scholarly journals Regulation of protein synthesis by eIF4E phosphorylation in adult cardiocytes: the consequence of secondary structure in the 5′-untranslated region of mRNA

2004 ◽  
Vol 378 (1) ◽  
pp. 73-82 ◽  
Author(s):  
William J. TUXWORTH ◽  
Atif N. SAGHIR ◽  
Laura S. SPRUILL ◽  
Donald R. MENICK ◽  
Paul J. McDERMOTT
Keyword(s):  
Protein Synthesis ◽  
Secondary Structure ◽  
Untranslated Region ◽  
Primary Cultures ◽  
Initiation Factor ◽  
Luciferase Reporter ◽  
Translational Efficiency ◽  
Mrna Levels ◽  
Hypertrophic Growth ◽  
Reporter Mrna

In adult cardiocytes, eIF4E (eukaryotic initiation factor 4E) activity and protein synthesis are increased concomitantly in response to stimuli that induce hypertrophic growth. We tested the hypothesis that increases in eIF4E activity selectively improve the translational efficiency of mRNAs that have an excessive amount of secondary structure in the 5´-UTR (5´-untranslated region). The activity of eIF4E was modified in primary cultures of adult cardiocytes using adenoviral gene transfer to increase either the amount of eIF4E or the extent of endogenous eIF4E phosphorylation. Subsequently, the effects of eIF4E on translational efficiency were assayed following adenoviral-mediated expression of luciferase reporter mRNAs that were either ‘stronger’ (less structure in the 5´-UTR) or ‘weaker’ (more structure in the 5´-UTR) with respect to translational efficiency. The insertion of G+C-rich repeats into the 5´-UTR doubled the predicted amount of secondary structure and was sufficient to reduce translational efficiency of the reporter mRNA by 48±13%. Translational efficiency of the weaker reporter mRNA was not significantly improved by overexpression of wild-type eIF4E when compared with the stronger reporter mRNA. In contrast, overexpression of the eIF4E kinase Mnk1 [MAP (mitogen-activated protein) kinase signal-integrating kinase 1] was sufficient to increase the translational efficiency of either reporter mRNA, independent of the amount of secondary structure in their respective 5´-UTRs. The increases in translational efficiency produced by Mnk1 occurred in association with corresponding decreases in mRNA levels. These findings indicate that the positive effect of eIF4E phosphorylation on translational efficiency in adult cardiocytes is coupled with the stability of mRNA.

Increased expression of eukaryotic initiation factor 4E during growth of neonatal rat cardiocytes in vitro

1998 ◽  
Vol 274 (6) ◽  
pp. H2133-H2142 ◽  
Author(s):  
Antoine A. Makhlouf ◽  
Paul J. McDermott
Keyword(s):  
Type I Collagen ◽  
Neonatal Rat ◽  
Initiation Factor ◽  
Translational Efficiency ◽  
Type I ◽  
Mrna Levels ◽  
Eukaryotic Initiation Factor ◽  
Translational Initiation ◽  
Hypertrophic Growth ◽  
Eukaryotic Initiation Factor 4E

Eukaryotic initiation factor 4E (eIF-4E) is rate limiting for translational initiation. The purpose of this study was to determine whether eIF-4E levels are increased during cardiocyte growth produced by increased load in the form of electrically stimulated contraction. Neonatal rat cardiocytes were cultured on a matrix of aligned type I collagen. The cardiocytes aligned in parallel to the direction of the collagen fibrils and exhibited an elongated, rod-shaped morphology. Cardiocytes were electrically stimulated to contract at 3 Hz (alternating polarity, 5-ms pulse width). Nonstimulated cardiocytes were quiescent and used as controls. Electrically stimulated contraction produced hypertrophic growth as determined by the following criteria: 1) increased protein content, 2) increased RNA content, 3) accelerated rate of protein synthesis, and 4) threefold increase in promoter activity of the atrial natriuretic factor gene. Cardiocyte growth was associated with an increase in eIF-4E mRNA levels that reached 48 ± 9% after 2 days of electrically stimulated contraction. eIF-4E protein levels were increased by more than twofold over the same time period. We conclude that an adaptive increase in eIF-4E is an important mechanism for maintaining translational efficiency during cardiocyte growth.

