Adri Thomas
Utrecht University, Developmental Biology, Faculty Member
Research Interests: Leukemia, Transcription Factors, Translation, Xenopus, Cell Differentiation, and 22 moreHumans, Animals, Male, Post-Transcriptional Gene Regulation, Differentiation, Fibroblast Growth Factor, Secondary Structure, Embryonic Development, Protein Expression, Transforming Growth Factor Beta, Androgen Receptor, Open Reading Frame, Growth Factor, Protein Binding, DNA binding proteins, UTR, Translation initiation, Ataxia Telangiectasia, Internal ribosome entry site, Protein Biosynthesis, Biochemistry and cell biology, and Insulin Like Growth Factor
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p50, a member of the Y-box binding transcription factor family, is tightly associated with eukaryotic mRNAs and is responsible for general translational regulation. Here we show that p50, in addition to its previously described ability to... more
p50, a member of the Y-box binding transcription factor family, is tightly associated with eukaryotic mRNAs and is responsible for general translational regulation. Here we show that p50, in addition to its previously described ability to melt mRNA secondary structure, is capable of promoting rapid annealing of complementary nucleic acid strands. p50 accelerates annealing of RNA and DNA duplexes up to 1500-fold within a wide range of salt concentrations and temperatures. Phosphorylation of p50 selectively inhibits DNA annealing. Moreover, p50 catalyzes strand exchange between double-stranded and single-stranded RNAs yielding a product bearing a more extended double-stranded structure. Strikingly, p50 displays both RNA-melting and -annealing activities in a dose-dependent manner; a relatively low amount of p50 promotes formation of RNA duplexes, whereas an excess of p50 causes unwinding of double-stranded forms. Our results suggest that the alteration of nucleic acid conformation is a basic mechanism of the p50-dependent regulation of gene expression.
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p50, the major core protein bound to mammalian mRNAs, has been reported to stimulate translation at low p50/mRNA ratios and inhibit translation at high p50/mRNA ratios. This study aims to address the molecular mechanisms underlying these... more
p50, the major core protein bound to mammalian mRNAs, has been reported to stimulate translation at low p50/mRNA ratios and inhibit translation at high p50/mRNA ratios. This study aims to address the molecular mechanisms underlying these phenomena using the in vitro assembly of 48 S preinitiation complexes from fully purified translational components in the presence or absence of p50 as analyzed by the toeprint assay. With limited concentrations of eIF2, eIF3, and eIF4F, p50 (but not pyrimidine tract-binding protein, which was taken for comparison) strongly stimulates formation of the 48 S preinitiation complexes with beta-globin mRNA. This stimulation is observed when just a few molecules of p50 are bound per molecule of the mRNA. When the amount of p50 in solution is increased over some threshold p50/mRNA ratio, a remarkable repression is observed that can still be relieved by adding more eIF2 and eIF4F. At even higher concentrations of p50, the inhibitory effect becomes irreversible. The threshold ratio depends upon the extent of secondary structure of the 5'-untranslated region linked to the beta-globin coding region. Chemical probing has confirmed that the binding of p50 to mRNA involves only the sugar-phosphate backbone of the mRNA leaving nucleotide bases free for interaction with other messenger ribonucleoprotein (mRNP) components. These data are best compatible with the functional role of p50 as a "manager" of mRNA-protein interactions in mammalian mRNPs.
