Coordinate regulation of ribosomal protein mRNA level in regenerating rat liver. Study with the corresponding mouse cloned cDNAs.

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.     

Deficiency of dietary EAA preferentially inhibits mRNA translation of ribosomal proteins in liver of meal-fed rats

The goal of these studies was to investigate the mechanisms by which amino acid supply regulates global rates of protein synthesis as well as the translation of ribosomal protein (rp) mRNAs in liver. In the experiments conducted, male weanling rats were trained over a 2-wk period to consume their daily food intake within 3 h. On day 14, rats were fed the control diet or an isocaloric, isonitrogenous diet lacking glycine, tryptophan, leucine, or the branched-chain amino acids (BCAA) for 1 h. Feeding Trp-, Leu-, or BCAA-deficient diets resulted in significant reductions in serum insulin, hepatic protein synthesis, eukaryotic initiation factor 2B (eIF2B) activity, and phosphorylation of eIF4E-binding protein 1 (4E-BP1) and ribosomal protein S6 kinase (S6K1). Phosphorylation of eIF2alpha was inversely related to eIF2B activity under all conditions. Alterations in the hepatic synthesis of rp were assessed by changes in the distribution of rp (S4, S8, L26) mRNAs across sucrose density gradients and compared with non-rp (beta-actin, albumin) mRNAs. In all dietary treatments, non-rp mRNAs were mostly polysome associated. Conversely, the proportion of rp mRNAs residing in polysomes was two- to fivefold less in rats fed diets lacking tryptophan, leucine, or BCAA compared with rats fed the control diet. Total hepatic abundance of all mRNAs examined did not differ among treatment groups. For all parameters examined, there were no differences between rats fed the glycine-deficient diet and rats fed the control diet. The data suggest that essential amino acid (EAA) deficiency inhibits global rates of liver protein synthesis via a block in translation initiation. Additionally, the translation of rp mRNAs is preferentially repressed in association with decreased S6K1 phosphorylation.     

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.     

Overview: phosphorylation and translation control

Protein synthesis is controlled by the phosphorylation of proteins comprising the translational apparatus. At least 12 initiation factor polypeptides, 3 elongation factors and a ribosomal protein are implicated. Stimulation of translation correlates with enhanced phosphorylation of eIF-4F, eIF-4B, eIF-2B, eIF-3 and ribosomal protein S6, whereas inhibition correlates with phosphorylation of eEF-2 and the alpha-subunit of eIF-2. Strong evidence for regulatory roles exists for eIF-2, eIF-4F and eEF-2, whereas changes in other factor activities due to phosphorylation remain to be demonstrated. Regulation of the specific activity of the translational apparatus by phosphorylation appears to be a general mechanism for the control of rates of global protein synthesis, and may also play a role in modulating the translation of specific mRNAs.     

Modification of eukaryotic initiation factor 4F during infection by influenza virus.

Influenza virus infection of cells is accompanied by a striking shutoff of cellular protein synthesis, resulting in the exclusive translation of viral mRNAs. The mechanism for control of cellular protein synthesis by influenza virus is poorly understood, but several translation properties of influenza virus mRNAs which are potentially involved have been described. Influenza virus mRNAs possess the surprising ability to translate in the presence of inhibitory levels of inactive (phosphorylated) eukaryotic initiation factor 2 (eIF-2). In addition, influenza virus mRNAs were shown to be capable of translating in cells during the late phase of adenovirus infection but not in cells infected by poliovirus. Since both adenovirus and poliovirus facilitate virus-specific translation by impairing the activity of initiation factor eIF-4F (cap-binding protein complex) but through different mechanisms, we investigated the translation properties of influenza virus mRNAs in more detail. We show that influenza virus infection is associated with the significant dephosphorylation and inactivation of eIF-4E (cap-binding protein), a component of eIF-4F, and accordingly that influenza virus mRNAs possess a moderate ability to translate by using low levels of eIF-4F. We also confirm the ability of influenza virus mRNAs to translate in the presence of high levels of inactive (phosphorylated) eIF-2 but to a more limited extent than reported previously. We suggest a potential mechanism for the regulation of protein synthesis by influenza virus involving a decreased requirement for large pools of active eIF-4F and eIF-2.     

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.     