Translational mechanisms accelerate the rate of protein synthesis during canine pressure-overload hypertrophy

1999 ◽  
Vol 277 (6) ◽  
pp. H2176-H2184 ◽  
Author(s):  
Yoshitatsu Nagatomo ◽  
Blase A. Carabello ◽  
Masayoshi Hamawaki ◽  
Shintaro Nemoto ◽  
Takeshi Matsuo ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Balloon Catheter ◽  
Pressure Overload ◽  
Left Ventricular ◽  
Ascending Aorta ◽  
Translational Efficiency ◽  
Mrna Levels ◽  
Hypertrophic Growth ◽  
Translational Mechanisms

This study examined how translational mechanisms regulate the rate of cardiac protein synthesis during canine pressure overload in vivo. Acute aortic stenosis (AS) was produced by inflating a balloon catheter in the ascending aorta for 6 h; sustained AS was created by controlled banding of the ascending aorta. AS caused significant hypertrophy as reflected by increased left ventricular (LV) mass after 5 and 10 days. To monitor LV protein synthesis in vivo, myosin heavy chain (MHC) synthesis was measured by continuous infusion of radiolabeled leucine. Acute AS accelerated the rate of myosin synthesis without a corresponding increase in ribosomal RNA, indicating an increase in translational efficiency. Total MHC synthesis (mg MHC/LV per day) was significantly increased at 5 and 10 days of sustained AS. Total MHC degradation was not significantly altered at 5 days of AS but increased at 10 days of AS in concordance with a new steady state with respect to growth. Translational capacity (mg total RNA/LV) was significantly increased after 5 and 10 days of AS and was preceded by an increase in the rate of ribosome formation. MHC mRNA levels remained unchanged during AS. These findings demonstrate that cardiac protein synthesis is accelerated in response to pressure overload by an initial increase in translational efficiency, followed by an adaptive increase in translational capacity during sustained hypertrophic growth.

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 Role of the 5′-untranslated region of mRNA in the synthesis of S-adenosylmethionine decarboxylase and its regulation by spermine

1994 ◽  
Vol 302 (3) ◽  
pp. 765-772 ◽  
Author(s):  
L M Shantz ◽  
R Viswanath ◽  
A E Pegg
Keyword(s):  
Secondary Structure ◽  
Untranslated Region ◽  
Fold Increase ◽  
Negative Regulator ◽  
Mrna Levels ◽  
Coding Region ◽  
Polyamine Biosynthesis ◽  
Adenosylmethionine Decarboxylase ◽  
Spermine Synthase ◽  
Synthase Inhibitor

S-Adenosylmethionine decarboxylase (AdoMetDC), a rate-limiting enzyme in polyamine biosynthesis, is regulated by polyamines at the levels of both transcription and translation. Two unusual features of AdoMetDC mRNA are a long (320 nt) 5′-untranslated region (5′UTR), which is thought to contain extensive secondary structure, and a short (15 nt) open reading frame (ORF) within the 5′UTR. We have studied the effects of altering these elements on both the expression of AdoMetDC and its regulation by n-butyl-1,3-diaminopropane (BDAP), a spermine synthase inhibitor. Human AdoMetDC cDNAs containing alterations in the 5′UTR, as well as chimaeric constructs in which the AdoMetDC 5′UTR was inserted ahead of the luciferase-coding region, were transfected into COS-7 cells. Construct pSAM320, which contains all of the 5′UTR, the AdoMetDC protein-coding region and the 3′UTR, was expressed poorly (2-fold over the endogenous activity). Deletion of virtually the entire 5′UTR, leaving nt -12 to -1, increased expression 59-fold, suggesting that 5′UTR acts as a negative regulator. The same effect was seen when the 27 nt at the extreme 5′ end were removed (pSAM293, 47-fold increase), or when the internal ORF which is present in this region was destroyed by changing the ATG to CGA (pSAM320-ATG, 38-fold increase). The expression and regulation of pSAM44 (made by deleting nt -288 to -12), which has very little predicted secondary strucutre, was very similar to that of pSAM320 indicating that the terminal 27 nt including the internal ORF rather than extensive secondary structure may be responsible for the low basal levels of AdoMetDC expression. These results, confirmed using luciferase constructs, suggest that the negative effect on expression is predominantly due to the internal ORF. Depletion of spermine by BDAP increased the expression from pSAM320 more than 5-fold without affecting AdoMetDC mRNA levels. Expression from pSAM293 was unchanged by spermine depletion, whereas that from pSAM320-ATG was increased 2.5-fold. These results indicate the presence of a spermine response element in the first 27 nt of the 5′UTR that may include but is not entirely due to the internal ORF.

