Ribosomal protein S6 kinase activity controls the ribosome biogenesis transcriptional program.

S6 kinases (S6Ks) are mechanistic target of rapamycin substrates that participate in cell growth control. S6Ks phosphorylate ribosomal protein S6 (rpS6) and additional proteins involved in the translational machinery, although the functional roles of these modifications remain elusive. Here we analyze the S6K-dependent transcriptional and translational regulation of gene expression by comparing whole-genome microarray of total and polysomal mouse liver RNA after feeding. We show that tissue lacking S6Ks 1 and 2 (S6K1 and S6K2), displays a defect in the ribosome biogenesis (RiBi) transcriptional program after feeding. Over 75% of RiBi factors are controlled by S6K, including Nop56, Nop14, Gar1, Rrp9, Rrp15, Rrp12 and Pwp2 nucleolar proteins. Importantly, the reduced activity of RiBi transcriptional promoters in S6K1;S6K2(-/-) cells is also observed in rpS6 knock-in mutants that cannot be phosphorylated. As ribosomal protein synthesis is not affected by these mutations, our data reveal a distinct and specific aspect of RiBi under the control of rpS6 kinase activity, that is, the RiBi transcriptional program.

Ribosomal protein S6 is a selective mediator of TRAIL-apoptotic signaling.

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is a potent inducer of apoptosis in tumor cells and holds a promise as a therapeutic agent against cancer. To elucidate the death signaling evoked by TRAIL, we performed a functional genetic screening and rescued TRAIL-resistant Jurkat clones harboring ribosomal protein S6 (rpS6) cDNA in anti-sense frame. Reduction of rpS6 expression in Jurkat and HeLa cells attenuated apoptosis induced by TRAIL, but not those by other cell death signals, including tumor necrosis factor-alpha and cycloheximide, etoposide, doxorubicin, tunicamycin and staurosporine. Death receptor (DR) 4, but not DR5, was downregulated in rpS6 knockdown cells. Conversely, the sensitivity to TRAIL was increased by the ectopic expression of wild-type rpS6 and further by phospho-defective rpS6 mutant (S6-SS235,6AA), but not by phospho-mimic rpS6 mutant (S6-SS235,6DD). Also, unphosphorylatable rpS6 knock-in mouse embryo fibroblasts (rpS6(P-/-) MEFs) were more sensitive to TRAIL than control MEFs. In addition, SKHep-1 tumor cells, which express less phospho-rpS6 and are more sensitive to TRAIL than other tumor cells, became effectively desensitized to TRAIL after rpS6 knockdown. These results suggest that rpS6, especially in its unphosphorylated form, is a selective mediator of TRAIL-induced apoptosis.

Amino acid-induced translation of TOP mRNAs is fully dependent on phosphatidylinositol 3-kinase-mediated signaling, is partially inhibited by rapamycin, and is independent of S6K1 and rpS6 phosphorylation.

Vertebrate TOP mRNAs contain an oligopyrimidine tract at their 5' termini (5'TOP) and encode components of the translational machinery. Previously it has been shown that they are subject to selective translational repression upon growth arrest and that their translational behavior correlates with the activity of S6K1. We now show that the translation of TOP mRNAs is rapidly repressed by amino acid withdrawal and that this nutritional control depends strictly on the integrity of the 5'TOP motif. However, neither phosphorylation of ribosomal protein (rp) S6 nor activation of S6K1 per se is sufficient to relieve the translational repression of TOP mRNAs in amino acid-starved cells. Likewise, inhibition of S6K1 activity and rpS6 phosphorylation by overexpression of dominant-negative S6K1 mutants failed to suppress the translational activation of TOP mRNAs in amino acid-refed cells. Furthermore, TOP mRNAs were translationally regulated by amino acid sufficiency in embryonic stem cells lacking both alleles of the S6K1 gene. Inhibition of mTOR by rapamycin led to fast and complete repression of S6K1, as judged by rpS6 phosphorylation, but to only partial and delayed repression of translational activation of TOP mRNAs. In contrast, interference in the phosphatidylinositol 3-kinase (PI3-kinase)-mediated pathway by chemical or genetic manipulations blocked rapidly and completely the translational activation of TOP mRNAs. It appears, therefore, that translational regulation of TOP mRNAs, at least by amino acids, (i) is fully dependent on PI3-kinase, (ii) is partially sensitive to rapamycin, and (iii) requires neither S6K1 activity nor rpS6 phosphorylation.

