◽  
Protein Synthesis Rate ◽  
Translational Efficiency ◽  
Mrna Levels ◽  
Control Of Gene Expression ◽  
Mrna Fate

Background: Regulation of protein output at the level of translation allows for a rapid adaptation to dynamic changes to the cell's requirements. This precise control of gene expression is achieved by complex and interlinked biochemical processes that modulate both the protein synthesis rate and stability of each individual mRNA. A major factor coordinating this regulation is the Ccr4-Not complex. Despite playing a role in most stages of the mRNA life cycle, no attempt has been made to take a global integrated view of how the Ccr4-Not complex affects gene expression. Results: This study has taken a holistic approach to investigate post-transcriptional regulation mediated by the Ccr4-Not complex assessing steady-state mRNA levels, ribosome position, mRNA stability and protein production transcriptome-wide. Depletion of the scaffold protein CNOT1 results in a global upregulation of mRNA stability and the preferential stabilisation of mRNAs enriched for GC-ending codons. We also uncover that mRNAs targeted to the ER for their translation have reduced translational efficiency when CNOT1 is depleted, specifically downstream of the signal sequence cleavage site. In contrast, translationally upregulated mRNAs are normally localised in p-bodies, contain disorder-promoting amino acids and encode nuclear localised proteins. Finally, using the unique complement of pulsed SILAC and ribosome profiling data we identify specific mRNAs with ribosome pause sites that are resolved following CNOT1 depletion. Conclusion: We define the key mRNA features that determine how the human Ccr4-Not complex differentially regulates mRNA fate and protein synthesis through a mechanism linked to codon composition, amino acid usage, and mRNA localisation.

scholarly journals Application of Domain- and Genotype-Specific Models to Infer Post-Transcriptional Regulation of Segmentation Gene Expression in Drosophila

Life ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 1232
Author(s):  
Maria A. Duk ◽  
Vitaly V. Gursky ◽  
Maria G. Samsonova ◽  
Svetlana Yu. Surkova
Keyword(s):  
Gene Expression ◽  
Transcriptional Regulation ◽  
Protein Expression ◽  
Drosophila Embryo ◽  
Synthesis Rate ◽  
Protein Synthesis Rate ◽  
Wild Type ◽  
Segmentation Gene ◽  
Segmentation Gene Expression ◽  
Post Transcriptional Regulation

Unlike transcriptional regulation, the post-transcriptional mechanisms underlying zygotic segmentation gene expression in early Drosophila embryo have been insufficiently investigated. Condition-specific post-transcriptional regulation plays an important role in the development of many organisms. Our recent study revealed the domain- and genotype-specific differences between mRNA and the protein expression of Drosophila hb, gt, and eve genes in cleavage cycle 14A. Here, we use this dataset and the dynamic mathematical model to recapitulate protein expression from the corresponding mRNA patterns. The condition-specific nonuniformity in parameter values is further interpreted in terms of possible post-transcriptional modifications. For hb expression in wild-type embryos, our results predict the position-specific differences in protein production. The protein synthesis rate parameter is significantly higher in hb anterior domain compared to the posterior domain. The parameter sets describing Gt protein dynamics in wild-type embryos and Kr mutants are genotype-specific. The spatial discrepancy between gt mRNA and protein posterior expression in Kr mutants is well reproduced by the whole axis model, thus rejecting the involvement of post-transcriptional mechanisms. Our models fail to describe the full dynamics of eve expression, presumably due to its complex shape and the variable time delays between mRNA and protein patterns, which likely require a more complex model. Overall, our modeling approach enables the prediction of regulatory scenarios underlying the condition-specific differences between mRNA and protein expression in early embryo.

Effects of exercise on protein synthesis and myosin heavy chain gene expression in hypothyroid rats

1994 ◽  
Vol 267 (1) ◽  
pp. E63-E67
Author(s):  
H. L. Katzeff ◽  
K. M. Ojamaa ◽  
I. Klein
Keyword(s):  
Gene Expression ◽  
Protein Synthesis ◽  
Thyroid Hormones ◽  
Cardiac Muscle ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Muscle Growth ◽  
Heart Weight ◽  
Synthesis Rate ◽  
Protein Synthesis Rate