TNF-binding protein ameliorates inhibition of skeletal muscle protein synthesis during sepsis

1999 ◽  
Vol 276 (4) ◽  
pp. E611-E619 ◽  
Author(s):  
Robert Cooney ◽  
Scot R. Kimball ◽  
Rebecca Eckman ◽  
George Maish ◽  
Margaret Shumate ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Protein Content ◽  
Muscle Protein ◽  
Muscle Protein Synthesis ◽  
Binding Protein ◽  
Initiation Factor ◽  
Translational Efficiency ◽  
Skeletal Muscle Protein ◽  
Muscle Weight ◽  
Initiation Factors

We examined the effects of TNF-binding protein (TNFBP) on regulatory mechanisms of muscle protein synthesis during sepsis in four groups of rats: Control; Control+TNFBP; Septic; and Septic+TNFBP. Saline (1.0 ml) or TNFBP (1 mg/kg, 1.0 ml) was injected daily starting 4 h before the induction of sepsis. The effect of TNFBP on gastrocnemius weight, protein content, and the rate of protein synthesis was examined 5 days later. Sepsis reduced the rate of protein synthesis by 35% relative to controls by depressing translational efficiency. Decreases in protein synthesis were accompanied by similar reductions in protein content and muscle weight. Treatment of septic animals with TNFBP for 5 days prevented the sepsis-induced inhibition of protein synthesis and restored translational efficiency to control values. TNFBP treatment of Control rats for 5 days was without effect on muscle protein content or protein synthesis. We also assessed potential mechanisms regulating translational efficiency. The phosphorylation state of p70S6 kinase was not altered by sepsis. Sepsis reduced the gastrocnemius content of eukaryotic initiation factor 2Bε (eIF2Bε), but not eIF2α. The decrease in eIF2Bε content was prevented by treatment of septic rats with TNFBP. TNFBP ameliorates the sepsis-induced changes in protein metabolism in gastrocnemius, indicating a role for TNF in the septic process. The data suggest that TNF may impair muscle protein synthesis by reducing expression of specific initiation factors during sepsis.

scholarly journals The 5′ Untranslated Region of Protein Kinase Cδ Directs Translation by an Internal Ribosome Entry Segment That Is Most Active in Densely Growing Cells and during Apoptosis

2002 ◽  
Vol 22 (17) ◽  
pp. 6089-6099 ◽  
Author(s):  
Bronwyn C. Morrish ◽  
Martin G. Rumsby
Keyword(s):  
Protein Kinase ◽  
Untranslated Region ◽  
Initiation Factor ◽  
Luciferase Reporter ◽  
Serum Starvation ◽  
Proliferating Cells ◽  
Internal Ribosome Entry ◽  
Internal Ribosome Entry Segment ◽  
Eukaryotic Initiation Factor 4G ◽  
Protein Kinase Cδ

ABSTRACT Protein kinase Cδ (PKCδ) is a member of the PKC family of phospholipid-dependent serine/threonine kinases and is involved in cell proliferation, apoptosis, and differentiation. Previous studies have suggested that different PKC isoforms might be translationally regulated. We report here that the 395-nt-long 5′ untranslated region (5′ UTR) of PKCδ is predicted to form very stable secondary structures with free energies (ΔG values) of around −170 kcal/mol. The 5′ UTR of PKCδ can significantly repress luciferase translation in rabbit reticulocyte lysate but does not repress luciferase translation in a number of transiently transfected cell lines. By using a bicistronic luciferase reporter, we show that the 5′ UTR of PKCδ contains a functional internal ribosome entry segment (IRES). The activity of the PKCδ IRES is greatest in densely growing cells and during apoptosis, when total protein synthesis and levels of full-length eukaryotic initiation factor 4G are reduced. However, the IRES activity of the 5′ UTR of PKCδ is not enhanced during serum starvation, another condition shown to inhibit cap-dependent translation, suggesting that its potency is dependent on specific cellular conditions. Accumulating data suggest that PKCδ has a function as proliferating cells reach high density and in early and later events of apoptosis. Our studies suggest a mechanism whereby PKCδ synthesis can be maintained under these conditions when cap-dependent translation is inhibited.