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Ab-neutralized HIV-1 can be captured by dendritic cells (DCs), which subsequently transfer infectious HIV-1 to susceptible CD4(+) T cells. In this study, we examined the capacity of early Abs, as well as recently identified broadly... more
Ab-neutralized HIV-1 can be captured by dendritic cells (DCs), which subsequently transfer infectious HIV-1 to susceptible CD4(+) T cells. In this study, we examined the capacity of early Abs, as well as recently identified broadly neutralizing Abs (bNAbs) targeting different envelope glycoprotein (Env) epitopes, to block HIV-1 transmission by immature and mature DCs to HIV-1-sensitive cells. Three bNAbs directed against the gp41 membrane proximal region of Env (2F5, 4E10, and 10E8) and three gp120 bNAbs targeting the CD4 binding site (b12, VRC01, and NIH45-46) were examined. In addition, eight glycan-dependent bNAbs targeting the V1V2 apex (PG9, PG16, and PGT145), the V3 loop (2G12, PGT121, and PGT128), and the gp120-gp41 interface of Env (PGT151 and 35O22) were tested. bNAbs that bound specific glycans showed, depending on the immature or mature state of the DC, diverse efficiencies in HIV-1 trans-infection. All bNAbs that bound the CD4 binding site blocked trans-infection, wherea...
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The Xenopus laevis Connexin-41 (Cx41) mRNA contains three upstream open reading frames (uORFs) in the 5'-untranslated region (UTR). We analyzed the translation efficiency of constructs containing the Cx41 5'-UTR linked to the... more
The Xenopus laevis Connexin-41 (Cx41) mRNA contains three upstream open reading frames (uORFs) in the 5'-untranslated region (UTR). We analyzed the translation efficiency of constructs containing the Cx41 5'-UTR linked to the green fluorescent protein reporter after injection of transcripts into one-cell stage Xenopus embryos. The translational efficiency of the wild-type Cx41 5'-UTR was only 2% compared with that of the beta-globin 5'-UTR. Mutation of each of the three uAUGs into AAG codons enhanced translation 82-, 9-, and 4-fold compared with the wild-type Cx41 5'-UTR. Based on these increased translation efficiencies, the percentages of ribosomes that recognized the uAUGs were calculated. Only 0.03% of the ribosomes that entered at the cap structure scanned the entire 5'-UTR and translated the main ORF. The results indicate that all uAUGs are recognized by the majority of the scanning ribosomes and that the three uAUGs strongly modulate translation effici...
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Leukoencephalopathy with vanishing white matter (VWM) is a childhood white matter disorder with an autosomal-recessive mode of inheritance. The clinical course is chronic progressive with episodes of rapid neurologic deterioration after... more
Leukoencephalopathy with vanishing white matter (VWM) is a childhood white matter disorder with an autosomal-recessive mode of inheritance. The clinical course is chronic progressive with episodes of rapid neurologic deterioration after febrile infections. The disease is caused by mutations in the genes encoding the subunits of eukaryotic initiation factor 2B (eIF2B), a protein complex that is essential for protein synthesis. In VWM, mutations in the eIF2B genes are thought to impair the ability of cells to regulate protein synthesis under normal and stress conditions. It has been suggested that the pathophysiology of VWM involves inappropriate activation of the unfolded protein response (UPR). The UPR is a protective mechanism activated by an overload of unfolded or malfolded proteins in the endoplasmic reticulum. Activation of one pathway of the UPR, in which eIF2B is involved, has already been described in brain tissue of patients with VWM. In the present study, we demonstrate activation of all 3 UPR pathways in VWM brain tissue using real-time quantitative polymerase chain reaction and immunohistochemistry. We show that activation occurs exclusively in the white matter, predominantly in oligodendrocytes and astrocytes. The selective involvement of these cells suggests that inappropriate UPR activation may play a key role in the pathophysiology of VWM.
