Ag-dependent activation of naive T cells induces dramatic changes in cellular metabolism that are... more Ag-dependent activation of naive T cells induces dramatic changes in cellular metabolism that are essential for cell growth, division, and differentiation. In recent years, the serine/threonine kinase mechanistic target of rapamycin (mTOR) has emerged as a key integrator of signaling pathways that regulate these metabolic processes. However, the role of specific downstream effectors of mTOR function in T cells is poorly understood. Ribosomal protein S6 (rpS6) is an essential component of the ribosome and is inducibly phosphorylated following mTOR activation in eukaryotic cells. In the current work, we addressed the role of phosphorylation of rpS6 as an effector of mTOR function in T cell development, growth, proliferation, and differentiation using knockin and TCR transgenic mice. Surprisingly, we demonstrate that rpS6 phosphorylation is not required for any of these processes either in vitro or in vivo. Indeed, rpS6 knockin mice are completely sensitive to the inhibitory effects of rapamycin and an S6 kinase 1 (S6K1)-specific inhibitor on T cell activation and proliferation. These results place the mTOR complex 1-S6K1 axis as a crucial determinant of T cell activation independently of its ability to regulate rpS6 phosphorylation.
Biochimica Et Biophysica Acta Gene Regulatory Mechanisms, 2014
TOP mRNAs encode proteins of the translational machinery.The hallmark of TOP mRNAs is a 5′ termin... more TOP mRNAs encode proteins of the translational machinery.The hallmark of TOP mRNAs is a 5′ terminal oligopyrimidine (5′ TOP) motif.TOP mRNAs are translationally repressed when subjected to a variety of stresses.Translational control of TOP mRNAs is mediated by the mTOR pathwayThe identity of the translational trans-acting factor of TOP mRNAs is still elusiveCells encountering hostile growth conditions, like those residing in the middle of a newly developing solid tumor, conserve resources and energy by downregulating protein synthesis. One mechanism in this response is the translational repression of multiple mRNAs that encode components of the translational apparatus. This coordinated translational control is carried through a common cis-regulatory element, the 5′ Terminal OligoPyrimidine motif (5′TOP), after which these mRNAs are referred to as TOP mRNAs. Subsequent to the initial structural and functional characterization of members of this family, the research of TOP mRNAs has progressed in three major directions: a) delineating the landscape of the family; b) establishing the pathways that transduce stress cues into selective translational repression; and c) attempting to decipher the most proximal trans-acting factor(s) and defining its mode of action — a repressor or activator. The present chapter critically reviews the development in these three avenues of research with a special emphasis on the two “top secrets” of the TOP mRNA family: the scope of its members and the identity of the proximal cellular regulator(s). This article is part of a Special Issue entitled: Translation and Cancer.
Cells encountering hostile growth conditions, like those residing in the middle of a newly develo... more Cells encountering hostile growth conditions, like those residing in the middle of a newly developing solid tumor, conserve resources and energy by downregulating protein synthesis. One mechanism in this response is the translational repression of multiple mRNAs that encode components of the translational apparatus. This coordinated translational control is carried through a common cis-regulatory element, the 5' Terminal OligoPyrimidine motif (5'TOP), after which these mRNAs are referred to as TOP mRNAs. Subsequent to the initial structural and functional characterization of members of this family, the research of TOP mRNAs has progressed in three major directions: a) delineating the landscape of the family; b) establishing the pathways that transduce stress cues into selective translational repression; and c) attempting to decipher the most proximal trans-acting factor(s) and defining its mode of action - a repressor or activator. The present chapter critically reviews the ...
International review of cell and molecular biology, 2008
The phosphorylation of ribosomal protein S6 (rpS6), which occurs in response to a wide variety of... more The phosphorylation of ribosomal protein S6 (rpS6), which occurs in response to a wide variety of stimuli on five evolutionarily conserved serine residues, has attracted much attention since its discovery more than three decades ago. However, despite a large body of information on the respective kinases and the signal transduction pathways, the role of this phosphorylation remained obscure. It is only recent that targeting the genes encoding rpS6, the phosphorylatable serine residues or the respective kinases that the unique role of rpS6 and its posttranslational modification have started to be elucidated. This review focuses primarily on the critical role of rpS6 for mouse development, the pathways that transduce various signals into rpS6 phosphorylation, and the physiological functions of this modification. The mechanism(s) underlying the diverse effects of rpS6 phosphorylation on cellular and organismal physiology has yet to be determined. However, a model emerging from the curre...