Interplay in lipoplexes between type of pDNA promoter and lipid composition determines transfection efficiency of human growth hormone in NIH3T3 cells in culture.

This study was aimed to investigate if and to what extent there is an interplay between lipoplex physicochemical properties and plasmid promoter type affecting transfection efficiency in vitro. To reduce the number of variables only one cell type (NIH3T3 cells), one gene (human growth hormone), one cationic lipid (DOTAP) in a plasmid >85% in supercoiled form, and the same medium conditions were used. The variables of the physicochemical properties included presence and type of helper lipid (DOPE, DOPC, or cholesterol, all in 1:1 mole ratio with DOTAP), size and lamellarity of the liposomes used for lipoplex preparation (large unilamellar vesicles, LUV, versus multilamellar vesicles, MLV), and DNA(-)/cationic lipid(+) charge ratio, all containing the same human growth hormone but differing in their promoter enhancer region. Two of the promoters were of viral origin: (a) SV40 promoter (simian virus early promoter) and (b) CMV promoter (cytomegalovirus early promoter); two were of mammalian cell origin: (c) PABP promoter (human poly(A)-binding protein promoter) and (d) S16 promoter (mouse ribosomal protein (rp) S16 promoter). Transfection studies showed that, irrespective of promoter type, large (> or =500 nm) MLV were superior to approximately 100 nm LUV; the extent of superiority was dependent on liposome lipid composition (larger for 100% DOTAP and DOTAP/DOPE than for DOTAP/DOPC and DOTAP/cholesterol). The optimal DNA(-)/DOTAP(+) charge ratio for all types of lipoplexes used was 0.2 or 0.5 (namely, when the lipoplexes were positively charged). Scoring the six best lipoplex formulations (out of 128 studied) revealed the following order: pCMV (DOTAP/DOPE) >> pSV (DOTAP/DOPE)=pCMV(DOTAP/cholesterol)=pS16 (100% DOTAP)=pS16 DOTAP/DOPE >> pCMV (DOTAP/DOPC). The lack of trivial consistency in the transfection efficiency score, the pattern of transfection efficiency, and statistical analysis of the data suggest that there is cross-talk between promoter type and lipoplex lipid composition, which may be related to the way the promoter is associated with the lipids.

Synthesis of the translational apparatus is regulated at the translational level.

The synthesis of many mammalian proteins associated with the translational apparatus is selectively regulated by mitogenic and nutritional stimuli, at the translational level. The apparent advantages of the regulation of gene expression at the translational level are the speed and the readily reversible nature of the response to altering physiological conditions. These two features enable cells to rapidly repress the biosynthesis of the translational machinery upon shortage of amino acids or growth arrest, thus rapidly blocking unnecessary energy wastage. Likewise, when amino acids are replenished or mitogenic stimulation is applied, then cells can rapidly respond in resuming the costly biosynthesis of the translational apparatus. A structural hallmark, common to mRNAs encoding many components of the translational machinery, is the presence of a 5' terminal oligopyrimidine tract (5'TOP), referred to as TOP mRNAs. This structural motif comprises the core of the translational cis-regulatory element of these mRNAs. The present review focuses on the mechanism underlying the translational control of TOP mRNAs upon growth and nutritional stimuli. A special emphasis is put on the pivotal role played by ribosomal protein S6 kinase (S6K) in this mode of regulation, and the upstream regulatory pathways, which might be engaged in transducing external signals into activation of S6K. Finally, the possible involvement of pyrimidine-binding proteins in the translational control of TOP mRNAs is discussed.

Overexpression of poly(A)-binding protein down-regulates the translation or the abundance of its own mRNA.

Poly(A)-binding protein (PABP) mRNA is subject to autoregulation through a 61 nucleotides long A-rich sequence in its 5' untranslated region (UTR). Here, we show that this mode of regulation is exerted in a cell type-specific manner. Thus, overexpression of PABP in mouse NIH 3T3 fibroblasts represses the translation of the respective endogenous mRNA or that of a chimeric mRNA containing just the 5' UTR of PABP mRNA. In contrast, ectopic expression of PABP in human embryonic kidney 293 cells down-regulates the abundance of the endogenous PABP mRNA, rather than affecting its translational efficiency. Transfection experiments with chimeric constructs suggest that the lack of translational autoregulation of endogenous PABP mRNA in these cells appears to reflect the presence of an overriding regulatory element outside the A-rich region.

TOP mRNAs are translationally inhibited by a titratable repressor in both wheat germ extract and reticulocyte lysate.