Research Interests: Protein Folding, Membrane Proteins, Immunohistochemistry, Neuropathology, Adolescent, and 25 moreTranscription Factors, Protein synthesis, Signal Transduction, Molecular chaperones, Humans, Child, Animals, Unfolded Protein Response, Endoplasmic Reticulum, Astrocyte, Infant, Alternative splicing, Clinical Sciences, Protein Complex Detection, White matter, Adult, Specific Activity, Biological markers, Heat Shock Proteins, Alternative Splicing, Protein Conformation, DNA binding proteins, Neurosciences, Protein Biosynthesis, and Quantitative polymerase chain reaction
Research Interests: Engineering, Technology, Biotechnology, Gene expression, Biological Sciences, and 12 moreSecretory Pathway, Animals, P-glycoprotein, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, Glycosylation, Amino Acid Sequence, Base Sequence, Recombinant Protein, Recombinant Proteins, Enzyme Linked Immunosorbent Assay, Secretion, and Immunoblotting
p50, the major core protein bound to mammalian mRNAs, has been reported to stimulate translation at low p50/mRNA ratios and inhibit translation at high p50/mRNA ratios. This study aims to address the molecular mechanisms underlying these... more
p50, the major core protein bound to mammalian mRNAs, has been reported to stimulate translation at low p50/mRNA ratios and inhibit translation at high p50/mRNA ratios. This study aims to address the molecular mechanisms underlying these phenomena using the in vitro assembly of 48 S preinitiation complexes from fully purified translational components in the presence or absence of p50 as analyzed by the toeprint assay. With limited concentrations of eIF2, eIF3, and eIF4F, p50 (but not pyrimidine tract-binding protein, which was taken for comparison) strongly stimulates formation of the 48 S preinitiation complexes with beta-globin mRNA. This stimulation is observed when just a few molecules of p50 are bound per molecule of the mRNA. When the amount of p50 in solution is increased over some threshold p50/mRNA ratio, a remarkable repression is observed that can still be relieved by adding more eIF2 and eIF4F. At even higher concentrations of p50, the inhibitory effect becomes irreversible. The threshold ratio depends upon the extent of secondary structure of the 5'-untranslated region linked to the beta-globin coding region. Chemical probing has confirmed that the binding of p50 to mRNA involves only the sugar-phosphate backbone of the mRNA leaving nucleotide bases free for interaction with other messenger ribonucleoprotein (mRNP) components. These data are best compatible with the functional role of p50 as a "manager" of mRNA-protein interactions in mammalian mRNPs.
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Complete cDNA sequences were obtained for ribosomal protein (rp) L15 and eukaryotic initiation factor eIF2alpha from the lepidopteran insect Spodoptera frugiperda, and for elongation factor eEF2 from S. exigua. The presence of a... more
Complete cDNA sequences were obtained for ribosomal protein (rp) L15 and eukaryotic initiation factor eIF2alpha from the lepidopteran insect Spodoptera frugiperda, and for elongation factor eEF2 from S. exigua. The presence of a 5' terminal oligopyrimidine (TOP) tract classified the lepidopteran rpL15 transcript as a TOP mRNA. For eEF2, two types of transcripts were observed, one of which had a 5'TOP tract. The transcript levels for rpL15, eEF2 and eIF2alpha decreased following baculovirus infection. Polysome analysis showed that the corresponding mRNAs remained to be translated until at least 16 h post-infection for both TOP and non-TOP mRNAs. Baculovirus-induced host shut-off therefore appears to be regulated at the level of RNA abundance rather than at the translational level.
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Leukoencephalopathy with vanishing white matter (VWM) is an inherited childhood white matter disorder, caused by mutations in the genes encoding eukaryotic initiation factor 2B (eIF2B). The present study showed that, while the eIF2B... more
Leukoencephalopathy with vanishing white matter (VWM) is an inherited childhood white matter disorder, caused by mutations in the genes encoding eukaryotic initiation factor 2B (eIF2B). The present study showed that, while the eIF2B activity was reduced in VWM lymphoblasts, the expression levels of the eIF2B subunits were similar to control lymphoblast lines. The mutations in eIF2B did not affect the interaction with eIF2. Strikingly, no apparent differences for the regulation of protein synthesis, measured by [35S]-methionine incorporation, were found between control and VWM lymphoblasts. Western blotting showed that, in some VWM cells, exposure to heat shock caused a decrease in the expression of specific eIF2B subunits. Most importantly, the increase in phosphorylation of eIF2alpha in response to heat shock was lower in VWM lymphoblasts than in control cells. These findings could form part of the explanation for the episodes of rapid and severe deterioration in VWM patients that are precipitated by febrile infections.