Recent studies are beginning to disclose a signaling network involved in regulating cell size. Al... more Recent studies are beginning to disclose a signaling network involved in regulating cell size. Although many links and effectors are still unknown, central components of this network include the mammalian target of rapamycin (mTOR) and its downstream effectors - the ribosomal protein S6 kinase (S6K) and the translational repressor eukaryotic initiation factor 4E-binding protein. Until recently, the role of S6K and its many substrates in cell-size control remained obscure; however, a knockin mouse carrying mutations at all phosphorylation sites in the primary S6K substrate, ribosomal protein S6 (rpS6), has provided insight into the physiological role of this protein phosphorylation event. In addition to its role in glucose homeostasis in the whole mouse, phosphorylation of rpS6 is essential for regulating the size of at least some cell types, but is dispensable for translational control of mRNAs with a 5' terminal oligopyrimidine tract (TOP mRNAs) - its previously assigned target...
International Review of Cell and Molecular Biology, 2015
The phosphorylation of ribosomal protein S6 (rpS6) has been described for the first time about fo... more The phosphorylation of ribosomal protein S6 (rpS6) has been described for the first time about four decades ago. Since then, numerous studies have shown that this modification occurs in response to a wide variety of stimuli on five evolutionarily conserved serine residues. However, despite a large body of information on the respective kinases and the signal transduction pathways, the physiological role of rpS6 phosphorylation remained obscure until genetic manipulations were applied in both yeast and mammals in an attempt to block this modification. Thus, studies based on both mice and cultured cells subjected to disruption of the genes encoding rpS6 and the respective kinases, as well as the substitution of the phosphorylatable serine residues in rpS6, have laid the ground for the elucidation of the multiple roles of this protein and its posttranslational modification. This review focuses primarily on newly identified kinases that phosphorylate rpS6, pathways that transduce various signals into rpS6 phosphorylation, and the recently established physiological functions of this modification. It should be noted, however, that despite the significant progress made in the last decade, the molecular mechanism(s) underlying the diverse effects of rpS6 phosphorylation on cellular and organismal physiology are still poorly understood.
When resting (G0) mouse 3T6 fibroblasts are serum stimulated to reenter the cell cycle, the rates... more When resting (G0) mouse 3T6 fibroblasts are serum stimulated to reenter the cell cycle, the rates of synthesis of rRNA and ribosomal proteins increase, resulting in an increase in ribosome content beginning about 6 h after stimulation. In this study, we monitored the content, metabolism, and translation of ribosomal protein mRNA (rp mRNA) in resting, exponentially growing, and serum-stimulated 3T6 cells. Cloned cDNAs for seven rp mRNAs were used in DNA-excess filter hybridization studies to assay rp mRNA. We found that about 85% of rp mRNA is polyadenylated under all growth conditions. The rate of labeling of rp mRNA relative to total polyadenylated mRNA changed very little after stimulation. The half-life of rp mRNA was about 11 h in resting cells and about 8 h in exponentially growing cells, values which are similar to the half-lives of total mRNA in resting and growing cells (about 9 h). The content of rp mRNA relative to total mRNA was about the same in resting and growing 3T6 c...
The translation of mammalian ribosomal protein (rp) mRNAs is selectively repressed in nongrowing ... more The translation of mammalian ribosomal protein (rp) mRNAs is selectively repressed in nongrowing cells. This response is mediated through a regulatory element residing in the 5' untranslated region of these mRNAs and includes a 5' terminal oligopyrimidine tract (5' TOP). To further characterize the translational cis-regulatory element, we monitored the translational behavior of various endogenous and heterologous mRNAs or hybrid transcripts derived from transfected chimeric genes. The translational efficiency of these mRNAs was assessed in cells that either were growing normally or were growth arrested under various physiological conditions. Our experiments have yielded the following results: (i) the translation of mammalian rp mRNAs is properly regulated in amphibian cells, and likewise, amphibian rp mRNA is regulated in mammalian cells, indicating that all of the elements required for translation control of rp mRNAs are conserved among vertebrate classes; (ii) selectiv...
