mTORC1 Transcriptional Regulation of Ribosome Subunits, Protein Synthesis, and Molecular Transport in Primary Human Trophoblast Cells

Mechanistic Target of Rapamycin Complex 1 (mTORC1) serves as positive regulator of placental nutrient transport and mitochondrial respiration. The role of mTORC1 signaling in modulating other placental functions is largely unexplored. We used gene array following silencing of raptor to identify genes regulated by mTORC1 in primary human trophoblast (PHT) cells. Seven hundred and thirty-nine genes were differentially expressed; 487 genes were down-regulated and 252 up-regulated. Bioinformatic analyses demonstrated that inhibition of mTORC1 resulted in decreased expression of genes encoding ribosomal proteins in the 60S and 40S ribosome subunits. Furthermore, down-regulated genes were functionally enriched in genes involved in eIF2, sirtuin and mTOR signaling, mitochondrial function, and glutamine and zinc transport. Stress response genes were enriched among up-regulated genes following mTORC1 inhibition. The protein expression of ribosomal proteins RPL26 (RPL26) and Ribosomal Protein S10 (RPS10) was decreased and positively correlated to mTORC1 signaling and System A amino acid transport in human placentas collected from pregnancies complicated by intrauterine growth restriction (IUGR). In conclusion, mTORC1 signaling regulates the expression of trophoblast genes involved in ribosome and protein synthesis, mitochondrial function, lipid metabolism, nutrient transport, and angiogenesis, representing novel links between mTOR signaling and multiple placental functions critical for normal fetal growth and development.

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Mechanistic Target of Rapamycin Complex 2 Regulation of the Primary Human Trophoblast Cell Transcriptome

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.670980 ◽

2021 ◽

Vol 9 ◽

Author(s):

Fredrick J. Rosario ◽

Amy Catherine Kelly ◽

Madhulika B. Gupta ◽

Theresa L. Powell ◽

Laura Cox ◽

...

Keyword(s):

Protein Expression ◽

Gene Array ◽

Target Of Rapamycin ◽

Human Trophoblast ◽

Mechanistic Target Of Rapamycin ◽

Expression Of Genes ◽

Genes Encoding ◽

Cell Transcriptome ◽

Pathway Analyses

Mechanistic Target of Rapamycin Complex 2 (mTORC2) regulates placental amino acid and folate transport. However, the role of mTORC2 in modulating other placental functions is largely unexplored. We used a gene array following the silencing of rictor to identify genes regulated by mTORC2 in primary human trophoblast (PHT) cells. Four hundred and nine genes were differentially expressed; 102 genes were down-regulated and 307 up-regulated. Pathway analyses demonstrated that inhibition of mTORC2 resulted in increased expression of genes encoding for pro-inflammatory IL-6, VEGF-A, leptin, and inflammatory signaling (SAPK/JNK). Furthermore, down-regulated genes were functionally enriched in genes involved in angiogenesis (Osteopontin) and multivitamin transport (SLC5A6). In addition, the protein expression of leptin, VEGFA, IL-6 was increased and negatively correlated to mTORC2 signaling in human placentas collected from pregnancies complicated by intrauterine growth restriction (IUGR). In contrast, the protein expression of Osteopontin and SLC5A6 was decreased and positively correlated to mTORC2 signaling in human IUGR placentas. In conclusion, mTORC2 signaling regulates trophoblast expression of genes involved in inflammation, micronutrient transport, and angiogenesis, representing novel links between mTOR signaling and multiple placental functions necessary for fetal growth and development.

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Intricate regulation of ribosome biogenesis genes in response to mTORC1 signaling

10.1101/2021.01.04.425249 ◽

2021 ◽

Author(s):

Sanjay Kumar ◽

Muneera Mashkoor ◽

Priya Balamurugan ◽

Anne Grove

Keyword(s):

Saccharomyces Cerevisiae ◽

Ribosomal Proteins ◽

Ribosome Biogenesis ◽

Target Genes ◽

Common Ancestor ◽

Gene Promoters ◽

Rrna Processing ◽

Processing Factor ◽

Mtorc1 Signaling ◽

Genes Encoding

SummaryGenes encoding ribosomal proteins are repressed in response to inhibition of mTORC1. In Saccharomyces cerevisiae, this involves dissociation of the activator Ifh1p in a process that depends on Utp22p, a protein that also functions in pre-rRNA processing. Ifh1p has a paralog, Crf1p, which can mediate mTORC1 inhibition by acting as a repressor. Ifh1p and Crf1p derive from a common ancestor, which may have acted as both an activator and a repressor. We report here that UTP22 and RRP7, which encodes another pre-rRNA processing factor, are controlled by mTORC1; both gene promoters are bound by Ifh1p, which dissociates on mTORC1 inhibition. Notably, Crf1p acts as an activator as evidenced by reduced expression in a crf1Δ strain. By contrast, Crf1p is required to repress expression of HMO1, which encodes a cofactor involved in communicating mTORC1 activity to target genes. Our data therefore indicate that Crf1p exhibits the dual repressor/activator functions of the Ifh1p-Crf1p ancestor.