Hypothyroidism suppresses muscle growth and alters myosin heavy chain (MHC) gene expression. To study the role of thyroid hormones in exercise-induced muscle growth and protein synthesis, we measured skeletal and cardiac muscle protein synthesis and MHC gene expression in hypothyroid rats allowed to exercise voluntarily. Female Sprague-Dawley rats (200-210 g) were separated into four groups for 28 days of treatment: control, hypothyroid (TX), hypothyroid plus running-wheel exercise (TX+Ex), and hypothyroid plus 25% overfed (TX+OF). Fractional protein synthesis rates (% incorporation/day) were measured using [3H]phenylalanine incorporation 10 min postinjection. The heart weight-to-body weight ratios of the TX and the TX+OF groups showed marked cardiac atrophy over the 28-day period (2.76 +/- 0.12 and 2.50 +/- 0.22 vs. 3.37 +/- 0.18 mg/g, respectively; P < 0.01). However, the TX+Ex group prevented heart, gastrocnemius, and soleus muscle atrophy over the same time period. Heart, gastrocnemius, and soleus muscles had markedly suppressed protein synthesis rates in the TX and TX+OF groups vs. the euthyroid controls (mean fall -72%; P < 0.01, analysis of variance). However, exercise increased protein synthesis rate by 50% (P < 0.05) compared with TX alone in all three muscle groups. Exercise did not modify hypothyroid-induced alterations of cardiac myosin isoform expression. Exercise-mediated effects on skeletal and cardiac muscle growth but not cardiac MHC gene expression appear to be independent of thyroid hormones.

Salubrinal Increases Human Fetal Hemoglobin Production Through a Post-Transcriptional Mechanism

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 250-250
Author(s):  
Cynthia K Hahn ◽  
Christopher H. Lowrey
Keyword(s):  
Gene Expression ◽  
Protein Synthesis ◽  
Steady State ◽  
Stress Response ◽  
Fetal Hemoglobin ◽  
Translational Efficiency ◽  
Mrna Levels ◽  
Globin Mrna ◽  
Hbf Induction ◽  
Hemoglobin Production

Abstract Abstract 250 Sickle cell disease and β-thalassemia continue to cause significant morbidity and mortality. Strategies to increase fetal hemoglobin (HbF) levels can ameliorate symptoms and improve the lives of patients with these diseases. While most previous studies have focused on induction of γ-globin gene expression as an approach to induce HbF, there is evidence that HbF may be post-transcriptionally regulated. For example, butyrate was shown to increase the translational efficiency of γ-globin mRNA and 5-azacytidine (5-Aza) induces HbF to a much greater degree than γ-globin mRNA steady state levels. These findings suggest that translational regulation may play an underappreciated yet important role in controlling HbF levels and that investigating the molecular mechanisms involved in this control may provide new therapeutic targets for HbF induction. We hypothesized that the Integrated Stress Response (ISR) pathway is involved in differentially regulating fetal and adult hemoglobin production. The ISR pathway has been shown to modulate globin protein synthesis in response to heme availability and other stresses. In the absence of heme, the heme-regulated inhibitor kinase phosphorylates eIF2α, downregulates general protein synthesis, but enables translation of a limited number of transcripts that are critical for coordinating the stress response. To test our hypothesis, we first evaluated the effects of salubrinal (Sal), a small molecule that activates ISR signaling by selectively inhibiting p-eIF2α dephosphorylation, in K562 cells. 3μM and 6μM Sal increased p-eIF2α and activated ISR signaling as evidenced by increased ATF4 and GADD34 protein levels and increased gene expression of ATF3 and CHOP, two transcriptional targets of ATF4. Once we verified that Sal increased p-eIF2α and ISR signaling, we extended testing to primary human erythroid cells to evaluate its effect on hemoglobin production. We first determined a dose range of Sal that increased p-eIF2α in primary cells without reducing cell viability. Both 3μM and 6μM Sal increased p-eIF2α and only reduced cell number by 15% when applied on days 15 and 18 of differentiation, the period of maximal hemoglobin synthesis. Next, we determined that 3μM and 6μM Sal slightly reduced γ-globin and β-globin steady state mRNA levels but did not change the γ/(γ+β) ratio relative to control. In contrast, Sal significantly induced HbF when evaluated by HPLC at the end of differentiation on day 20. Compared to untreated cells, 3μM Sal increased the percent HbF from 2.7% to 5.0% (1.8 fold) and 6μM Sal resulted in 12.9% HbF (4.7 fold) (n=4, p<0.05). The enhanced %HbF was due to increased HbF but also reduced HbA, providing evidence that HbF and HbA may be differentially or reciprocally regulated at the translational level. Importantly, Sal treatment did not significantly reduce the total hemoglobin content relative to the untreated control and did not alter cellular differentiation when assessed by flow cytometry for CD71 and CD235a. These results suggest that Sal increases HbF by a post-transcriptional mechanism potentially through ISR activation. Sal treatments earlier in the differentiation process (days 9 and 12) before considerable amounts of hemoglobin are synthesized failed to significantly increase HbF, further supporting this conclusion. We then evaluated whether Sal treatment could enhance HbF induction by known activators of γ-globin transcription, such as 5-Aza and hydroxyurea (HU). 200nM 5-Aza alone increased %HbF from 2.7% to 12.4%. When 200nM 5-Aza was combined with 3μM and 6μM Sal, the %HbF increased to 18.0% and 22.8%, respectively. Similarly, 10μM HU alone increased HbF from 2.9% to 4.9%, but co-treatment with 3μM and 6μM Sal increased HbF to 7.7% and 15.0%, respectively. For both HU and 5-Aza, combined treatment with Sal did not alter the γ/(γ+β) ratio from what was seen with HU or 5-Aza alone. Taken together, these results indicate that the novel method of HbF induction by Sal enhances the effect of transcriptional activators of γ-globin. In the future, utilization of transcriptional and translational mechanisms of HbF induction may provide an opportunity for combination therapy to achieve therapeutic HbF levels at reduced doses, thereby reducing toxicity. Disclosures: No relevant conflicts of interest to declare.