Mammalian liver development is accompanied by a transition from rapid growth in the fetus to a qu... more Mammalian liver development is accompanied by a transition from rapid growth in the fetus to a quiescent state in the adult. However, extensive proliferation can be induced in the adult liver by partial hepatectomy. In this study, we examined the regulation of ribosomal protein (rp) gene expression in the developing and regenerating rat liver. Our results indicate that the translation of rp mRNAs is selectively repressed by about 70% upon development from fetal to adult life, as illustrated by the decrease in ribosomal loading. In addition, the relative abundance of these mRNAs, like that of several other, but not all, housekeeping mRNAs, declines during development through a posttranscriptional mechanism. When liver cells commence growth following partial hepatectomy, translation of rp mRNAs is resumed to near-maximal capacity, as judged by their very efficient recruitment into polysomes. The concomitant increase in the abundance rp mRNAs under these circumstances is achieved by a ...
The translational efficiency of mammalian ribosomal protein mRNAs correlates with the growth stat... more The translational efficiency of mammalian ribosomal protein mRNAs correlates with the growth status of the cells and its control is mediated through a 5' terminal oligopyrimidine tract (5' TOP) common to all these mRNAs. In the present study, we demonstrate that the plant translational apparatus, as represented by wheat-germ extract, discriminates against mammalian mRNAs containing this motif to the same extent as do quiescent mammalian cells. Moreover, mutations in the 5' TOP, which abolish the growth-dependent translational control of the respective mRNAs in mammalian cells, render these mRNAs refractory to discrimination in the plant cell-free system. This selective discrimination reflects neither the specific instability of 5' TOP-containing mRNAs during the incubation in vitro nor a lower competitive potential for the cap-binding protein. The lower in vitro translational efficiency of these mRNAs is an inherent feature which is independent of whether they were derived from polysomes or messenger ribonucleoprotein particles of the transfected mammalian cells. The conservation of the discriminatory property of the translational apparatus between the animal and plant kingdoms is discussed from mechanistic and evolutionary points of view.
Cloned mouse ribosomal protein (rp) cDNAs exhibit extensive homology with the corresponding rat s... more Cloned mouse ribosomal protein (rp) cDNAs exhibit extensive homology with the corresponding rat sequences. The size of the rp-mRNAs and complexity of the rp-genes are very similar in the two species. Using the mouse rp-recombinant DNAs we find that the relative abundance of rat L7, L13, L18, L30, L32/33 and S16 mRNAs increases after partial hepatectomy. Their maximal level is about twice that of normal rat liver, and is achieved 12-18 h after the operation, while the relative abundance of albumin mRNA decreases to half the normal values 12 h after partial hepatectomy. This concomitant increase in the relative content of these rp-mRNAs indicates coordinate regulation of their level in the rat. The dissimilar behavior of L10 and L19 rp-mRNA suggests additional control mechanisms of rp-mRNA levels in the regenerating rat liver.
TOP mRNAs are translationally controlled by mitogenic, growth, and nutritional stimuli through a ... more TOP mRNAs are translationally controlled by mitogenic, growth, and nutritional stimuli through a 5'-terminal oligopyrimidine tract. Here we show that LiCl can alleviate the translational repression of these mRNAs when progression through the cell cycle is blocked at G(0), G(1)/S, or G(2)/M phases in different cell lines and by various physiological and chemical means. This derepressive effect of LiCl does not involve resumption of cell division. Unlike its efficient derepressive effect in mitotically arrested cells, LiCl alleviates inefficiently the repression of TOP mRNAs in amino acid-deprived cells and has no effect in lymphoblastoids whose TOP mRNAs are constitutively repressed even when they are proliferating. LiCl is widely used as a relatively selective inhibitor of glycogen synthase kinase-3. However, inhibition per se of this enzyme by more specific drugs failed to derepress the translation of TOP mRNAs, implying that relief of the translational repression of TOP mRNAs by LiCl is carried out in a glycogen synthase kinase-3-independent manner. Moreover, this effect is apparent, at least in some cell lines, in the absence of S6-kinase 1 activation and ribosomal protein S6 phosphorylation, thus further supporting the notion that translational control of TOP mRNAs does not rely on either of these variables.
