scholarly journals Cap-independent mRNA translation is upregulated in long-lived endocrine mutant mice

2019 ◽  
Vol 63 (2) ◽  
pp. 123-138 ◽  
Author(s):  
Ulas Ozkurede ◽  
Rishabh Kala ◽  
Cameron Johnson ◽  
Ziqian Shen ◽  
Richard A Miller ◽  
...  
Keyword(s):  
Growth Hormone ◽  
Growth Hormone Receptor ◽  
Protein A ◽  
Mrna Translation ◽  
Mutant Mice ◽  
Mrna Levels ◽  
Healthy Longevity ◽  
Independent Mechanism ◽  
Independent Translation

It has been hypothesized that transcriptional changes associated with lower mTORC1 activity in mice with reduced levels of growth hormone and insulin-like growth factor 1 are responsible for the longer healthy lifespan of these mutant mice. Cell lines and tissues from these mice show alterations in the levels of many proteins that cannot be explained by corresponding changes in mRNAs. Such post-transcriptional modulation may be the result of preferential mRNA translation by the cap-independent translation of mRNA bearing the N6-methyl-adenosine (m6A) modification. The long-lived endocrine mutants – Snell dwarf, growth hormone receptor deletion and pregnancy-associated plasma protein-A knockout – all show increases in the N6-adenosine-methyltransferases (METTL3/14) that catalyze 6-methylation of adenosine (m6A) in the 5′ UTR region of select mRNAs. In addition, these mice have elevated levels of YTH domain-containing protein 1 (YTHDF1), which recognizes m6A and promotes translation by a cap-independent mechanism. Consistently, multiple proteins that can be translated by the cap-independent mechanism are found to increase in these mice, including DNA repair and mitochondrial stress response proteins, without changes in corresponding mRNA levels. Lastly, a drug that augments cap-independent translation by inhibition of cap-dependent pathways (4EGI-1) was found to elevate levels of the same set of proteins and able to render cells resistant to several forms of in vitro stress. Augmented translation by cap-independent pathways facilitated by m6A modifications may contribute to the stress resistance and increased healthy longevity of mice with diminished GH and IGF-1 signals.

scholarly journals 5’ UTR recruitment of eIF4GI or DAP5 drives cap-independent translation for a subset of human mRNAs

2018 ◽  
Author(s):  
Solomon A. Haizel ◽  
Usha Bhardwaj ◽  
Ruben L. Gonzalez ◽  
Somdeb Mitra ◽  
Dixie J. Goss
Keyword(s):  
Tumor Hypoxia ◽  
Mrna Translation ◽  
Luciferase Reporter ◽  
Binding Studies ◽  
Mechanistic Insight ◽  
Specific Subset ◽  
Independent Mechanism ◽  
Insight Into ◽  
Independent Translation

AbstractDuring unfavorable human cellular conditions (e.g., tumor hypoxia, viral infection, etc.), canonical, cap-dependent mRNA translation is suppressed. Nonetheless, a subset of physiologically important mRNAs (e.g., HIF-1α, FGF-9, and p53) is still translated by an unknown, cap-independent mechanism. Additionally, expression levels of eIF4G and its homolog, death associated protein 5 (DAP5), are elevated. Using fluorescence anisotropy binding studies, luciferase reporter-based in vitro translation assays, and mutational analyses, here we demonstrate that eIF4GI and DAP5 specifically bind to the 5’ UTRs of these cap-independently translated mRNAs. Surprisingly, we find that the eIF4E binding domain of eIF4GI increases not only the binding affinity, but also the selectivity among these mRNAs. We further demonstrate that the affinities of eIF4GI and DAP5 binding to these 5’ UTRs correlate with the efficiency with which these factors drive cap-independent translation of these mRNAs. Integrating the results of our binding and translation assays, we show that eIF4GI and/or DAP5 are critical for recruitment of a specific subset of mRNAs to the ribosome and provide mechanistic insight into their cap-independent translation.

