Translational blockade imposed by cytokine-derived UA-rich sequences.

The messenger RNAs specifying certain proteins involved in the inflammatory response and certain oncoproteins contain a conserved UA-rich sequence in the 3' untranslated region. This sequence, which is composed of several interspersed repeats of the octanucleotide UUAUUUAU, has been shown to destabilize mRNA in some eukaryotes. However, this effect is not seen when mRNAs are transferred to Xenopus oocytes, which made it possible to separate stability from translational regulation. For interferon, granulocyte-macrophage colony-stimulating factor, and c-fos RNAs, the UA-rich sequence was observed to preclude mRNA translation.

A GG nucleotide sequence of the 3' untranslated region of amyloid precursor protein mRNA plays a key role in the regulation of translation and the binding of proteins.

The alternative polyadenylation of the mRNA encoding the amyloid precursor protein (APP) involved in Alzheimer's disease generates two molecules, with the first of these containing 258 additional nucleotides in the 3' untranslated region (3'UTR). We have previously shown that these 258 nucleotides increase the translation of APP mRNA injected in Xenopus oocytes (5). Here, we demonstrate that this mechanism occurs in CHO cells as well. We also present evidence that the 3'UTR containing 8 nucleotides more than the short 3'UTR allows the recovery of an efficiency of translation similar to that of the long 3'UTR. Moreover, the two guanine residues located at the 3' ends of these 8 nucleotides play a key role in the translational control. Using gel retardation mobility shift assay, we show that proteins from Xenopus oocytes, CHO cells, and human brain specifically bind to the short 3'UTR but not to the long one. The two guanine residues involved in the translational control inhibit this specific binding by 65%. These results indicate that there is a correlation between the binding of proteins to the 3'UTR of APP mRNA and the efficiency of mRNA translation, and that a GG motif controls both binding of proteins and translation.

Translation of the human c-myc P0 tricistronic mRNA involves two independent internal ribosome entry sites.

The human c-myc proto-oncogene is transcribed from four alternative promoters (P0, P1, P2, and P3) giving rise to mRNAs having 5' leader sequences of various length. The c-myc P0 mRNA contains three open reading frames (ORFs), the last one encoding c-Myc1 and c-Myc2 proteins generated by alternative translation initiated at CUG and AUG codons. The middle ORF (MYCHEX1) and the 5' ORF (ORF1) code for proteins 188 and 114 amino acids in length, respectively. We and others previously identified an internal ribosome entry site (IRES) in P0 and P2 c-myc mRNAs, promoting the cap-independent translation of c-Myc1 and c-Myc2. Here, we report the presence of a second IRES (named IRES1) promoting the cap-independent translation of MYCHEX1 in c-myc P0 mRNA. Using deletion analysis, we mapped an 80-nt region essential for IRES1 activity. c-myc P0 mRNA is thus the first eukaryotic polycistronic mRNA described for which translation initiation of two different open reading frames (MYCHEX1 and c-Myc1/c-Myc2) involves internal ribosome entry.

Complex regulation of tumor necrosis factor mRNA turnover in lipopolysaccharide-activated macrophages.

The turnover of tumor necrosis factor (TNF) mRNA in permanently transfected macrophages of the RAW 264.7 cell line was studied directly (by Northern blot analysis using a probe specific for TNF) and indirectly (through studies of the turnover of various reporter mRNAs, either containing or lacking the TNF 3' untranslated region (UTR)). The TNF mRNA was found to be very unstable in RAW 264.7 cells. Instability appeared to result from two distinguishable nucleolytic processes. The major degradative process involved was not specific for the TNF 3' UTR of reporter mRNAs, and was inhibited by actinomycin D pretreatment. It appeared to be expressed constitutively, in that cell activation by lipopolysaccharide (LPS) did not modify message stability. When cells were treated with actinomycin D, a minor nucleolytic activity was 'uncovered'. This minor activity was noted to increase with time following LPS activation. It also exhibited specificity, in that reporter mRNAs bearing the 3' UTR of TNF were more susceptible to degradation in the presence of actinomycin D than were constructs lacking the 3' UTR of TNF. Thus, TNF mRNA turnover appears complex, and depends upon at least two separable degradative pathways. The TNF 3' UTR apparently contributes only modestly to the instability of this mRNA under normal conditions.

Glutathione depletion increases tyrosinase activity in human melanoma cells.