Ag-dependent activation of naive T cells induces dramatic changes in cellular metabolism that are... more Ag-dependent activation of naive T cells induces dramatic changes in cellular metabolism that are essential for cell growth, division, and differentiation. In recent years, the serine/threonine kinase mechanistic target of rapamycin (mTOR) has emerged as a key integrator of signaling pathways that regulate these metabolic processes. However, the role of specific downstream effectors of mTOR function in T cells is poorly understood. Ribosomal protein S6 (rpS6) is an essential component of the ribosome and is inducibly phosphorylated following mTOR activation in eukaryotic cells. In the current work, we addressed the role of phosphorylation of rpS6 as an effector of mTOR function in T cell development, growth, proliferation, and differentiation using knockin and TCR transgenic mice. Surprisingly, we demonstrate that rpS6 phosphorylation is not required for any of these processes either in vitro or in vivo. Indeed, rpS6 knockin mice are completely sensitive to the inhibitory effects of rapamycin and an S6 kinase 1 (S6K1)-specific inhibitor on T cell activation and proliferation. These results place the mTOR complex 1-S6K1 axis as a crucial determinant of T cell activation independently of its ability to regulate rpS6 phosphorylation.
Biochimica Et Biophysica Acta Gene Regulatory Mechanisms, 2014
TOP mRNAs encode proteins of the translational machinery.The hallmark of TOP mRNAs is a 5′ termin... more TOP mRNAs encode proteins of the translational machinery.The hallmark of TOP mRNAs is a 5′ terminal oligopyrimidine (5′ TOP) motif.TOP mRNAs are translationally repressed when subjected to a variety of stresses.Translational control of TOP mRNAs is mediated by the mTOR pathwayThe identity of the translational trans-acting factor of TOP mRNAs is still elusiveCells encountering hostile growth conditions, like those residing in the middle of a newly developing solid tumor, conserve resources and energy by downregulating protein synthesis. One mechanism in this response is the translational repression of multiple mRNAs that encode components of the translational apparatus. This coordinated translational control is carried through a common cis-regulatory element, the 5′ Terminal OligoPyrimidine motif (5′TOP), after which these mRNAs are referred to as TOP mRNAs. Subsequent to the initial structural and functional characterization of members of this family, the research of TOP mRNAs has progressed in three major directions: a) delineating the landscape of the family; b) establishing the pathways that transduce stress cues into selective translational repression; and c) attempting to decipher the most proximal trans-acting factor(s) and defining its mode of action — a repressor or activator. The present chapter critically reviews the development in these three avenues of research with a special emphasis on the two “top secrets” of the TOP mRNA family: the scope of its members and the identity of the proximal cellular regulator(s). This article is part of a Special Issue entitled: Translation and Cancer.
Cells encountering hostile growth conditions, like those residing in the middle of a newly develo... more Cells encountering hostile growth conditions, like those residing in the middle of a newly developing solid tumor, conserve resources and energy by downregulating protein synthesis. One mechanism in this response is the translational repression of multiple mRNAs that encode components of the translational apparatus. This coordinated translational control is carried through a common cis-regulatory element, the 5' Terminal OligoPyrimidine motif (5'TOP), after which these mRNAs are referred to as TOP mRNAs. Subsequent to the initial structural and functional characterization of members of this family, the research of TOP mRNAs has progressed in three major directions: a) delineating the landscape of the family; b) establishing the pathways that transduce stress cues into selective translational repression; and c) attempting to decipher the most proximal trans-acting factor(s) and defining its mode of action - a repressor or activator. The present chapter critically reviews the ...
International review of cell and molecular biology, 2008
The phosphorylation of ribosomal protein S6 (rpS6), which occurs in response to a wide variety of... more The phosphorylation of ribosomal protein S6 (rpS6), which occurs in response to a wide variety of stimuli on five evolutionarily conserved serine residues, has attracted much attention since its discovery more than three decades ago. However, despite a large body of information on the respective kinases and the signal transduction pathways, the role of this phosphorylation remained obscure. It is only recent that targeting the genes encoding rpS6, the phosphorylatable serine residues or the respective kinases that the unique role of rpS6 and its posttranslational modification have started to be elucidated. This review focuses primarily on the critical role of rpS6 for mouse development, the pathways that transduce various signals into rpS6 phosphorylation, and the physiological functions of this modification. The mechanism(s) underlying the diverse effects of rpS6 phosphorylation on cellular and organismal physiology has yet to be determined. However, a model emerging from the curre...