FXYD-11 associates with Na+-K+-ATPase in the gill of Atlantic salmon: regulation and localization in relation to changed ion-regulatory status

2010 ◽  
Vol 299 (5) ◽  
pp. R1212-R1223 ◽  
Author(s):  
Christian K. Tipsmark ◽  
Yasser A. Mahmmoud ◽  
Russell J. Borski ◽  
Steffen S. Madsen
Keyword(s):  
Growth Hormone ◽  
Atlantic Salmon ◽  
Protein A ◽  
Regulatory Subunit ◽  
Kinetic Properties ◽  
Mrna Levels ◽  
Α Subunit ◽  
Protein Levels ◽  
Parallel Increase

The Na+-K+-ATPase is the primary electrogenic component driving transepithelial ion transport in the teleost gill; thus regulation of its level of activity is of critical importance for osmotic homeostasis. In the present study, we examined the dynamics of the gill-specific FXYD-11 protein, a putative regulatory subunit of the pump, in Atlantic salmon during seawater (SW) acclimation, smoltification, and treatment with cortisol, growth hormone, and prolactin. Dual-labeling immunohistochemistry showed that branchial FXYD-11 is localized in Na+-K+-ATPase immunoreactive cells, and coimmunoprecipitation experiments confirmed a direct association between FXYD-11 and the Na+-K+-ATPase α-subunit. Transfer of freshwater (FW)-acclimated salmon to SW induced a parallel increase in total α-subunit and FXYD-11 protein expression. A similar concurrent increase was seen during smoltification in FW. In FW fish, cortisol induced an increase in both α-subunit and FXYD-11 abundance, and growth hormone further stimulated FXYD-11 levels. In SW fish, prolactin induced a decrease in FXYD-11 and α-subunit protein levels. In vitro cortisol (18 h, 10 μg/ml) stimulated FXYD-11, but not FXYD-9, mRNA levels in gills from FW and SW salmon. The data show that Na+-K+-ATPase expressed in branchial mitochondrion-rich cells is accompanied by FXYD-11, and that regulation of the two proteins is highly coordinated. The demonstrated association of FXYD-11 and α-subunit strengthens our hypothesis that FXYD-11 has a role in modulating the pump's kinetic properties. The presence of putative phosphorylation sites on the intracellular domain of FXYD-11 suggests the possibility that this protein also may transmit external signals that regulate Na+-K+-ATPase activity.

scholarly journals Calcitriol Suppresses HIF-1 and HIF-2 Transcriptional Activity by Reducing HIF-1/2α Protein Levels via a VDR-Independent Mechanism

Cells ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 2440
Author(s):  
Ioanna-Maria Gkotinakou ◽  
Eleni Kechagia ◽  
Kalliopi Pazaitou-Panayiotou ◽  
Ilias Mylonis ◽  
Panagiotis Liakos ◽  
...  
Keyword(s):  
Vitamin D ◽  
Transcriptional Activity ◽  
Mrna Translation ◽  
Biologically Active ◽  
Mrna Levels ◽  
Akt Signaling Pathway ◽  
Protein Levels ◽  
Anticancer Properties ◽  
Independent Mechanism ◽  
Negative Effect

Hypoxia-inducible transcription factors 1 and 2 (HIFs) are major mediators of cancer development and progression and validated targets for cancer therapy. Although calcitriol, the biologically active metabolite of vitamin D, was attributed with anticancer properties, there is little information on the effect of calcitriol on HIFs and the mechanism underling this activity. Here, we demonstrate the negative effect of calcitriol on HIF-1/2α protein levels and HIF-1/2 transcriptional activity and elucidate the molecular mechanism of calcitriol action. We also reveal that the suppression of vitamin D receptor (VDR) expression by siRNA does not abrogate the negative regulation of HIF-1α and HIF-2α protein levels and HIF-1/2 transcriptional activity by calcitriol, thus testifying that the mechanism of these actions is VDR independent. At the same time, calcitriol significantly reduces the phosphorylation of Akt protein kinase and its downstream targets and suppresses HIF-1/2α protein synthesis by inhibiting HIF1A and EPAS1 (Endothelial PAS domain-containing protein 1) mRNA translation, without affecting their mRNA levels. On the basis of the acquired data, it can be proposed that calcitriol reduces HIF-1α and HIF-2α protein levels and inhibits HIF-1 and HIF-2 transcriptional activity by a VDR-independent, nongenomic mechanism that involves inhibition of PI3K/Akt signaling pathway and suppression of HIF1A and EPAS1 mRNA translation.