The Translation Initiation Factor eIF4E Is Overexpressed in Multiple Myeloma and Is Critical for Myeloma Cell Proliferation and Survival

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1853-1853 ◽  
Author(s):  
Shirong Li ◽  
MeiHua Jin ◽  
Ailing Liu ◽  
Markus Y. Mapara ◽  
Suzanne Lentzsch
Keyword(s):  
Multiple Myeloma ◽  
Protein Synthesis ◽  
Translation Initiation ◽  
Clinical Care ◽  
Protein Translation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Mrna Levels ◽  
Myeloma Cells ◽  
Rpmi 8226

Abstract Abstract 1853 Methods: The translation initiation factor eIF4E is central to protein synthesis in general, and overexpression and/or activation of eIF4E is associated with a malignant phenotype by regulating oncogenic protein translation. Several previous publications indicate that aberrant control of protein synthesis contributes to lymphoma genesis but the exact role of protein translation in multiple myeloma (MM) is less clear. Therefore, understanding the mechanisms that control protein synthesis is an emerging new research area in MM with significant potential for developing innovative therapies. The goal of this study was to determine the role and regulation of eIF4E, as well as the effects of protein translation controlling drugs in MM. Results: By western blot analysis as well as RT-PCR we found that eIF4E protein and mRNA levels are significantly elevated (up to 20 fold) in MM cell lines (H929, RPMI-8226, MM.1S and OPM2) and primary myeloma cells compared to normal plasma cells. Silencing of eIF4E gene expression in RPMI-8226 MM cells by a stable and inducible shRNA system significantly decreased viability of myeloma cells (by ∼ 43%) but not of HEK 293 suggesting a higher dependency of MM cells to protein translation. Next we evaluated different drugs including pomalidomide, rapamycin, pp242, 4EGI-1 and ribavirin, that are known to inhibit protein synthesis for their effects on protein translation in MM. By m7GTP pull down assays we evaluated the effects of the different drugs on eIF4E expression and activity. Rapamycin blocked the phosphorylation of 4EBP1 and eIF4E release, and subsequently inhibited eIF4G binding. The compound 4EGI-1 decreased the interaction between eIF4E and eIF4G. Pomalidomide decreased eIF4E protein expression. All drugs inhibited MM cell DNA synthesis measured by 3H-Thymidine incorporation. Treatment with pomalidomide (10uM), rapamycin (40nM), pp242 (10uM), 4EGI1 (50uM) or ribavirin (50uM) for 48h significantly decreased (p<0.05) proliferation by 43–62% indicating that drugs controlling protein translation inhibit MM growth. We also found that all drugs decreased expression of eIF4E dependent targets such as cyclin D1 and c-myc. Conclusion: Here we show that eIF4E, a key player in translational control, is highly expressed in MM cells and critical for MM growth and survival. Therefore our study helps to understand the function and regulatory mechanism of eIF4E in MM. Further the evaluation of drugs targeting protein translation provides the basis for the optimization of current MM treatment or to open up new strategies such as targeting protein translation in future MM therapy. Disclosures: Lentzsch: Celgene Corp: Consultancy, Research Funding; Onyx: Consultancy; Genzyme: Consultancy; prIME Oncology: Honoraria; Imedex: Honoraria; Clinical Care Options: Honoraria.

scholarly journals Signal Transduction Mechanisms for Glucagon-Induced Somatolactin Secretion and Gene Expression in Nile Tilapia (Oreochromis niloticus) Pituitary Cells

2021 ◽  
Vol 11 ◽  
Author(s):  
Chaoyi Zhang ◽  
Anji Lian ◽  
Yue Xu ◽  
Quan Jiang
Keyword(s):  
Gene Expression ◽  
Oreochromis Niloticus ◽  
Nile Tilapia ◽  
Primary Cultures ◽  
Hormone Secretion ◽  
Hek293 Cells ◽  
Luciferase Reporter ◽  
Pituitary Hormone ◽  
Pituitary Cells ◽  
Mrna Levels