Recent studies are beginning to disclose a signaling network involved in regulating cell size. Al... more Recent studies are beginning to disclose a signaling network involved in regulating cell size. Although many links and effectors are still unknown, central components of this network include the mammalian target of rapamycin (mTOR) and its downstream effectors - the ribosomal protein S6 kinase (S6K) and the translational repressor eukaryotic initiation factor 4E-binding protein. Until recently, the role of S6K and its many substrates in cell-size control remained obscure; however, a knockin mouse carrying mutations at all phosphorylation sites in the primary S6K substrate, ribosomal protein S6 (rpS6), has provided insight into the physiological role of this protein phosphorylation event. In addition to its role in glucose homeostasis in the whole mouse, phosphorylation of rpS6 is essential for regulating the size of at least some cell types, but is dispensable for translational control of mRNAs with a 5' terminal oligopyrimidine tract (TOP mRNAs) - its previously assigned target...
International Review of Cell and Molecular Biology, 2015
The phosphorylation of ribosomal protein S6 (rpS6) has been described for the first time about fo... more The phosphorylation of ribosomal protein S6 (rpS6) has been described for the first time about four decades ago. Since then, numerous studies have shown that this modification occurs in response to a wide variety of stimuli on five evolutionarily conserved serine residues. However, despite a large body of information on the respective kinases and the signal transduction pathways, the physiological role of rpS6 phosphorylation remained obscure until genetic manipulations were applied in both yeast and mammals in an attempt to block this modification. Thus, studies based on both mice and cultured cells subjected to disruption of the genes encoding rpS6 and the respective kinases, as well as the substitution of the phosphorylatable serine residues in rpS6, have laid the ground for the elucidation of the multiple roles of this protein and its posttranslational modification. This review focuses primarily on newly identified kinases that phosphorylate rpS6, pathways that transduce various signals into rpS6 phosphorylation, and the recently established physiological functions of this modification. It should be noted, however, that despite the significant progress made in the last decade, the molecular mechanism(s) underlying the diverse effects of rpS6 phosphorylation on cellular and organismal physiology are still poorly understood.
When resting (G0) mouse 3T6 fibroblasts are serum stimulated to reenter the cell cycle, the rates... more When resting (G0) mouse 3T6 fibroblasts are serum stimulated to reenter the cell cycle, the rates of synthesis of rRNA and ribosomal proteins increase, resulting in an increase in ribosome content beginning about 6 h after stimulation. In this study, we monitored the content, metabolism, and translation of ribosomal protein mRNA (rp mRNA) in resting, exponentially growing, and serum-stimulated 3T6 cells. Cloned cDNAs for seven rp mRNAs were used in DNA-excess filter hybridization studies to assay rp mRNA. We found that about 85% of rp mRNA is polyadenylated under all growth conditions. The rate of labeling of rp mRNA relative to total polyadenylated mRNA changed very little after stimulation. The half-life of rp mRNA was about 11 h in resting cells and about 8 h in exponentially growing cells, values which are similar to the half-lives of total mRNA in resting and growing cells (about 9 h). The content of rp mRNA relative to total mRNA was about the same in resting and growing 3T6 c...
The translation of mammalian ribosomal protein (rp) mRNAs is selectively repressed in nongrowing ... more The translation of mammalian ribosomal protein (rp) mRNAs is selectively repressed in nongrowing cells. This response is mediated through a regulatory element residing in the 5' untranslated region of these mRNAs and includes a 5' terminal oligopyrimidine tract (5' TOP). To further characterize the translational cis-regulatory element, we monitored the translational behavior of various endogenous and heterologous mRNAs or hybrid transcripts derived from transfected chimeric genes. The translational efficiency of these mRNAs was assessed in cells that either were growing normally or were growth arrested under various physiological conditions. Our experiments have yielded the following results: (i) the translation of mammalian rp mRNAs is properly regulated in amphibian cells, and likewise, amphibian rp mRNA is regulated in mammalian cells, indicating that all of the elements required for translation control of rp mRNAs are conserved among vertebrate classes; (ii) selectiv...