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Faculty Opinions recommendation of Effect of age on basal muscle protein synthesis and mTORC1 signaling in a large cohort of young and older men and women.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.725380242.793518281 ◽

2016 ◽

Author(s):

James Timmons ◽

Iain Gallagher

Keyword(s):

Protein Synthesis ◽

Muscle Protein ◽

Muscle Protein Synthesis ◽

Older Men ◽

Large Cohort ◽

Men And Women ◽

Mtorc1 Signaling ◽

Older Men And Women ◽

Effect Of Age

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Leucine or carbohydrate supplementation reduces AMPK and eEF2 phosphorylation and extends postprandial muscle protein synthesis in rats

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00242.2011 ◽

2011 ◽

Vol 301 (6) ◽

pp. E1236-E1242 ◽

Cited By ~ 46

Author(s):

Gabriel J. Wilson ◽

Donald K. Layman ◽

Christopher J. Moulton ◽

Layne E. Norton ◽

Tracy G. Anthony ◽

...

Keyword(s):

Protein Synthesis ◽

Test Meal ◽

Muscle Protein ◽

Muscle Protein Synthesis ◽

Adenosine Monophosphate ◽

Energy Status ◽

Sprague Dawley ◽

Translation Elongation ◽

Cellular Energy ◽

Mtorc1 Signaling

Muscle protein synthesis (MPS) increases after consumption of a protein-containing meal but returns to baseline values within 3 h despite continued elevations of plasma amino acids and mammalian target of rapamycin (mTORC1) signaling. This study evaluated the potential for supplemental leucine (Leu), carbohydrates (CHO), or both to prolong elevated MPS after a meal. Male Sprague-Dawley rats (∼270 g) trained to consume three meals daily were food deprived for 12 h, and then blood and gastrocnemius muscle were collected 0, 90, or 180 min after a standard 4-g test meal (20% whey protein). At 135 min postmeal, rats were orally administered 2.63 g of CHO, 270 mg of Leu, both, or water (sham control). Following test meal consumption, MPS peaked at 90 min and then returned to basal ( time 0) rates at 180 min, although ribosomal protein S6 kinase and eIF4E-binding protein-1 phosphorylation remained elevated. In contrast, rats administered Leu and/or CHO supplements at 135 min postmeal maintained peak MPS through 180 min. MPS was inversely associated with the phosphorylation states of translation elongation factor 2, the “cellular energy sensor” adenosine monophosphate-activated protein kinase-α (AMPKα) and its substrate acetyl-CoA carboxylase, and increases in the ratio of AMP/ATP. We conclude that the incongruity between MPS and mTORC1 at 180 min reflects a block in translation elongation due to reduced cellular energy. Administering Leu or CHO supplements ∼2 h after a meal maintains cellular energy status and extends the postprandial duration of MPS.

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KLK11 promotes the activation of mTOR and protein synthesis to facilitate cardiac hypertrophy

BMC Cardiovascular Disorders ◽

10.1186/s12872-021-02053-y ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Yi Wang ◽

Hongjuan Liao ◽

Yueheng Wang ◽

Jinlin Zhou ◽

Feng Wang ◽

...

Keyword(s):