Vertebrate TOP mRNAs contain a 5' terminal oligopyrimidine tract (5' TOP), which is subject to selective translational repression in non-growing cells or in cell-free translation systems. In the present study, we monitored in vitro the effect of increasing amounts of a 16 nucleotides long oligoribonucleotide representing the 5' terminus of mouse ribosomal protein S16 mRNA on the translation of TOP and non-TOP mRNAs. Our results demonstrate that the wild-type sequence (but not its mutant counterparts) derepresses the translation of mRNAs containing 5' TOP motifs, but failed to stimulate the translation of non-TOP mRNAs, even if the latter differed only by a single nucleotide from their 5' TOP-containing counterparts. Similar results have been obtained with both wheat germ extract and rabbit reticulocyte lysate. It appears, therefore, that translational repression of TOP mRNAs is achieved in vitro by the accumulation of a titratable repressor rather than by the loss of an activator and that this repressor recognizes multiple TOP mRNAs with a diverse set of 5' TOP motifs.

The expression of poly(A)-binding protein gene is translationally regulated in a growth-dependent fashion through a 5'-terminal oligopyrimidine tract motif.

Poly(A)-binding protein (PABP) is an important regulator of gene expression that has been implicated in control of translation initiation. Here we report the isolation and the initial structural and functional characterization of the human PABP gene. Delineation of the promoter region revealed that it directs the initiation of transcription at consecutive C residues within a stretch of pyrimidines. A study of the translational behavior of the corresponding mRNA demonstrates that it is translationally repressed upon growth arrest of cultured mouse fibroblasts and translationally activated in regenerating rat liver. Furthermore, transfection experiments show that the first 32 nucleotides of PABP mRNA are sufficient to confer growth-dependent translational control on a heterologous mRNA. Substitution of the C residue at the cap site by purines abolishes the translational control of the chimeric mRNA. These features have established PABP mRNA as a new member of the terminal oligopyrimidine tract mRNA family. Members of this family are known to encode for components of the translational apparatus and to contain an oligopyrimidine tract at the 5' terminus (5'TOP). This motif mediates their translational control in a growth-dependent manner.

Growth-dependent and PKC-mediated translational regulation of the upstream stimulating factor-2 (USF2) mRNA in hematopoietic cells.

Upstream stimulating factor (USF2) is a basic helix-loop-helix leucine zipper transcription factor, which is found in most tissues. A critical role for USF2 in cellular proliferation has been proposed based on its importance in the regulation of various cyclins and P53 and its capability to antagonize c-myc. In this paper we report that IL-3, which is a major growth factor for mast cells, induces USF2 protein synthesis in murine mast cells (MC-9). Surprisingly, it does not significantly affect the level of USF2 mRNA in these cells at any of the time points tested. Using polysomal fractionation and RNA analysis we then demonstrated that this translational regulation is mostly the result of increased USF2 translational efficiency. Moreover, protein kinase C (PKC) inhibitors prevented both the induction of USF2 protein synthesis and the increase in USF2 translational efficiency in IL-3-activated mast cells. Two other hematopoietic cell lines were used to determine whether the translational regulation of USF2 is of a more general nature: mouse lymphosarcoma cells whose proliferation is inhibited by dexamethasone; and mouse erythroleukemia cells that differentiate upon exposure to hexamethylen bisacetamide. In both cell types, USF2 translation was repressed in the non-dividing cells. This strongly implies that USF2 is translationally repressed in quiescent hematopoietic cells. Considering the proposed role of USF in proliferation it seems that translational regulation of USF2 might have an important role in cellular growth.

Substitution of just five nucleotides at and around the transcription start site of rat beta-actin promoter is sufficient to render the resulting transcript a subject for translational control.

Vertebrate mRNAs with a 5' terminal oligopyrimidine tract (5' TOP), including those encoding ribosomal proteins and elongation factors, are candidates for translational control in a growth-dependent fashion. The present study was designed to determine the minimal cis-regulatory element involved in this mode of regulation. We selected rat beta-actin mRNA, a typical translationally uncontrolled transcript, as a subject for gain-of-function analysis. Mutations at and around its cap site leading to the formation of a 7 pyrimidines long 5' TOP render the resulting transcript translationally repressed upon growth arrest of lymphosarcoma cells. In contrast, growth-dependent translational control of this mRNA in fibroblasts requires, in addition, a GC motif downstream of the 5' TOP. A similar motif is present in all ribosomal prtein mRNAs shown to be translationally controlled.

