Natural UAG suppressor glutamine tRNA is elevated in mouse cells infected with Moloney murine leukemia virus.

Kuchino, Yoshiyuki; Beier, Hildburg; Akita, Noriko; Nishimura, Susumu

doi:10.1073/pnas.84.9.2668

Cited by 77 publications

(53 citation statements)

References 21 publications

(25 reference statements)

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“…This lack of leucine incorporation highlights the specificity of the MuLV suppression system. While Kuchino et al (17) reported that infection with Mo-MuLV alters the cellular profile of glutamine tRNAs, we found no difference between tRNAs of infected and uninfected cells (1). In contrast to the proposal of Kuchino et al (17), our results (1, 2), as well as those of Panganiban (18), strongly suggested that suppression is mediated by a cellular tRNA that is neither altered nor induced as a result of virus infection.…”

Section: Discussioncontrasting

confidence: 50%

“…Kuchino et al (17) isolated a minor glutamine tRNA that was reported to suppress the UAG codon of TMV RNA in a rabbit reticulocyte lysate. It is not clear that this tRNA can also suppress the MuLV UAG codon as suggested (17), however, since the MuLV and TMV systems are not entirely equivalent. Thus, in contrast to the situation with MuLV (Fig.…”

Section: Discussionmentioning

confidence: 97%

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Identification of amino acids inserted during suppression of UAA and UGA termination codons at the gag-pol junction of Moloney murine leukemia virus.

Feng

Copeland

Oroszlan

et al. 1990

Proc. Natl. Acad. Sci. U.S.A.

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Expression of the murine leukemia virus poi gene occurs by translational readthrough of an in-frame UAG codon between the gag and poi coding regions. In a previous study, we mutated the UAG codon to UAA or UGA and demonstrated that both of these termination codons could be suppressed in reticulocyte lysates and in infected cells with the same efficiency as UAG. We now report the identity of the amino acids inserted in vitro in response to UAA and UGA in fusion products containing the gag-pol junction region. The results show that UAA, like UAG, directs the incorporation of glutamine, whereas UGA directs the incorporation of three amino acids, arginine, cysteine, and tryptophan. To our knowledge, this is the first report indicating misreading of UAA as glutamine and UGA as arginine and cysteine in higher eukaryotes. Interestingly, although our protein synthesis system presumably contains other known UAG and UGA suppressors, these tRNAs did not suppress the termination codons in our experiments. Thus, it seems possible that the sequence surrounding the gag-pol junction not only promotes suppression but also helps determine which tRNAs function in suppression.Readthrough suppression of termination codons is a mechanism of translational regulation that is used by a number of viruses infecting higher eukaryotes. The analysis of this phenomenon thus provides new information on the functional capabilities of the translational apparatus of these organisms. We have been investigating suppression in the murine leukemia virus (MuLV) system (1, 2). MuLV, like other mammalian type C retroviruses, regulates pol gene expression by translational readthrough ofan in-frame UAG codon between the gag and pol coding regions (3-5). Suppression of this termination codon occurs with a frequency of about 5% (3)(4)(5) and is mediated by insertion of a glutamine residue at the position corresponding to the UAG (6). The readthrough product is a 200-kDa gag-pol fusion protein, which is cleaved at a late stage in virus assembly to give rise to protease, reverse transcriptase, and integrase (3).To determine whether the signals that govern readthrough are unique for UAG, we used oligonucleotide-directed mutagenesis to change the UAG at the Moloney (Mo)-MuLV gag-pol junction to UAA or UGA (2). Interestingly, we found that these termination codons could be efficiently suppressed both in reticulocyte lysates and in infected cells (2). These results demonstrated that the suppression signals are not specific for UAG and that mammalian cells and cell extracts contain tRNAs capable of suppressing UGA and UAA codons which appear in a retroviral context. Similar findings have been reported by other investigators working with tobacco mosaic virus (TMV) (7), Sindbis virus (8), and MuLV (9).In the present study we have identified the amino acids inserted in response to UAA and UGA by using an in vitro MuLV suppression system (1). We find that UAA, like UAG, directs the incorporation of glutamine into protein, whereas UGA directs the incorporation ...

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Section: Discussioncontrasting

confidence: 50%

Section: Discussionmentioning

confidence: 97%

Identification of amino acids inserted during suppression of UAA and UGA termination codons at the gag-pol junction of Moloney murine leukemia virus.

Feng

Copeland

Oroszlan

et al. 1990

Proc. Natl. Acad. Sci. U.S.A.