The aim of the present work was to estimate the effect of intracellular glutathione depletion on melanogenesis in human melanoma cells. We determined tyrosine hydroxylation activity, the rate-limiting step of the pathway, and 14C-melanin formation, an assay reflecting the global eumelanogenic pathway. Intracellular glutathione was depleted by treatment with buthionine-S-sulfoximine, a well-known inhibitor of gamma-glutamylcysteine synthetase. The intracellular depletion of glutathione was substantial after 20 h of incubation with 50 microM buthionine-S-sulfoximine, although a significant effect could be observed after 6 h. Tyrosine hydroxylase activity increased in parallel with glutathione depletion, to reach 160% with respect to the control values during 24 h of buthionine-S-sulfoximine treatment. We have found the response to buthionine-S-sulfoximine to be dose dependent and the two different human cell lines HBL and LND1 to have similar, if not identical, responses. 14C-melanin formation assay revealed even greater activation, up to 400% of the control values. This indicates that glutathione depletion may have two distinct effects: first, a direct one on tyrosinase activity and, second, an effect on the promotion of eumelanogenesis. The stimulation of tyrosine hydroxylase can be explained by a possible inactivation of the enzyme by endogenous thiol compounds rather than by a direct effect of buthionine-S-sulfoximine itself on tyrosinase. The data suggest that thiol compounds may play a role for stimulation of melanogenesis by ultraviolet radiation.

Identification of a translation inhibitory element (TIE) in the 3' untranslated region of the human interferon-beta mRNA.

We have previously reported that the 3' untranslated region (UTR) of the human interferon-beta mRNA has an inhibitory effect on the mRNA translation both in vitro, in a rabbit reticulocyte lysate, and in vivo, in the Xenopus oocyte. In the present study, we identify the sequence in the 3' UTR which is responsible for this translation inhibition. We show that this sequence is located between the 100th and 161st nucleotides downstream from the translation stop codon. It contains several repeats of the A + U-rich consensus octanucleotide UUAUUUAU, which is also present in the 3' UTR of several mRNAs involved in the inflammatory response. We also demonstrate here that the inhibitory effect of the sequence on the mRNA translation does not depend on its position in relation to the termination codon. However, no inhibition of translation is observed when this sequence is inserted in the 5' UTR of the mRNA. The removal of the translation inhibitory sequence not only improves the mRNA translation in Xenopus oocytes but it also strongly decreases the IFN-beta mRNA stability in those cells. This suggests that, in this system at least, the mRNA degradation is linked to its translational efficiency.

The IFI-56K and IFI-54K interferon-inducible human genes belong to the same gene family.

The IFI-56K and IFI-54K human genes are coordinately regulated by interferon, double-stranded RNA and viruses in a number of cell lines. These genes encode polypeptides of 56 and 54 kDa, respectively, whose function remains to be determined. We analysed the possible structural relatedness between these syntenic and similarly regulated genes. We found that they are very closely related at the protein, mRNA and promoter levels. This suggests that the IFI-56K and IFI-54K genes are members of a gene family, which probably arose from duplication of an ancestor gene.

Fertilization of Xenopus eggs imposes a complete translational arrest of mRNAs containing 3'UUAUUUAU elements.

The early embryonic development of Xenopus is mainly governed by post-transcriptional regulations until the mid-blastula transition. In this report, we present evidence demonstrating that fertilization of Xenopus eggs triggers a complete translational arrest of mRNAs containing UA-rich elements in their 3'-untranslated region. This control is maintained at least until the mid-blastula transition. Neither maturation nor pseudo-fertilization of the egg is sufficient for triggering this control, suggesting that components originating from the male gamete are involved in the mechanism. Moreover, this control is exerted whether the mRNA is polyadenylated or not.

Microinjection of catalytic subunit of cyclic AMP-dependent protein kinase triggers acute morphological changes in thyroid epithelial cells.

In dog thyroid epithelial cells in primary culture, thyrotropin acting through cyclic AMP induced rapid morphological changes associated with complete disruption of actin containing stress fibers. This modification preceded cell retraction and rounding up. These morphological effects were also induced by glass capillary microinjection of purified catalytic subunit of cAMP-dependent protein kinase. This provides the first direct evidence in intact cells that catalytic subunit, which is released upon activation of cAMP-dependent protein kinases, is responsible for cAMP-dependent morphological transformation.

Antiviral activity of a chemically stabilized 2-5A analog upon microinjection into HeLa cells.

2-5A[ppp(A2'p)n5'A] has been implicated as a mediator in the antiviral action of interferon. Its direct evaluation as an indicator of virus replication is hampered by two limitations: its inability to penetrate intact cells, and its rapid intracellular degradation by (2'-5')phosphodiesterase. These problems could be overcome by using a microinjection technique whereby a phosphodiesterase-resistant analog of 2-A, in which the 2'-terminals adenosine residue is replaced by 2-(9-adenyl)-6-hydroxy-methyl-4-hexylmorpholine, was injected into individual HeLa cells before infection with mengovirus or vesicular stomatitis virus (VSV). This comparative assay with two representatives of different virus classes in a single experimental system pointed to the high sensitivity of VSV to inhibition by 2-5A oligonucleotides, in contrast with the low sensitivity of mengovirus. Microinjection of the hexylmorpholine 2-5A analog led to a much greater reduction in mengovirus yield than did microinjection of 2-5A itself.