The 5' terminal oligopyrimidine tract confers translational control on TOP mRNAs in a cell type- and sequence context-dependent manner.

TOP mRNAs are vertebrate transcripts which contain a 5'terminal oligopyrimidine tract (5'TOP), encode for ribosomal proteins and elongation factors 1alpha and 2, and are candidates for growth-dependent translational control mediated through their 5'TOP. In the present study we show that elongation factor 2 (EF2) mRNA is translationally regulated in a growth-dependent manner in cells of hematopoietic origin, but not in any of three different non-hematopoietic cell lines studied. Human beta1-tubulin mRNA is a new member of the family which contains all the hallmarks of a typical TOP mRNA, yet its translation is refractory to growth arrest of any of the examined cell lines. Transfection experiments indicate that the first 29 and 53 nucleotides of the mRNAs encoding EF2 and beta1-tubulin, respectively, contain all the translational cis-regulatory elements sufficient for ubiquitously conferring growth-dependent translational control on a reporter mRNA. These results suggest that the distinct translational regulation of TOP mRNAs reflects downstream sequences which can override the regulatory features of the 5'TOP in a cell type-specific manner. This notion is further supported by the fact that mutations within the region immediately downstream of the 5'TOP of rpS16 mRNA confer onto the resulting transcripts growth-dependent translational control with a cell type specificity similar to that displayed by EF2 mRNA.

The translational cis-regulatory element of mammalian ribosomal protein mRNAs is recognized by the plant translational apparatus.

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.

Overexpression of initiation factor eIF-4E does not relieve the translational repression of ribosomal protein mRNAs in quiescent cells.

Translation of ribosomal protein (rp) mRNA is selectively repressed in mouse erythroleukemia (MEL) cells, which cease to proliferate upon differentiation, and in NIH 3T3 cells, for which growth is arrested by either serum starvation, contact inhibition, or treatment with the DNA polymerase inhibitor, aphidicolin. The efficiency of translation of rp mRNAs correlates with the expression of the gene encoding the cap binding protein, eIF-4E, as indicated by the fact that the abundance of the corresponding mRNA and protein also fluctuates in a growth-dependent manner. To examine the hypothesis that eIF-4E plays a role in regulation of the translation efficiency of rp mRNAs, we utilized an NIH 3T3-derived eIF-4E-overexpressing cell line. These cells overproduce eIF-4E to the extent that even under conditions of growth arrest, the abundance of the respective protein in its active (phosphorylated) form is higher than that found in exponentially growing NIH 3T3 cells. Nevertheless, this surplus amount of eIF-4E does not prevent the translational repression of rp mRNAs when the growth of these cells is arrested by blocking DNA synthesis with aphidicolin or hydroxyurea. In complementary experiments we used an in vitro translation system to compare the competitive potential of mRNAs, containing the translational cis-regulatory element (5' terminal oligopyrimidne tract) and mRNAs lacking such a motif, for the cap binding protein. Our results demonstrate that both types of mRNAs, regardless of their translational response to growth arrest, exhibit similar sensitivity to the cap analogue m7G(5')ppp(5')G. It appears, therefore, that the presence of the regulatory sequence at the 5' terminus of rp mRNAs does not lessen its competitive potential for the cap binding protein and that the growth-dependent decrease in the activity of eIF-4E does not play a key role in the repression of translation of rp mRNAs.

Vertebrate mRNAs with a 5'-terminal pyrimidine tract are candidates for translational repression in quiescent cells: characterization of the translational cis-regulatory element.

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) selective translational control is not confined to rp mRNAs, as mRNAs encoding the naturally occurring ubiquitin-rp fusion protein and elongation factor 1 alpha, which contain a 5' TOP, also conform this mode of regulation; (iii) rat rpP2 mRNA contains only five pyrimidines in its 5' TOP, yet this mRNA is translationally controlled in the same fashion as other rp mRNAs with a 5' TOP of eight or more pyrimidines; (iv) full manifestation of this mode of regulation seems to require both the 5' TOP and sequences immediately downstream; and (v) an intact translational regulatory element from rpL32 mRNA fails to exert its regulatory properties even when preceded by a single A residue.

Selective translational control and nonspecific posttranscriptional regulation of ribosomal protein gene expression during development and regeneration of rat liver.

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 posttranscriptional mechanism. The apparent fluctuations in the translation efficiency of rp mRNAs are accompanied by parallel changes in the expression of the genes encoding the initiation factors eIF-4E and eIF-4A. Our results indicate that selective translational control of rp mRNAs in mammals is not confined to manipulated cells in culture but constitutes an important regulatory mechanism operating in vivo in the course of liver development and regeneration.