361 GROWTH HORMONE RECEPTOR MUTANT PIGS PRODUCED BY USING THE CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS (CRISPR) AND CRISPR-ASSOCIATED SYSTEMS IN IN VITRO-PRODUCED ZYGOTES

2015 ◽  
Vol 27 (1) ◽  
pp. 269 ◽  
Author(s):  
M. Kurome ◽  
M. Dahlhoff ◽  
S. Bultmann ◽  
S. Krebs ◽  
H. Blum ◽  
...  
Keyword(s):  
Growth Hormone ◽  
Hormone Receptor ◽  
Growth Hormone Receptor ◽  
Large Animal Model ◽  
Large Animal ◽  
Single Mutation ◽  
Wild Type ◽  
Ghr Gene ◽  
Normal Fertilization

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) technology is considered as an efficient strategy for generating gene edited large animals, such as pigs. Compared to somatic cell nuclear transfer, this new technology offers a relatively simple way to generate mutant pigs by direct injection of RNA into the cytoplasm of zygotes. Moreover, the use of in vitro produced zygotes would provide a highly effective and practical method for the production of porcine disease models for biomedical research. Here we examined the production efficiency of growth hormone receptor (GHR) mutant pigs by the combination of the CRISPR/Cas system and in vitro produced zygotes. In vitro maturation (IVM) of oocytes was performed as described previously (Kurome et al., Meth. Mol. Biol., in press). In all experiments, the same batch of frozen sperm was used. After IVM, around 20 oocytes with expanded cumulus cells were incubated with 5 × 104 spermatozoa in a 100-μL drop of porcine fertilization medium for 7 h. In vitro-produced embryos were assessed by the ratio of normal fertilization (eggs with 2 pronuclei) and blastocyst formation at Day 7. The Cas9 mRNA and a single guide RNA, recognising a short sequence of 20 base pairs in exon 3 of the GHR gene, were injected directly into the cytoplasm of the embryos 8.5 to 9.5 h after IVF. Injected embryos were transferred laparoscopically to recipient pigs, and 86.4% (57/66) of sperm-penetrated oocytes (66/96) exhibited normal fertilization. Incidence of polyspermy was relatively low (9/66, 13.6%). Developmental ability of in vitro-produced embryos to the blastocyst stage was 17.4% (24/138). In total, 426 RNA-injected embryos were transferred into 2 recipients, one of which became pregnant and gave birth to 8 piglets. All piglets were clinically healthy and developed normally. In 3 out of 8 piglets (37.5%), mutations were introduced. Next-generation sequencing revealed that all of them were mosaics: one with a single mutation (22% wild-type/78% mutant) and 2 piglets with 2 different mutations (80% wild-type/2% mutant_1/18% mutant_2 and 94% wild-type/4% mutant_1/2% mutant_2). Four out of 5 mutations caused a frameshift in the GHR gene. Our study reports for the first time generation of GHR mutant pigs by the use of the CRISPR/Cas system in in vitro-produced zygotes. Because all GHR mutant offspring were mosaic, Cas9 activation probably occurred after the 1-cell stage under our experimental conditions. The founder animal with the highest proportion of mutant GHR alleles will be used for breeding to establish a large animal model for Laron syndrome.This work is supported by the German Research Council (TR-CRC 127).

scholarly journals 5′-UTR recruitment of the translation initiation factor eIF4GI or DAP5 drives cap-independent translation of a subset of human mRNAs

2020 ◽  
Vol 295 (33) ◽  
pp. 11693-11706 ◽  
Author(s):  
Solomon A. Haizel ◽  
Usha Bhardwaj ◽  
Ruben L. Gonzalez ◽  
Somdeb Mitra ◽  
Dixie J. Goss
Keyword(s):  
Translation Initiation ◽  
Tumor Hypoxia ◽  
Mrna Translation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Luciferase Reporter ◽  
Binding Studies ◽  
Specific Subset ◽  
Independent Translation

During unfavorable conditions (e.g. tumor hypoxia or viral infection), canonical, cap-dependent mRNA translation is suppressed in human cells. Nonetheless, a subset of physiologically important mRNAs (e.g. hypoxia-inducible factor 1α [HIF-1α], fibroblast growth factor 9 [FGF-9], and p53) is still translated by an unknown, cap-independent mechanism. Additionally, expression levels of eukaryotic translation initiation factor 4GI (eIF4GI) and of its homolog, death-associated protein 5 (DAP5), are elevated. By examining the 5′ UTRs of HIF-1α, FGF-9, and p53 mRNAs and using fluorescence anisotropy binding studies, luciferase reporter-based in vitro translation assays, and mutational analyses, we demonstrate here that eIF4GI and DAP5 specifically bind to the 5′ UTRs of these cap-independently translated mRNAs. Surprisingly, we found that the eIF4E-binding domain of eIF4GI increases not only the binding affinity but also the selectivity among these mRNAs. We further demonstrate that the affinities of eIF4GI and DAP5 binding to these 5′ UTRs correlate with the efficiency with which these factors drive cap-independent translation of these mRNAs. Integrating the results of our binding and translation assays, we conclude that eIF4GI or DAP5 is critical for recruitment of a specific subset of mRNAs to the ribosome, providing mechanistic insight into their cap-independent translation.

