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.

Localization of transcriptional regulatory elements and nuclear factor binding sites in mouse ribosomal protein gene rpL32.

The DNA sequences required for expression of the ribosomal protein gene rpL32 were identified by transient-expression assays of chimeric rpL32-chloramphenicol acetyltransferase genes. These studies showed that maximal rpL32 expression requires sequences in a 150- to 200-base-pair region spanning the transcriptional start site. Three discrete regions of importance were identified: one between positions -79 and -69 and two others located downstream of the transcriptional start site. Progressive 5' or 3' deletions caused stepwise decreases in expression, which suggested a complex interplay of redundant or compensatory elements. Gel mobility shift assays were used to identify trans-acting nuclear factors which bind to segments of the rpL32 promoter that are known to be important for transcription. Evidence for several distinct nuclear factors is presented. The binding sites for these factors were localized to the following regions: -79 to -69, -36 to -19, -19 to +11, +11 to +46 in exon I, and within the first 31 base pairs of intron 1. One of these factors may bind to multiple sites within the promoter region. Interestingly, the factor that binds to a sequence motif in the first exon also binds to similar motifs in a comparable region of the c-myc gene.

Glucocorticoids selectively inhibit translation of ribosomal protein mRNAs in P1798 lymphosarcoma cells.

When P1798 murine lymphosarcoma cells are exposed to 10(-7) M dexamethasone, there is a dramatic inhibition of rRNA synthesis, which is completely reversible when the hormone is withdrawn. In the present experiments we examined whether dexamethasone treatment causes any alteration in the accumulation or utilization of mRNAs that encode ribosomal proteins (rp mRNAs). No effect on the accumulation of six different rp mRNAs was detected. However, the translation of five of six rp mRNAs was selectively inhibited in the presence of the hormone, as judged by a substantial decrease in ribosomal loading. Normal translation of rp mRNA was resumed within a few hours after hormone withdrawal. In untreated or fully recovered cells, the distribution of rp mRNAs between polyribosomes and free ribonucleoprotein is distinctly bimodal, suggesting that rp mRNAs are subject to a particular form of translational control in which they are either translationally inactive or fully loaded with ribosomes. A possible relationship between this mode of translational control and the selective suppression of rp mRNA translation by glucocorticoids is discussed.

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.

Regulation of ribosomal protein mRNA content and translation in growth-stimulated mouse fibroblasts.

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 cells. Furthermore, the total amount of rp mRNA did not begin to increase until about 6 h after stimulation. Since an increase in rp mRNA content did not appear to be responsible for the increase in ribosomal protein synthesis, we determined the efficiency of translation of rp mRNA under different conditions. We found that about 85% of pulse-labeled rp mRNA was associated with polysomes in exponentially growing cells. In resting cells, however, only about half was associated with polysomes, and about 30% was found in the monosomal fraction. The distribution shifted to that found in growing cells within 3 h after serum stimulation. Similar results were obtained when cells were labeled for 10.5 h. About 70% of total polyadenylated mRNA was in the polysome fraction in all growth states regardless of labeling time, indicating that the shift in mRNA distribution was species specific. These results indicate that the content and metabolism of rp mRNA do not change significantly after growth stimulation. The rate of ribosomal protein synthesis appears to be controlled during the resting-growing transition by an alteration of the efficiency of translation of rp mRNA, possibly at the level of protein synthesis initiation.

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.

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.

Evolutionary conservation of ribosomal protein mRNA sequences: application for expansion of corresponding cDNA and gene libraries.

Cloned cDNAs, containing ribosomal protein sequences from mouse (five cDNAs) or Xenopus laevis (six cDNAs), were used to estimate the evolutionary conservation, from insects to mammals, of the corresponding mRNA sequences. Northern blot analysis reveals a variable degree of homology between these sequences in different eukaryotes. Thus, among the ribosomal protein cDNA clones utilized, some exhibit complete, others partial, and a few no interphyla cross-hybridization. Melting profile analysis was employed to quantitate this homology. It is proposed that for expansion of eukaryotic ribosomal cDNA and gene libraries, one can exploit the interspecies homology of the corresponding sequences. However, the diverse evolutionary conservation of individual ribosomal protein gene sequences should be taken into account.