Mammalian liver development is accompanied by a transition from rapid growth in the fetus to a qu... more Mammalian liver development is accompanied by a transition from rapid growth in the fetus to a quiescent state in the adult. However, extensive proliferation can be induced in the adult liver by partial hepatectomy. In this study, we examined the regulation of ribosomal protein (rp) gene expression in the developing and regenerating rat liver. Our results indicate that the translation of rp mRNAs is selectively repressed by about 70% upon development from fetal to adult life, as illustrated by the decrease in ribosomal loading. In addition, the relative abundance of these mRNAs, like that of several other, but not all, housekeeping mRNAs, declines during development through a posttranscriptional mechanism. When liver cells commence growth following partial hepatectomy, translation of rp mRNAs is resumed to near-maximal capacity, as judged by their very efficient recruitment into polysomes. The concomitant increase in the abundance rp mRNAs under these circumstances is achieved by a ...
The translational efficiency of mammalian ribosomal protein mRNAs correlates with the growth stat... more The translational efficiency of mammalian ribosomal protein mRNAs correlates with the growth status of the cells and its control is mediated through a 5' terminal oligopyrimidine tract (5' TOP) common to all these mRNAs. In the present study, we demonstrate that the plant translational apparatus, as represented by wheat-germ extract, discriminates against mammalian mRNAs containing this motif to the same extent as do quiescent mammalian cells. Moreover, mutations in the 5' TOP, which abolish the growth-dependent translational control of the respective mRNAs in mammalian cells, render these mRNAs refractory to discrimination in the plant cell-free system. This selective discrimination reflects neither the specific instability of 5' TOP-containing mRNAs during the incubation in vitro nor a lower competitive potential for the cap-binding protein. The lower in vitro translational efficiency of these mRNAs is an inherent feature which is independent of whether they were derived from polysomes or messenger ribonucleoprotein particles of the transfected mammalian cells. The conservation of the discriminatory property of the translational apparatus between the animal and plant kingdoms is discussed from mechanistic and evolutionary points of view.
Cloned mouse ribosomal protein (rp) cDNAs exhibit extensive homology with the corresponding rat s... more Cloned mouse ribosomal protein (rp) cDNAs exhibit extensive homology with the corresponding rat sequences. The size of the rp-mRNAs and complexity of the rp-genes are very similar in the two species. Using the mouse rp-recombinant DNAs we find that the relative abundance of rat L7, L13, L18, L30, L32/33 and S16 mRNAs increases after partial hepatectomy. Their maximal level is about twice that of normal rat liver, and is achieved 12-18 h after the operation, while the relative abundance of albumin mRNA decreases to half the normal values 12 h after partial hepatectomy. This concomitant increase in the relative content of these rp-mRNAs indicates coordinate regulation of their level in the rat. The dissimilar behavior of L10 and L19 rp-mRNA suggests additional control mechanisms of rp-mRNA levels in the regenerating rat liver.
TOP mRNAs are translationally controlled by mitogenic, growth, and nutritional stimuli through a ... more TOP mRNAs are translationally controlled by mitogenic, growth, and nutritional stimuli through a 5'-terminal oligopyrimidine tract. Here we show that LiCl can alleviate the translational repression of these mRNAs when progression through the cell cycle is blocked at G(0), G(1)/S, or G(2)/M phases in different cell lines and by various physiological and chemical means. This derepressive effect of LiCl does not involve resumption of cell division. Unlike its efficient derepressive effect in mitotically arrested cells, LiCl alleviates inefficiently the repression of TOP mRNAs in amino acid-deprived cells and has no effect in lymphoblastoids whose TOP mRNAs are constitutively repressed even when they are proliferating. LiCl is widely used as a relatively selective inhibitor of glycogen synthase kinase-3. However, inhibition per se of this enzyme by more specific drugs failed to derepress the translation of TOP mRNAs, implying that relief of the translational repression of TOP mRNAs by LiCl is carried out in a glycogen synthase kinase-3-independent manner. Moreover, this effect is apparent, at least in some cell lines, in the absence of S6-kinase 1 activation and ribosomal protein S6 phosphorylation, thus further supporting the notion that translational control of TOP mRNAs does not rely on either of these variables.
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