Oligopyrimidine tract at the 5' end of mammalian ribosomal protein mRNAs is required for their translational control.

Mammalian ribosomal protein (rp) mRNAs are subject to translational control, as illustrated by their selective release from polyribosomes in growth-arrested cells and their underrepresentation in polysomes in normally growing cells. In the present experiments, we have examined whether the translational control of rp mRNAs is attributable to the distinctive features of their 5' untranslated region, in particular to the oligopyrimidine tract adjacent to the cap structure. Murine lymphosarcoma cells were transfected with chimeric genes consisting of selected regions of rp mRNA fused to non-rp mRNA segments, and the translational efficiency of the resulting chimeric mRNAs was assessed in cells that either were growing normally or were growth-arrested by glucocorticoid treatment. We observed that translational control of rpL32 mRNA was abolished when its 5' untranslated region was replaced by that of beta-actin. At the same time, human growth hormone (hGH) mRNA acquired the typical behavior of rp mRNAs when it was preceded by the first 61 nucleotides of rpL30 mRNA or the first 29 nucleotides of rpS16 mRNA. Moreover, the translational control of rpS16-hGH mRNA was abolished by the substitution of purines into the pyrimidine tract or by shortening it from eight to six residues with a concomitant cytidine----uridine change at the 5' terminus. These results indicate that the 5'-terminal pyrimidine tract plays a critical role in the translational control mechanism. Possible factors that might interact with this translational cis regulatory element are discussed.

The mammalian genome contains a high proportion of processed pseudogenes corresponding to ribosomal protein L19.

The mammalian genome contains multiple copies of ribosomal protein (rp) L19-related sequences. Screening of mouse and rat genomic libraries with cloned rpL19 cDNA yielded seventeen independent lambda Charon 4A recombinant phages containing twelve and five genes for mouse and rat rpL19, respectively. Structural analysis indicated that all of these rpL19 genes contain the entire coding sequence (approx. 700 bp) but lack introns. The nucleotide sequence of a mouse gene (rpL19-17), exhibiting the highest homology with the mouse rpL19 cDNA, revealed genetic lesions which would preclude the translation of an intact protein, from a putative transcript. Based on these features we propose that these clones represent processed genes of which most, if not all, are pseudogenes.

The mouse ribosomal protein L7 gene. Its primary structure and functional analysis of the promoter region.

The expressed gene coding for mouse ribosomal protein L7 (rpL7) was structurally and functionally characterized. It consists of seven exons, spans 3107 base pairs, and its coding sequence initiates within exon 1. The primary structure of mouse rpL7 (270 amino acids), as inferred from the nucleotide sequence of the exons of the gene, and from the cDNA, is 12 residues longer than the rat counterpart. The rpL7 gene shares common structural features with most other mammalian ribosomal protein genes analyzed thus far. These include the lack of a canonical TATA box and a major transcription initiation site at a cytidine residue embedded in a stretch of 14 pyrimidines, flanked by C + G-rich regions. Transient expression assays revealed that the promoter region of rpL7 gene bears several regulatory elements, both upstream to the capsite and within the transcribed portion of the gene. One internal regulatory element was assigned to the first intron and a second one to a 20-base pair region spanning the first exon-intron junction. The activity of a deletion mutant of rpL32 gene, lacking its internal elements can be rescued by insertion, in the sense orientation, of the corresponding elements from the rpL7 gene. The unique spatial organization of the regulatory elements in rpL7 gene, as well as in other murine ribosomal protein genes examined thus far, might indicate that this common architecture is involved in the mechanism coordinating their expression.

Glucocorticoids repress ribosome biosynthesis in lymphosarcoma cells by affecting gene expression at the level of transcription, posttranscription and translation.

Growth arrest of P1798 murine lymphosarcoma cells by glucocorticoids is accompanied by a remarkable decrease in transcription of rRNA and translation of mRNAs encoding basic ribosomal proteins (rps). Here we report that the expression of other genes involved in ribosome biogenesis is repressed in dexamethasone-treated P1798 cells. These include posttranscriptionally regulated decline in the abundance of the mRNA and primary transcript of nucleolin; abrupt drop in the transcription rate of U3 small nucleolar RNA; and inhibition of translation of mRNAs coding for P2 and L5, acidic and basic rps, respectively. Normal expression of these genes is resumed upon hormonal withdrawal.