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... J Biol Chem 273: 32340-32346 Berlanga JJ, Santoyo J, De Haro C (1999) Characterization of a ... J Biol Chem 270: 14597-14603 Joshi-Barve S, De Benedetti A, Rhoads RE (1992 ... J Biol Chem 267: 21038-21043 Kimball SR, Fabian JR, Pavitt... more
... J Biol Chem 273: 32340-32346 Berlanga JJ, Santoyo J, De Haro C (1999) Characterization of a ... J Biol Chem 270: 14597-14603 Joshi-Barve S, De Benedetti A, Rhoads RE (1992 ... J Biol Chem 267: 21038-21043 Kimball SR, Fabian JR, Pavitt GD, Hinnebusch AG, Jefferson LS ...
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The properties of the architecturally complex Xenopus laevis TGFbeta5, PDGF-A and PDGF-alpha receptor 5'UTRs were investigated. 5' extended cDNAs were obtained by 5'RACE, resulting in long 5'UTRs (478-710 nt) with multiple... more
The properties of the architecturally complex Xenopus laevis TGFbeta5, PDGF-A and PDGF-alpha receptor 5'UTRs were investigated. 5' extended cDNAs were obtained by 5'RACE, resulting in long 5'UTRs (478-710 nt) with multiple upstream AUGs (3-13), andthe potential to fold into stable structures. Injection studies suggested that the cloned PDGF-alphaR 5'UTR contains an intron. Splicing at potential 5' and 3' splice sites would result in a non-complex 5'UTR of 142 nt. The above mentioned 5'UTR characteristics are inhibitory for ribosomal scanning. Indeed, relative to the beta-globin 5'UTR, the complex 5'UTRs strongly repressed initiation of protein synthesis in pre-MBT Xenopus embryos. However, later in embryogenesis, the inhibition was partly relieved. The results show temporal translational control by these 5'UTRs. Transgenic embryos showed that the 5'UTRs allowed translation throughout the embryo; spatial control could not be observe...
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A number of growth factors, whose spatio-temporal expression is essential for embryonic development, are encoded by mRNAs with a complex 5'UTR. Human insulin-like growth factor 2 mRNA contains a long (592 nucleotides) 5'UTR... more
A number of growth factors, whose spatio-temporal expression is essential for embryonic development, are encoded by mRNAs with a complex 5'UTR. Human insulin-like growth factor 2 mRNA contains a long (592 nucleotides) 5'UTR (IGFl1) with one upstream open reading frame and stable stem-loop structures, elements which might be important for controlled translation. To investigate whether these unusual features of IGFl1 can control translation initiation during embryogenesis, we examined the initiation efficiency on this 5'UTR during development of Xenopus laevis. The results demonstrate that IGFl1 strongly represses translation of a reporter in early embryos, compared with the Xenopus beta-globin 5'UTR. The inhibition is alleviated soon after the midblastula transition, suggesting a stimulatory effect of the transcription start. However, a similar stimulation of IGFl1-driven translation is seen in embryos in which de novo transcription was inhibited by actinomycin D. Fur...
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The p10 gene of Autographa californica nucleopolyhedrovirus has two putative AATAAA polyadenylation signals. The downstream signal is used predominantly, as was determined by analysing 3' cDNA ends. This downstream motif is followed... more
The p10 gene of Autographa californica nucleopolyhedrovirus has two putative AATAAA polyadenylation signals. The downstream signal is used predominantly, as was determined by analysing 3' cDNA ends. This downstream motif is followed by a GT-rich sequence, known to be important for efficient polyadenylation in mammalian systems. To analyse the importance of polyadenylation for p10 gene expression, recombinant viruses with altered 3' untranslated regions (UTRs) were tested using chloramphenicol acetyltransferase (CAT) as a reporter. Surprisingly, after inactivation of the downstream AATAAA motif, CAT expression remained at the same high level as observed with a wild-type 3' UTR. Polyadenylation occurred 24-28 nucleotides further downstream, probably due to an ATTAAA sequence motif. Replacing the p 10 3' UTR with the SV40 early terminator sequence as part of an hsp70-lacZ-SV40 gene cassette, which is commonly used in baculovirus expression vectors, resulted in a reducti...