Glucagon (GCG) plays a stimulatory role in pituitary hormone regulation, although previous studies have not defined the molecular mechanism whereby GCG affects pituitary hormone secretion. To this end, we identified two distinct proglucagons, Gcga and Gcgb, as well as GCG receptors, Gcgra and Gcgrb, in Nile tilapia (Oreochromis niloticus). Using the cAMP response element (CRE)-luciferase reporter system, tilapia GCGa and GCGb could reciprocally activate the two GCG receptors expressed in human embryonic kidney 293 (HEK293) cells. Quantitative real-time PCR analysis revealed that differential expression of the Gcga and Gcgb and their cognate receptors Gcgra and Gcgrb was found in the various tissues of tilapia. In particular, the Gcgrb is abundantly expressed in the neurointermediate lobe (NIL) of the pituitary gland. In primary cultures of tilapia NIL cells, GCGb effectively stimulated SL release, with parallel rises in the mRNA levels, and co-incubation with the GCG antagonist prevented GCGb-stimulated SL release. In parallel experiments, GCGb treatment dose-dependently enhanced intracellular cyclic adenosine monophosphate (cAMP) accumulation with increasing inositol 1,4,5-trisphosphate (IP3) concentration and the resulting in transient increases of Ca2+ signals in the primary NIL cell culture. Using selective pharmacological approaches, the adenylyl cyclase (AC)/cAMP/protein kinase A (PKA) and phospholipase C (PLC)/IP3/Ca2+/calmodulin (CaM)/CaMK-II pathways were shown to be involved in GCGb-induced SL release and mRNA expression. Together, these results provide evidence for the first time that GCGb can act at the pituitary level to stimulate SL release and gene expression via GCGRb through the activation of the AC/cAMP/PKA and PLC/IP3/Ca2+/CaM/CaMK-II cascades.

IGF-I/IGFBP-3 binary complex modulates sepsis-induced inhibition of protein synthesis in skeletal muscle

2000 ◽  
Vol 279 (5) ◽  
pp. E1145-E1158 ◽  
Author(s):  
Elisabeth Svanberg ◽  
Robert A. Frost ◽  
Charles H. Lang ◽  
Jorgen Isgaard ◽  
Leonard S. Jefferson ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Binding Protein ◽  
Initiation Factor ◽  
Translational Efficiency ◽  
Binary Complex ◽  
Igf I ◽  
Inhibition Of Protein Synthesis ◽  
Values Assessment ◽  
Igf Binding ◽  
Igfbp 3

The present study evaluated the ability of insulin-like growth factor I (IGF-I) complexed with IGF binding protein-3 (IGFBP-3) to modulate the sepsis-induced inhibition of protein synthesis in gastrocnemius. Beginning 16 h after the induction of sepsis, either the binary complex or saline was injected twice daily via a tail vein, with measurements made 3 and 5 days later. By day 3, sepsis had reduced plasma IGF-I concentrations ∼50% in saline-treated rats. Administration of the binary complex provided exogenous IGF-I to compensate for the sepsis-induced diminished plasma IGF-I. Sepsis decreased rates of protein synthesis in gastrocnemius relative to controls by limiting translational efficiency. Treatment of septic rats with the binary complex for 5 days attenuated the sepsis-induced inhibition of protein synthesis and restored translational efficiency to control values. Assessment of potential mechanisms regulating translational efficiency showed that neither the sepsis-induced change in gastrocnemius content of eukaryotic initiation factor 2B (eIF2B), the amount of eIF4E associated with 4E binding protein-1 (4E-BP1), nor the phosphorylation state of 4E-BP1 or eIF4E were altered by the binary complex. Overall, the results are consistent with the hypothesis that decreases in plasma IGF-I are partially responsible for enhanced muscle catabolism during sepsis.

Role of eIF4E in stimulation of protein synthesis by IGF-I in perfused rat skeletal muscle

2000 ◽  
Vol 278 (1) ◽  
pp. E58-E64 ◽  
Author(s):  
Thomas C. Vary ◽  
Leonard S. Jefferson ◽  
Scot R. Kimball
Keyword(s):  
Skeletal Muscle ◽  
Protein Synthesis ◽  
Initiation Factor ◽  
Translational Efficiency ◽  
Rat Skeletal Muscle ◽  
Phosphorylation State ◽  
Factor I ◽  
Igf I ◽  
Stimulation Of

Insulin-like growth factor I (IGF-I) promotes anabolism by stimulating protein synthesis in skeletal muscle. In the present study, we have examined mechanisms by which IGF-I stimulates protein synthesis in skeletal muscle with a perfused rat hindlimb preparation. IGF-I (10 nM) stimulated protein synthesis over 2.7-fold. Total RNA content was unaffected, but translational efficiency was increased by IGF-I. We next examined the effect of IGF-I on eukaryotic initiation factor (eIF) 4E as a mechanism regulating translation initiation. IGF-I did not alter either the amount of eIF4E associated with the eIF4E binding protein 4E-BP1 or the phosphorylation state of 4E-BP1. Likewise, the phosphorylation state of eIF4E was unaltered by IGF-I. In contrast, the amount of eIF4E bound to eIF4G was increased threefold by IGF-I. We conclude that IGF-I regulates protein synthesis in skeletal muscle by enhancing formation of the active eIF4E ⋅ eIF4G complex.

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