Regulation of intestinal cholecystokinin gene expression by glucocorticoids

1996 ◽  
Vol 151 (1) ◽  
pp. 137-145 ◽  
Author(s):  
C Ratineau ◽  
C Roche ◽  
F Chuzel ◽  
M Cordier-Bussat ◽  
M Blanc ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Synthesis ◽  
Steady State ◽  
Cell Lines ◽  
Actinomycin D ◽  
Competitive Inhibitor ◽  
Male Rats ◽  
Mrna Levels ◽  
Intestinal Origin

Abstract The effect of glucocorticoids on the expression of intestinal cholecystokinin (CCK) was investigated both in vivo and in cell culture systems. In vivo, 2-day administration of methylprednisolone to adult male rats induced a decrease in CCK-like immunoreactivity (CCK-LI) and CCK mRNA levels in mucosal extracts. In two CCK-producing cell lines, RIN 1056E and STC-1 of pancreatic and intestinal origin respectively, dexamethasone induced dose-dependent decreases in both CCK-LI and steady-state CCK mRNA levels. The decrease in CCK mRNA was totally prevented by incubation of cells with an excess of RU 38486, a competitive inhibitor for the binding of glucocorticoids to their receptor. Actinomycin D, used to prevent RNA synthesis, did not modify CCK mRNA stability in dexamethasone-pretreated cells as compared with cells not exposed to dexamethasone. When cells were first incubated with actinomycin D, subsequent addition of dexamethasone left the steady-state CCK mRNA levels unaltered in both cell lines. Nuclear run-on assays performed in RIN 1056E cells showed that glucocorticoids decreased the rate of transcription of the CCK gene. In addition, cycloheximide, used to prevent protein synthesis, abolished the inhibitory effects of dexamethasone on steady-state CCK mRNA levels. These results demonstrate that glucocorticoids down-regulate CCK gene expression in the rat intestinal mucosa and in two CCK-producing cell lines. The effect is blocked by a glucocorticoid receptor antagonist. Inhibition of CCK gene expression may result from a decrease in the transcription rate, and probably involves one or several steps that depend on protein synthesis. Journal of Endocrinology (1996) 151, 137–145

Impaired Muscle Mitochondrial Function in Familial Partial Lipodystrophy

2021 ◽  
Author(s):  
Vinaya Simha ◽  
Ian R Lanza ◽  
Surendra Dasari ◽  
Katherine A Klaus ◽  
Nathan Le Brasseur ◽  
...  
Keyword(s):  
Gene Expression ◽  
Skeletal Muscle ◽  
Protein Synthesis ◽  
Muscle Strength ◽  
Mitochondrial Function ◽  
Muscle Protein ◽  
Muscle Protein Synthesis ◽  
Synthesis Rate ◽  
Protein Synthesis Rate ◽  
Partial Lipodystrophy