Gene expression profile analysis in human hepatocellular carcinoma by cDNA microarray

We performed gene expression profiling of normal and hepatocellular carcinoma (HCC) liver tissues using a high-density microarray that contained 3,063 human cDNA. The results of a microarray hybridization experiment from eight different HCC tissues were analyzed and classified by the Cluster program. Among these differentially-expressed genes, the galectin-3, serine/threonine kinase SGK, translation factor eIF-4A, -4B, -3, fibroblast growth factor receptor, and ribosomal protein L35A were up-regulated; the mRNAs of Nip3, decorin, and the insulin-like growth factor binding protein-3 were down-regulated in HCC. The differential expression of these genes was further confirmed by an RT-PCR analysis. In addition, our data suggest that the gene expression profile of HCC varies according to the histological types.     

The mTOR signaling pathway mediates control of ribosomal protein mRNA translation in rat liver

Previous studies have shown that oral administration of leucine to fasted rats results in a preferential increase in liver in the translation of mRNAs containing an oligopyrimidine sequence at the 5'-end of the message (i.e. a TOP sequence). TOP mRNAs include those encoding the ribosomal proteins (rp) and translation elongation factors. In cells in culture, the preponderance of evidence suggests that translation of TOP mRNAs is regulated by the mammalian target of rapamycin (mTOR), a protein kinase that signals through ribosomal protein S6 kinase (S6K1) to rpS6. However, the results of previous studies were recently challenged by several reports suggesting that translation of TOP mRNAs is independent of mTOR, S6K1, and S6 phosphorylation. The purpose of the present study was to evaluate the role of mTOR in the stimulation of TOP mRNA translation by leucine in vivo. Fasted rats were treated with the mTOR inhibitor, rapamycin, prior to oral administration of leucine. It was found that rapamycin severely attenuated leucine-induced signaling through mTOR in liver. In addition, rapamycin prevented the enhanced translation of TOP mRNAs in rats administered leucine, as assessed by a decrease in the proportion of TOP mRNAs associated with polysomes (i.e. those mRNAs being actively translated). Instead, in rapamycin-treated rats, ribosomal protein mRNAs accumulated in the fraction containing monosomes (mRNA bound to one ribosome). The results suggest that in liver in vivo, mTOR-dependent signaling is critical for maximal stimulation of TOP mRNA translation.     

mRNAs containing extensive secondary structure in their 5' non-coding region translate efficiently in cells overexpressing initiation factor eIF-4E.

Cellular eukaryotic mRNAs (except organellar) contain at the 5' terminus the structure m7(5')Gppp(5')N (where N is any nucleotide), termed cap. Cap recognition by eukaryotic initiation factor eIF-4F plays an important role in regulating the overall rate of translation. eIF-4F is believed to mediate the melting of mRNA 5' end secondary structure and facilitate 43S ribosome binding to capped mRNAs. eIF-4E, the cap-binding subunit of eIF-4F, plays an important role in cell growth; its overexpression results in malignant transformation of rodent cells, and its phosphorylation is implicated in signal transduction pathways of mitogens and growth factors. The molecular mechanism by which eIF-4E transforms cells is not known. Here, we report that overexpression of eIF-4E facilitates the translation of mRNAs containing excessive secondary structure in their 5' non-coding region. This effect may represent one mechanism by which eIF-4E regulates cell growth and transforms cells in culture.     

Translational initiation factor expression and ribosomal protein gene expression are repressed coordinately but by different mechanisms in murine lymphosarcoma cells treated with glucocorticoids.

P1798 murine lymphosarcoma cells cease to proliferate upon exposure to 10(-7) M dexamethasone and exhibit a dramatic inhibition of rRNA and ribosomal protein synthesis (O. Meyuhas, E. Thompson, Jr., and R. P. Perry, Mol. Cell Biol. 7:2691-2699, 1987). These workers demonstrated that ribosomal protein synthesis is regulated primarily at the level of translation, since dexamethasone did not alter mRNA levels but shifted the mRNAs from active polysomes into inactive messenger ribonucleoproteins. We have examined the effects of dexamethasone on the biosynthesis of initiation factor proteins in the same cell line. The relative protein synthesis rates of eIF-4A and eIF-2 alpha were inhibited by about 70% by the hormone, a reduction comparable to that for ribosomal proteins. The mRNA levels of eIF-4A, eIF-4D, and eIF-2 alpha also were reduced by 60 to 70%, indicating that synthesis rates are proportional to mRNA concentrations. Analysis of polysome profiles showed that the average number of ribosomes per initiation factor polysome was only slightly reduced by dexamethasone, and little or no mRNA was present in messenger ribonucleoproteins. The results indicate that initiation factor gene expression is coordinately regulated with ribosomal protein synthesis but is controlled primarily by modulating mRNA levels rather than mRNA efficiency.