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Protein yields in the baculovirus expression system do not always correlate with the presence of abundant amounts of corresponding mRNAs. Therefore, a novel aspect of the baculovirus expression system was studied: initiation of... more
Protein yields in the baculovirus expression system do not always correlate with the presence of abundant amounts of corresponding mRNAs. Therefore, a novel aspect of the baculovirus expression system was studied: initiation of translation of very late mRNAs of Autographa californica multicapsid nucleopolyhedrovirus. The untranslated regions (UTRs) of the p10 mRNA of this baculovirus were studied by in vitro translation and after transfection into Spodoptera frugiperda insect cells. Lysates from insect cells were optimized for translation of in vitro transcripts containing p10 sequences. The lysates were used to measure the effects of various deletions in either the 5' or 3'UTR on protein synthesis. Transcripts containing the p10 5'UTR were translated efficiently. Large deletions in the 5'UTR severely decreased this efficiency. Deletions in the 3'UTR negatively affected expression of the reporter gene in vivo; however, no effect on translational efficiency in the...
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The majority of cellular mRNAs have relatively short and unstructured 5' untranslated regions (UTRs) that allow efficient translation, such as the beta-globin mRNA. An exception to this rule is the group of growth factor mRNAs which,... more
The majority of cellular mRNAs have relatively short and unstructured 5' untranslated regions (UTRs) that allow efficient translation, such as the beta-globin mRNA. An exception to this rule is the group of growth factor mRNAs which, in general, have long 5' UTRs with a high G + C content. An example is insulin-like growth factor II (IGF-II), which is encoded by four mRNAs, arising from four different promoters. Transcripts having the human IGF-II leader 1 are only expressed in adult liver where IGF-II protein synthesis is solely under direction of this 5' UTR. We investigated the translational efficiency in vitro of this 5' UTR, linked to the chloramphenicol acetyltransferase (CAT) encoding region. As expected from the primary structure of IGF-II leader 1, translational efficiency was very low compared with beta-globin 5' UTR-CAT mRNA. Addition of cell extract from undifferentiated P19 embryonal carcinoma (EC) cells preferentially stimulated translation of an IG...
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eIF-3 from wheat germ is a large multicomponent factor. It sediments at 15 S and is comprised of ten different polypeptides with an Mr value ranging from 26 000 to 135 000; five out of the ten seem to be present in a 1 : 1 stoichiometric... more
eIF-3 from wheat germ is a large multicomponent factor. It sediments at 15 S and is comprised of ten different polypeptides with an Mr value ranging from 26 000 to 135 000; five out of the ten seem to be present in a 1 : 1 stoichiometric ratio, whereas the others appear to occur approximately in a 0.5 to 1 ratio. The factor is active in a partially purified cell-free system derived from wheat germ and in a mammalian model assay system for the synthesis of methionyl puromycin.
We describe a re-examination of the cell-free protein synthetic activity of eukaryotic ribosomes having proteins phosphorylated to different extents. Ribosomal 40 S subunits were isolated both from a variety of cells in which there is... more
We describe a re-examination of the cell-free protein synthetic activity of eukaryotic ribosomes having proteins phosphorylated to different extents. Ribosomal 40 S subunits were isolated both from a variety of cells in which there is relatively little phosphorylation of ribosomal protein S6, and from cells subjected in vivo to different stimuli that promote the extensive phosphorylation of protein S6. The ability of these subunits to bind Met-tRNA as well as the second amino acyl-tRNA (Val-tRNA) was compared in the presence of highly purified initiation factors, elongation factor EF-1 at various concentrations of 60S subunits, 9 S globin mRNA and potassium ions. The ability of the subunits to synthesize polyphenylalanine was also studied using highly purified elongation factors. In no case was any significant difference in activity observed between ribosomes with protein S6 phosphorylated to different extents. Similar, though less extensive, studies were preformed comparing 60 S ri...