P31, a mammalian housekeeping protein encoded by a multigene family containing a high proportion of pseudogenes.

The primary structure of P31, a 13.300 kDa mammalian 'housekeeping' protein, was deduced from the nucleotide sequence of a rat recombinant cDNA clone. The P31 mRNA has 950 nucleotides and codes for a protein of 121 amino acids. This mRNA is present in several mouse tissues and in other mammals. Nuclei of rapidly growing cells contain mature P31 mRNA and a distinctive set of larger transcripts. P31 is encoded by a multigene family. Five members of the family were isolated from a mouse genomic library and restriction mapping and S1 nuclease analysis of four of them (P31-4, P31-12, P31-13 and P31-14) suggest that they are processed genes. The very low homology of the fifth (P31-8) with respect to the corresponding cDNA sequence indicates that it is a pseudogene. Although the features of the mRNA and the genes encoding P31 exhibit extensive similarity with those of ribosomal proteins, neither the identity nor the biological function of the protein has yet been determined.

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.

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.

Isolation and characterization of four mouse ribosomal-protein-L18 genes that appear to be processed pseudogenes.

We describe the isolation and initial characterization of six independent lambda Charon 4A recombinant phages which carry four mouse genes for ribosomal protein L18 (L18-15, L18-17, L18-18, L18-30). Structural analysis indicates that these L18 genes contain the entire coding sequence (approx. 600 bp), but lack introns and differ both in their flanking and coding sequences. Based on these features we propose that these are processed pseudogenes. However, we have no indications whether these genes are expressed at either the transcriptional or translational levels. Quantitative estimation of sequence divergence of these genes suggest that the order of their evolutionary emergence (integration) is: first L18-17 and L18-18, then L18-15 and finally L18-30. We discuss the implication of the high proportion of L18 processed genes in this multigene family with respect to the coordinate regulation of r-proteins.

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.

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.

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.

The primary structure of rat ribosomal protein L18a.

The amino acid sequence of rat ribosomal protein L18a was deduced from the sequence of nucleotides in a recombinant cDNA. Ribosomal protein L18a contains 175 amino acids and has a molecular mass of 20,047 Da. Hybridization of the cDNA to digests of rat nuclear DNA and to a preparation of poly(A)+ mRNA suggests that there are 8-11 copies of the L18a gene and that the mRNA for the protein is about 700 nucleotides in length. Rat L18a is related to Schizosaccharomyces pombe L17 and perhaps to Halobacterium marismortui L19.

Conservation from rat to human of cytosolic phosphoenolpyruvate carboxykinase and the control of its gene expression.

Structural conservation of cytosolic phosphoenolpyruvate carboxykinase protein and mRNA sequence was found in all species examined from rodents to human. The mitochondrial isoenzyme, in all species tested, represents a distinct protein. Moreover, irrespective of the ratio of cytosolic to mitochondrial isoenzyme, cytosolic phosphoenolpyruvate carboxykinase activity in the human as in the rat is controlled at the level of gene expression and through the same multiple hormonal stimulation. This evolutionary conservation of the cytosolic phosphoenolpyruvate carboxykinase structure and mode of regulation supports the enzymes' physiological importance in mammals.

Glucocorticoids induce transcription of ribosomal protein genes in rat liver.

Transcription of rat liver ribosomal RNA is induced by glucocorticoids. In order to determine whether the expression of ribosomal protein genes is coordinately regulated, we measured the effect of dexamethasone on their transcription. Administration of this hormone to adrenalectomized rats led, within 1 h, to a 2.2-fold enhancement of transcription of liver ribosomal protein genes. To define the dexamethasone-responsive element, we isolated and tested mouse L32 gene sequences for the ability to confer glucocorticoid induction to the bacterial chloramphenicol acetyltransferase (CAT) gene in L cells. An 80 base pair region of the L32 gene, between nucleotide position -69 and +11, with respect to the start site of transcription, was sufficient for induction of the CAT gene by dexamethasone. Despite these stimulating effects, we have failed to detect elevation in the abundance of the ribosomal protein mRNAs both in rat liver and in mouse L cells. Possible interpretations for this seemingly ineffectual process are discussed.

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.

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.