Full-length sequence and expression of the 42 kDa 2-5A synthetase induced by human interferon.

Interferon-induced 2-5A synthetases are probably involved in some antiviral actions of interferon. In human cells two different mRNAs (1.6, 1.8 kb long) coding for this protein are transcribed from the same gene and are produced by differential splicing. The relationship between the two mRNAs of different size and the active enzyme is not clear, nor is the cellular localization of the latter known. We have cloned a cDNA corresponding to the 1.6 kb RNA. This cDNA was sequenced and its complete coding region was subcloned into pSP64. The resulting plasmid was used to direct the synthesis of micrograms of capped RNA transcript after linearization in the 3'-non-coding region. A 39 kDa protein was synthesized when this RNA was translated in rabbit reticulocyte lysate. When this capped RNA was introduced by microinjection into Xenopus oocytes, production of 2-5A synthetase was clearly observed in the cytoplasm and 10-30% of the enzyme accumulated with time in the nucleoplasm. Analysis of cytoplasmic homogenates of these oocytes on a glycerol gradient revealed that the enzyme is fully active in the monomeric form.

TRP-1 expression correlates with eumelanogenesis in human pigment cells in culture.

We have investigated the relationship in human cultured normal and malignant melanocytes between the accumulation of mRNAs encoding tyrosinase and tyrosinase-related protein-1 (TRP-1), the activity of tyrosinase and the presence of melanin. Tyrosinase mRNA correlates with tyrosinase activity and with the presence of pheomelanin, eumelanin or both melanin types. In contrast TRP-1 mRNA is only detectable in cells containing eumelanin, which suggests a role for TRP-1 in the eumelanin synthesis pathway.

Translational control of cytokine expression by 3' UA-rich sequences.

Several messenger RNAs which are transiently expressed contain a conserved uridine-adenosine-rich sequence in their 3' untranslated region. Many of these mRNas encode cytokines, growth factors or oncoproteins. This UA-rich sequence is composed of several interpsersed repeats of the octanucleotide UUAUUUAU and plays a key role in the post-transcriptional regulation of these mRNAs. Known as instability determinants, these UA-rich elements can also strongly affect mRNA translational efficiency. In this report, we review the data which illustrate this translational regulation and give insight the underlying mechanism.

Effect of tRNA pool balance on rate and uniformity of elongation during translation of fibroin mRNA in a reticulocyte cell-free system.

Unsuccessful attempts to synthesize complete fibroin chains in vitro were previously made in heterologous cell-free system [3]. In the present work, we succeeded to obtain complete translation of purified fibroin mRNA in a rabbit reticulocyte lysate. Whilst this work was being completed [1], similar results were published by Lizardi et al. [4]. The synthesis of full-sized molecules of fibroin (M.W. 360,000) was achieved by adding tRNA from the posterior silk gland to the cell-free system. With tRNA from other sources, both the translation rate and the amount of complete fibroin chains dropped. This effect of tRNA is situated at the elongation levels. Analysis of cell-free synthesized products by polyacrylamide gel electrophoresis shows that smaller discrete polypeptides are accumulated after 120 minutes of incubation. These polypeptides correspond to growing fibroin chains. This pattern of translation products suggests that elongation might decelerate at specific sites of the fibroin mRNA. These results show that a tRNA pool adjusted to mRNA codon frequency is required to obtain the maximal average elongation rate. A stochastic model based on random acceptance of tRNA at the ribosomal A site for the codon-anticodon recognition process can explain this phenomenon. It can also explain the occurrence of the unfinished discrete fibroin polypeptides during in vitro translation.

Very low level of major BCR-ABL expression in blood of some healthy individuals.

The nested reverse transcriptase polymerase chain reaction (RT-PCR) provides a powerful tool for detection of minimal residual disease in CML. The RT-PCR used in the present study for detection of the major bcr-abl fusion gene, the hallmark and presumably the cause of CML, was optimized by: (a) increasing the amount of total RNA involved in the reverse transcription reaction to correspond to total RNA extracted from 10(8) cells; (b) using a specific abl primer in this reverse transcriptase reaction, and (c) reamplifying 10% of the RT-PCR product in a nested amplification. This optimized RT-PCR permitted to detect up to 1 copy of RNA bcr-abl synthesized in vitro, mixed with yeast RNA in a quantity equivalent to 10(8) white blood cells (WBC). Using the highly sensitive RT-PCR, a systematic study of the possible expression of bcr-abl RNA in WBC of healthy adults, children and umbilical cord blood (UCB) revealed the presence of bcr-abl transcripts in blood cells of 22/73 adults, 1/22 children but not in 22 samples of UCB. The comparison of these three groups indicated a significant tendency for the anomaly to increase in frequency with age.