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eIF-4C has a pronounced stimulatory effect on initiation complex formation with native 80-S ribosomes (80-Sn) as the only source of ribosomal subunits, but only a small effect when washed 40-S subunits are used. eIF-4C is accessary to... more
eIF-4C has a pronounced stimulatory effect on initiation complex formation with native 80-S ribosomes (80-Sn) as the only source of ribosomal subunits, but only a small effect when washed 40-S subunits are used. eIF-4C is accessary to eIF-3 in dissociating 80-Sn ribosomes. eIF-4C is present on 40-Sn but absent on 40-Sn dimers, which occur in preparations of native ribosomes and are as such inactive in protein synthesis. eIF-4C dissociates 40-Sn dimers into active monomers. These results can be explained by assuming that the presence of eIF-4C on 40-Sn prevents: (a) premature association with 60-S ribosomal subunits and (b) dimerisation, thus increasing the rate and extent of initiation complex formation.
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Many DNA constructs are generated for protein expression studies. Translational properties and mRNA stability are crucial aspects that have to be accounted for during DNA construction. An optimized vector for protein overexpression... more
Many DNA constructs are generated for protein expression studies. Translational properties and mRNA stability are crucial aspects that have to be accounted for during DNA construction. An optimized vector for protein overexpression studies is described considering elements in the mature mRNA that influence translatability and stability. Recommendations regarding vector construction for Xenopus laevis embryo injection are provided, based on literature and experimental data. The 5'untranslated region (5'UTR) should be non-regulated, short, unstructured, and without AUG codons. The sequence around the start codon should match the initiation context of the species studied (ACCAUGG, for vertebrates), and the open reading frame should be cloned with its own stop codon, followed by a G or A residue. Furthermore, the 3'UTR should be non-regulated, and a strong polyadenylation signal must be included in DNA vectors. In RNA template vectors, the presence of a poly(A) or AC tail is...
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Research Interests: Kinetics, Virology, Enzyme Inhibitors, Protein synthesis, Influenza virus, and 16 moreBiological Sciences, Humans, Mice, Animals, Peptides, Phosphorylation, Poliomyelitis, Viral Infection, HeLa cells, Amino Acid Sequence, Virus diseases, Protein Binding, Protein Kinase Inhibitors, Translation initiation, Okadaic acid, and Encephalomyocarditis virus
The complex architecture of human insulin-like growth factor (IGF) II-leader 1 of 592 nucleotides (nt), with one open reading frame (ORF), and the potential to fold into stable structures makes efficient linear ribosomal scanning... more
The complex architecture of human insulin-like growth factor (IGF) II-leader 1 of 592 nucleotides (nt), with one open reading frame (ORF), and the potential to fold into stable structures makes efficient linear ribosomal scanning difficult to comprehend. Indeed, leader 1-driven reporter expression is low in rabbit reticulocyte lysate. Contrarily, leader 1 is very efficient in cells. Therefore, we tested whether this 5'UTR uses an alternative mechanism for translation initiation in vivo, internal entry or ribosomal shunting. Internal initiation was tested by introducing leader 1 into the intercistronic region of a bicistronic vector. Second cistron expression, driven by leader 1, was lower than by the intercistronic beta-globin 5'UTR, indicating that leader 1 does not contain an internal ribosomal entry site (IRES). Shunting was tested by inserting hairpin (HP) structures, capable of blocking ribosomal scanning, at eight positions in leader 1. After transfection, these mutant 5'UTRs were incapable of directing reporter expression. Less stable HPs at the same positions increased the activity to 50% of wild-type activity, indicating that insertions at these positions are not disastrous for initiation. These data indicate that the translational machinery encounters major parts of leader 1. As scanning seems unlikely, and internal entry and shunting were shown not to occur, we discuss a modified scanning mechanism for architecturally complex 5'UTRs.