scholarly journals Dose-response effects of a new growth hormone receptor antagonist (B2036-PEG) on circulating, hepatic and renal expression of the growth hormone/insulin-like growth factor system in adult mice

2000 ◽  
Vol 167 (2) ◽  
pp. 295-303 ◽  
Author(s):  
JW van Neck ◽  
NF Dits ◽  
V Cingel ◽  
IA Hoppenbrouwers ◽  
SL Drop ◽  
...  
Keyword(s):  
Growth Hormone ◽  
Growth Factor ◽  
Receptor Antagonist ◽  
Growth Hormone Receptor ◽  
Binding Protein ◽  
Mrna Levels ◽  
Insulin Like Growth Factor ◽  
Gh Receptor ◽  
Igf I ◽  
Igfbp 3

The effects of growth hormone (GH) in regulating the expression of the hepatic and renal GH and insulin-like growth factor (IGF) system were studied by administering a novel GH receptor antagonist (GHRA) (B2036-PEG) at different doses (0, 1.25, 2.5, 5 and 10 mg/kg/day) to mice for 7 days. No differences were observed in the groups with respect to body weight, food consumption or blood glucose. However, a dose-dependent decrease was observed in circulating IGF-I levels and in hepatic and renal IGF-I levels at the highest doses. In contrast, in the 5 and 10 mg/kg/day GHRA groups, circulating and hepatic transcriptional IGF binding protein-3 (IGFBP-3) levels were not modified, likely resulting in a significantly decreased IGF-I/IGFBP-3 ratio. Hepatic GH receptor (GHR) and GH binding protein (GHBP) mRNA levels increased significantly in all GHRA dosage groups. Endogenous circulatory GH levels increased significantly in the 2.5 and 5 mg/kg/day GHRA groups. Remarkably, increased circulating IGFBP-4 and hepatic IGFBP-4 mRNA levels were observed in all GHRA administration groups. Renal GHR and GHBP mRNA levels were not modified by GHRA administration at the highest doses. Also, renal IGFBP-3 mRNA levels remained unchanged in most GHRA administration groups, whereas IGFBP-1, -4 and -5 mRNA levels were significantly increased in the 5 and 10 mg/kg/day GHRA administration groups. In conclusion, the effects of a specific GHR blockade on circulating, hepatic and renal GH/IGF axis reported here, may prove useful in the future clinical use of GHRAs.

scholarly journals Disruption of insulin-like growth factor-II imprinting during embryonic development rescues the dwarf phenotype of mice null for pregnancy-associated plasma protein-A

2005 ◽  
Vol 186 (2) ◽  
pp. 325-331 ◽  
Author(s):  
Laurie K Bale ◽  
Cheryl A Conover
Keyword(s):  
Body Size ◽  
Growth Factor ◽  
Plasma Protein ◽  
Fetal Development ◽  
Protein A ◽  
Receptor Activation ◽  
Mutant Mice ◽  
Insulin Like Growth Factor ◽  
Dwarf Phenotype

Pregnancy-associated plasma protein-A (PAPP-A), an insulin-like growth factor-binding protein (IGFBP) protease, increases insulin-like growth factor (IGF) activity through cleavage of inhibitory IGFBP-4 and the consequent release of IGF peptide for receptor activation. Mice homozygous for targeted disruption of the PAPP-A gene are born as proportional dwarfs and exhibit retarded bone ossification during fetal development. Phenotype and in vitro data support a model in which decreased IGF-II bioavailability during embryogenesis results in growth retardation and reduction in overall body size. To test the hypothesis that an increase in IGF-II during embryogenesis would overcome the growth deficiencies, PAPP-A-null mice were crossed with ΔH19 mutant mice, which have increased IGF-II expression and fetal overgrowth due to disruption of IgfII imprinting. ΔH19 mutant mice were 126% and PAPP-A-null mice were 74% the size of controls at birth. These size differences were evident at embryonic day 16.5. Importantly, double mutants were indistinguishable from controls both in terms of size and skeletal development. Body size programmed during embryo development persisted post-natally. Thus, disruption of IgfII imprinting and consequent elevation in IGF-II during fetal development was associated with rescue of the dwarf phenotype and ossification defects of PAPP-A-null mice. These data provide strong genetic evidence that PAPP-A plays an essential role in determining IGF-II bioavailability for optimal fetal growth and development.

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