Tumor necrosis factor-alpha production induced by viruses and by lipopolysaccharides in macrophages: similarities and differences.

Tumor necrosis factor (TNF)-a gene expression can be induced primarily in cells of the monocyte-macrophage lineage by a variety of inducers, including lipopolysaccharides (LPS), phorbol esters, ultraviolet (UV) light, and viruses. In this paper, we analyzed the regulatory mechanisms of TNF-alpha production induced by infection with the Sendai" virus in RAW 264.7 macrophages. We show that in these cells TNF-a synthesis results mainly from TNF-alpha mRNA translational activation. Using CAT reporter genes, we identified the UA- rich (UAR) sequences localized in the TNF-alpha mRNA 3' untranslated region (UTR) as the main sequence involved in this regulation. This sequence has been previously shown to be the essential regulatory element involved in LPS- induced translational activation of TNF mRNA. Activation of TNF gene expression by viral infection presents other similarities with those induced by LPS. First, TNF production in response to viral infection is inhibited by the protein-tyrosine kinase inhibitor herbimycin A as it is in response to LPS. More specifically, we show here that TNF mRNA translational activation induced by viral infection or by LPS is inhibited by pretreating the cells with herbimycin A. Second, TNF production in response to viruses is tissue-specific and is abrogated in RAW 264.7x NIH3T3 hybrid cells, which lack the ability to produce TNF in response to LPS, as a consequence of a defect in the LPS signaling pathway. However, viral infection induces TNF production in LPS- unresponsive C3H/HeJ mouse-derived peritoneal macro phages indicating that viruses and LPS signaling pathways differ for at least one intermediate which is the product of the Lps gene. Finally, we show that this regulatory mechanism can be triggered by different classes of viruses.

Defective translation of tumor necrosis factor mRNA in lipopolysaccharide-tolerant macrophages.

Macrophage activation by lipopolysaccharide (LPS) results in the translational activation of tumor necrosis factor (TNF) mRNA. The initial phase of macrophage activation is followed by a refractory state called LPS tolerance characterized by an impaired TNF production in response to a secondary LPS challenge. LPS-tolerant macrophages contain high amounts of TNF mRNA, suggesting a translational regulation of TNF biosynthesis. The induction of LPS tolerance was studied in RAW 264.7 macrophages stably transfected with a chloramphenicol acetyl-transferase (CAT) reporter gene construct driven by a constitutive cytomegalovirus promoter and containing the 3' untranslated region of the murine TNF gene. We found that primary stimulation of transfected cells by LPS (1 ng/ml, 12 hr) resulted in a marked suppression (80%) of CAT accumulation in response to a secondary LPS challenge (1 microgram/ml, 6 hr). In contrast, the accumulation of CAT mRNA was not influenced by LPS tolerance. Using the same CAT reporter, we observed that the serine/threonine phosphatases 1 and 2A inhibitor okadaic acid induced TNF mRNA translation and that this activation was not inhibited by LPS-tolerance. In conclusion, these data indicate that deficient production of TNF in LPS-tolerant macrophages in response to a second LPS challenge is characterized by a defective translation of TNF mRNA. However, this hyporesponsiveness to LPS is specific, since translation of TNF mRNA induced by okadaic acid is not inhibited in LPS-tolerant macrophages.

Long-lasting decreases of type II calmodulin kinase expression in kindled rat brains.

The kindling model of epilepsy is associated with long-lasting changes in type II calmodulin kinase (CaM kinase) activity and immunoreactivity. In order to determine the mechanism of these alterations, we measured gene expression of CaM kinase using in situ hybridization in septally kindled rat brains and paired controls using a 35S-labeled riboprobe for the beta subunit of the enzyme. We found CaM kinase mRNA concentrated in the hippocampus and other limbic structures. Kindling decreased hippocampal CaM kinase mRNA by 30% in CA1, 34% in CA2, 35% in CA3 41% in CA4, and 29% in the dentate gyrus. Hybridization was also decreased by 21% in the cerebral cortex but not in the lateral septum. These changes are similar in distribution and direction to those previously measured by immunohistochemistry. These data suggest that altered CaM kinase activity and immunoreactivity associated with kindling reflect long-lasting alterations in gene expression of this important synaptic protein, and provide further evidence for its possible importance in the kindling phenomenon.