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“…In the light of earlier results which showed that the ciliated protozoan Tetrahymena [36] and even mouse cells [14] contain, under certain physiological conditions, an UAG-recognizing Gln-tRNA we searched for the existence of such a tRNA in sponge cells. Unfractionated G. cydonium tRNA was subjected to column chromatography on BD-cellulose (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Total tRNA (from 5 g sponge cells) was extracted from the collected cells using phenol [14]. The tRNA fraction (A260 = 38-44) was loaded onto a BD-cellulose column (5 cm x 0.5 cm) and eluted with a 120-ml linear gradient of 0.3 -1.2 M NaCl in 0.2 M sodium acetate, pH 6.0.…”

Section: Separation Of T R N Amentioning

confidence: 99%

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Regulated expression and phosphorylation of the 23–26‐kDa ras protein in the sponge Geodia cydonium

Robitzki¹,

Schröder²,

Ugarković³

et al. 1990

European Journal of Biochemistry

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We have cloned, sequenced and examined the sponge Geodia cydonium cDNA encoding a protein homologous to ras proteins. The sponge ras protein has a more conserved N-terminal region and a less conserved Cterminal region, especially in comparison to Dictyostelium discoideum; the similarity to human c-Ha-ras-I and to Saccharomyces cerevisiae is less pronounced. The sponge ras cDNA comprises five TAG triplets; at the translational level these UAG termination codons are suppressed by a Gln-tRNA. The sponge ras protein was isolated and partially purified (23 -26 kDa) and found to undergo phosphorylation at a threonine moiety, when dissociated cells were incubated in the presence of a homologous aggregation factor and insulin. Insulin-mediated phosphorylation of the ras protein resulted in a decrease in its Kd with GTP from 2 FM to 80 nM. The activated ras protein displayed high GTPase activity if the partially purified protein was incubated with homologous lectin and lectin receptor molecules. These results suggest that in the sponge, ras is activated by the insulin/insulin(insulin-like)-receptor system. This transition enables the ras protein to interact with the lectin-receptor/lectin complex, a process which may ultimately lead to an initiation of an intracellular signal-transduction chain.Sponges are the lowest multicellular eukaryotic organisms. Due to the relatively low specialization and concomitantly to the high differentiation and dedifferentiation potency of the cells, the sponge cell system has proven to be a useful model for studying the mechanism of cell-cell adhesion on molecular level. Results of detailed biochemical and cell biological studies with the main cell-adhesion molecules, the aggregation factor and the aggregation receptor, led to the formulation of the modulation theory of cell adhesion [I].The events of cell adhesion are contingent on a multiplicity of precisely coordinated intracellular signal-transduction pathways. Using the marine sponge Geodia cydonium we showed [2] that during the initial phase of cell-cell contact the aggregation factor causes a rapid stimulation of the phosphatidylinositol pathway, resulting in an activation of protein kinase C and subsequent phosphorylation of DNA topoisomerase 11. As one consequence of these processes, the cells undergo a phase of high-level DNA synthesis. However, at later stages, the aggregation factor loses its mitogenic activity; this function is then taken over by the matrix lectin. During this switch, the lectin receptor associates in the plasma membrane with the ras protooncogene product.In the present study we report that the recently discovered insulin and its receptor molecules in sponges [3] augment the level of phosphorylation of the ras protein, resulting in an increase in its intrinsic GTPase activity in response to an

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Structural characterization of modified nucleosides in tRNA hydrolysates by frit-fast atom bombardment liquid chromatography/mass spectrometry

Takeda

Nakamura

Yoshizumi

et al. 1994

Biol. Mass Spectrom.

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Feasibility for the structural characterization of modified nucleosides in transfer RNA at low microgram levels has been investigated by using continuous-flow frit-fast atom bombardment liquid chromatography/mass spectrometry (frit-FAB LC/MS). Sample of tRNA(Phe) from brewer's yeast (Saccharomyces cerevisiae) was used as a main model, and enzymatically hydrolysed by nuclease P1 and alkaline phosphatase. The resulting nucleoside mixture was separated by using a microbore reversed-phase LC column (150 mm x 0.5 mm i.d.) with an aqueous ammonium acetate-methanol gradient, and the mass spectra were acquired on both positive and negative ionization modes. The modified nucleosides were characterized by comparison of the relative LC elution times with authentic nucleosides, and further confirmed by the structural information from the frit-FAB mass spectra where both molecular and base ions were in general observed as intense peaks in both ionization modes. Typically, 0.06-0.2 A260 units (3-10 micrograms) of isoaccepting tRNA was enough to obtain full-scan mass spectra of modified nucleosides, often occurring at a frequency of one per tRNA molecule using positive ion detection. The LC/MS system was used to screen modified nucleosides in tRNA of the extremely thermophilic microorganism Pyrodictium occultum.

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Natural UAG suppressor glutamine tRNA is elevated in mouse cells infected with Moloney murine leukemia virus.

Cited by 77 publications

References 21 publications

Identification of amino acids inserted during suppression of UAA and UGA termination codons at the gag-pol junction of Moloney murine leukemia virus.

Identification of amino acids inserted during suppression of UAA and UGA termination codons at the gag-pol junction of Moloney murine leukemia virus.

Regulated expression and phosphorylation of the 23–26‐kDa ras protein in the sponge Geodia cydonium

Structural characterization of modified nucleosides in tRNA hydrolysates by frit-fast atom bombardment liquid chromatography/mass spectrometry

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