Protein Synthesis ◽

Angiotensin Ii ◽

Cardiac Hypertrophy ◽

Western Blot ◽

Real Time ◽

Real Time Pcr ◽

Mtor Signaling ◽

Heart Weight ◽

Cardiomyocyte Hypertrophy ◽

Quantitative Real Time Pcr

Abstract Background Cardiovascular diseases have become the leading cause of death worldwide, and cardiac hypertrophy is the core mechanism underlying cardiac defect and heart failure. However, the underlying mechanisms of cardiac hypertrophy are not fully understood. Here we investigated the roles of Kallikrein 11 (KLK11) in cardiac hypertrophy. Methods Human and mouse hypertrophic heart tissues were used to determine the expression of KLK11 with quantitative real-time PCR and western blot. Mouse cardiac hypertrophy was induced by transverse aortic constriction (TAC), and cardiomyocyte hypertrophy was induced by angiotensin II. Cardiac function was analyzed by echocardiography. The signaling pathway was analyzed by western blot. Protein synthesis was monitored by the incorporation of [3H]-leucine. Gene expression was analyzed by quantitative real-time PCR. Results The mRNA and protein levels of KLK11 were upregulated in human hypertrophic hearts. We also induced cardiac hypertrophy in mice and observed the upregulation of KLK11 in hypertrophic hearts. Our in vitro experiments demonstrated that KLK11 overexpression promoted whereas KLK11 knockdown repressed cardiomyocytes hypertrophy induced by angiotensin II, as evidenced by cardiomyocyte size and the expression of hypertrophy-related fetal genes. Besides, we knocked down KLK11 expression in mouse hearts with adeno-associated virus 9. Knockdown of KLK11 in mouse hearts inhibited TAC-induced decline in fraction shortening and ejection fraction, reduced the increase in heart weight, cardiomyocyte size, and expression of hypertrophic fetal genes. We also observed that KLK11 promoted protein synthesis, the key feature of cardiomyocyte hypertrophy, by regulating the pivotal machines S6K1 and 4EBP1. Mechanism study demonstrated that KLK11 promoted the activation of AKT-mTOR signaling to promote S6K1 and 4EBP1 pathway and protein synthesis. Repression of mTOR with rapamycin blocked the effects of KLK11 on S6K1 and 4EBP1 as well as protein synthesis. Besides, rapamycin treatment blocked the roles of KLK11 in the regulation of cardiomyocyte hypertrophy. Conclusions Our findings demonstrated that KLK11 promoted cardiomyocyte hypertrophy by activating AKT-mTOR signaling to promote protein synthesis.

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Upregulation of 5′-terminal oligopyrimidine mRNA translation upon loss of the ARF tumor suppressor

Scientific Reports ◽

10.1038/s41598-020-79379-8 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Kyle A. Cottrell ◽

Ryan C. Chiou ◽

Jason D. Weber

Keyword(s):

Protein Synthesis ◽

Tumor Cells ◽

Ribosomal Proteins ◽

Human Cancer ◽

Mrna Translation ◽

Ribosome Profiling ◽

Translational Efficiency ◽

Gene Encoding ◽

Terminal Oligopyrimidine ◽

Ribosome Export

AbstractTumor cells require nominal increases in protein synthesis in order to maintain high proliferation rates. As such, tumor cells must acquire enhanced ribosome production. How the numerous mutations in tumor cells ultimately achieve this aberrant production is largely unknown. The gene encoding ARF is the most commonly deleted gene in human cancer. ARF plays a significant role in regulating ribosomal RNA synthesis and processing, ribosome export into the cytoplasm, and global protein synthesis. Utilizing ribosome profiling, we show that ARF is a major suppressor of 5′-terminal oligopyrimidine mRNA translation. Genes with increased translational efficiency following loss of ARF include many ribosomal proteins and translation factors. Knockout of p53 largely phenocopies ARF loss, with increased protein synthesis and expression of 5′-TOP encoded proteins. The 5′-TOP regulators eIF4G1 and LARP1 are upregulated in Arf- and p53-null cells.

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40S ribosome profiling reveals distinct roles for Tma20/Tma22 (MCT-1/DENR) and Tma64 (eIF2D) in 40S subunit recycling

Nature Communications ◽

10.1038/s41467-021-23223-8 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

David J. Young ◽

Sezen Meydan ◽

Nicholas R. Guydosh

Keyword(s):

Protein Synthesis ◽

Neurological Disease ◽

Ribosome Profiling ◽

Minor Role ◽

Stop Codons ◽

Genes Encoding ◽

Ribosome Footprinting ◽

A Minor ◽

And Control ◽

In Cells

AbstractThe recycling of ribosomes at stop codons for use in further rounds of translation is critical for efficient protein synthesis. Removal of the 60S subunit is catalyzed by the ATPase Rli1 (ABCE1) while removal of the 40S is thought to require Tma64 (eIF2D), Tma20 (MCT-1), and Tma22 (DENR). However, it remains unclear how these Tma proteins cause 40S removal and control reinitiation of downstream translation. Here we used a 40S ribosome footprinting strategy to directly observe intermediate steps of ribosome recycling in cells. Deletion of the genes encoding these Tma proteins resulted in broad accumulation of unrecycled 40S subunits at stop codons, directly establishing their role in 40S recycling. Furthermore, the Tma20/Tma22 heterodimer was responsible for a majority of 40S recycling events while Tma64 played a minor role. Introduction of an autism-associated mutation into TMA22 resulted in a loss of 40S recycling activity, linking ribosome recycling and neurological disease.

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