Abstract Background Familial Partial Lipodystrophy (FPL), Dunnigan variety is characterized by skeletal muscle hypertrophy and insulin resistance besides fat loss from the extremities. The cause for the muscle hypertrophy, and its functional consequences is not known. Objective To compare muscle strength and endurance, besides muscle protein synthesis rate between subjects with FPL and matched controls (n = 6 in each group). In addition, we studied skeletal muscle mitochondrial function and gene expression pattern to help understand the mechanisms for the observed differences. Methods Body composition by DEXA, insulin sensitivity by minimal modelling, assessment of peak muscle strength and fatigue, skeletal muscle biopsy and calculation of muscle protein synthesis rate, mitochondrial respirometry, skeletal muscle transcriptome, proteome and gene set enrichment analysis. Results Despite increased muscularity, FPL subjects did not demonstrate increased muscle strength but had earlier fatigue on chest press exercise. Decreased mitochondrial state 3 respiration in the presence of fatty acid substrate was noted, concurrent to elevated muscle lactate and decreased long-chain acylcarnitine. Based on gene transcriptome, there was significant down regulation of many critical metabolic pathways involved in mitochondrial biogenesis and function. Moreover, the overall pattern of gene expression was indicative of accelerated aging in FPL subjects. A lower muscle protein synthesis and down regulation of gene transcripts involved in muscle protein catabolism was observed. Conclusion Increased muscularity in FPL is not due to increased muscle protein synthesis and is likely due to reduced muscle protein degradation. Impaired mitochondrial function and altered gene expression likely explain the metabolic abnormalities and skeletal muscle dysfunction in FPL subjects.

Hepatocellular expression of glucose-6-phosphatase is unaltered during hepatic regeneration

1998 ◽  
Vol 275 (4) ◽  
pp. G717-G722 ◽  
Author(s):  
Wisam F. Zakko ◽  
Carl L. Berg ◽  
John L. Gollan ◽  
Richard M. Green
Keyword(s):  
Gene Expression ◽  
Enzyme Activity ◽  
Protein Expression ◽  
Partial Hepatectomy ◽  
Initial Phase ◽  
Physiological Function ◽  
Liver Homogenate ◽  
Hepatic Regeneration ◽  
Mrna Levels ◽  
Microsomal Enzyme

Gluconeogenesis and glycogenolysis are essential hepatic functions required for glucose homeostasis. During the initial phase of hepatic regeneration, the immediate-early genes (IEG) are rapidly expressed, and the IEG RL-1 encodes for glucose-6-phosphatase (G-6- Pase). G-6- Pase is a microsomal enzyme essential for gluconeogenesis and glycogenolysis. This study employs a partial-hepatectomy model to examine the expression and activity of G-6- Pase. After partial hepatectomy, rat hepatic G-6- Pase gene expression is transcriptionally regulated, and mRNA levels are increased ≈30-fold. However, in contrast to this rapid gene induction, microsomal enzyme activity is unchanged after partial hepatectomy. Western blotting demonstrates that microsomal G-6- Pase protein expression is also unchanged after partial hepatectomy, and similar results are also noted in whole liver homogenate. Thus, despite marked induction in gene expression of the IEG G-6- Pase after partial hepatectomy, protein expression and enzyme activity remain unchanged. These data indicate that, although this hepatocyte IEG is transcriptionally regulated, the physiologically important level of regulation is posttranscriptional. This highlights the importance of correlating gene expression of IEG with protein expression and physiological function.

scholarly journals Viral Gene Expression Patterns in Human Herpesvirus 6B-Infected T Cells

2002 ◽  
Vol 76 (15) ◽  
pp. 7578-7586 ◽  
Author(s):  
Bodil Øster ◽  
Per Höllsberg
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Protein Synthesis ◽  
De Novo ◽  
Expression Patterns ◽  
Human Herpesvirus ◽  
Viral Gene Expression ◽  
Polymerase Activity ◽  
Viral Gene ◽  
De Novo Protein Synthesis

ABSTRACT Herpesvirus gene expression is divided into immediate-early (IE) or α genes, early (E) or β genes, and late (L) or γ genes on the basis of temporal expression and dependency on other gene products. By using real-time PCR, we have investigated the expression of 35 human herpesvirus 6B (HHV-6B) genes in T cells infected by strain PL-1. Kinetic analysis and dependency on de novo protein synthesis and viral DNA polymerase activity suggest that the HHV-6B genes segregate into six separate kinetic groups. The genes expressed early (groups I and II) and late (groups V and VI) corresponded well with IE and L genes, whereas the intermediate groups III and IV contained E and L genes. Although HHV-6B has characteristics similar to those of other roseoloviruses in its overall gene regulation, we detected three B-variant-specific IE genes. Moreover, genes that were independent of de novo protein synthesis clustered in an area of the viral genome that has the lowest identity to the HHV-6A variant. The organization of IE genes in an area of the genome that differs from that of HHV-6A underscores the distinct differences between HHV-6B and HHV-6A and may provide a basis for further molecular and immunological analyses to elucidate their different biological behaviors.

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