Béatrice Jolles

DNA structure studies by resonance Raman spectroscopy

Abstract The application of ultraviolet resonance Raman spectroscopy (RRS) in DNA structure studies is reviewed. In exciting the purine and pyrimidine bases of DNA in their allowed electronic transitions at wavelengths shorter than 300 nm, the resonance Raman effect offers a specific source of information at each level of complexity. For the nucleic bases and mononucleotides, it gives information on the excited state geometries of the molecules through their intensity excitation profiles. For oligo- and poly-nucleotides, the Raman hypo/hyperchromism observed under the resonance conditions is very sensitive to base-base stacking interactions: thus it can monitor slight changes in these interactions, due to changes in the polymer geometries. Lastly the resonance Raman effect provides structural information through vibrational couplings between different components of the macromolecular buildings. Thus resonance Raman spectroscopy is shown to be a powerful technique for studying DNA conformational changes and gives very promising results in the selective investigation of chromophores in such elaborate molecular complexes as nucleic bases within DNA duplexes.

Translation termination efficiency modulates ATF4 response by regulating ATF4 mRNA translation at 5′ short ORFs

The activating transcription factor 4 (ATF4) promotes transcriptional upregulation of specific target genes in response to cellular stress. ATF4 expression is regulated at the translational level by two short open reading frames (uORFs) in its 5′-untranslated region (5′-UTR). Here, we describe a mechanism regulating ATF4 expression in translation termination-deficient human cells. Using microarray analysis of total RNA and polysome-associated mRNAs, we show that depletion of the eucaryotic release factor 3a (eRF3a) induces upregulation of ATF4 and of ATF4 target genes. We show that eRF3a depletion modifies ATF4 translational control at regulatory uORFs increasing ATF4 ORF translation. Finally, we show that the increase of REDD1 expression, one of the upregulated targets of ATF4, is responsible for the mTOR pathway inhibition in eRF3a-depleted cells. Our results shed light on the molecular mechanisms connecting eRF3a depletion to mammalian target of rapamycin (mTOR) pathway inhibition and give an example of ATF4 activation that bypasses the signal transduction cascade leading to the phosphorylation of eIF2α. We propose that in mammals, in which the 5′-UTR regulatory elements of ATF4 mRNA are strictly conserved, variations in translation termination efficiency allow the modulation of the ATF4 response.

Studies on human eRF3-PABP interaction reveal the influence of eRF3a N-terminal glycin repeat on eRF3-PABP binding affinity and the lower affinity of eRF3a 12-GGC allele involved in cancer susceptibility

ABSTRACT The eukaryotic release factor 3 (eRF3) has been involved in the control of mRNA degradation through its association with the cytoplasmic Poly(A) Binding Protein, PABP. In mammals, eRF3 N-terminal domain contains two overlapping PAM2 motifs which specifically recognize the MLLE domain of PABP. In humans, eRF3a/GSPT1 gene contains a stable GGC repeat encoding a repeat of glycine residues in eRF3a N-terminus. There are five known eRF3a/GSPT1 alleles in the human population, encoding 7, 9, 10, 11 and 12 glycines. Several studies have reported that the presence of eRF3a 12-GGC allele is correlated with an increased risk of cancer development. Using surface plasmon resonance, we have studied the interaction of the various allelic forms of eRF3a with PABP alone or poly(A)-bound PABP. We found that the N-terminal glycine repeat of eRF3a influences eRF3a-PABP interaction and that eRF3a 12-GGC allele has a decreased binding affinity for PABP. Our comparative analysis on eRF3a alleles suggests that the presence of eRF3a 12-GGC allele could modify the coupling between translation termination and mRNA deadenylation.

A Resonance Raman Spectroscopic Study of the Quadruplex Form of Polyriboinosinic Acid

The four stranded form of polyriboinosinic acid, or poly(rl), formed under conditions of high ionic strength, has been studied principally by resonance Raman spectroscopy excited in the ultraviolet absorbent band of the hypoxanthine residues. UV Absorption and circular dichroism studies were made, principally in order to verify the presence of the quadruplex form at the low concentrations of poly(rl) used, and a trial experiment with the structural probe Tb3+ was also performed. Experimental evidence is found for highly stacked metastable forms present at low concentrations of polynucleotide, which are destroyed by heating in favor of the two well known forms.

Impact of alendronate and VEGF-antisense combined treatment on highly VEGF-expressing A431 cells.

Bisphosphonates, and more specially nitrogen-containing bisphosphonates, which are in current use for the treatment of bone diseases, demonstrate proapoptotic, antiproliferative, antiangiogenic and anti-invasive properties on tumor cells. The amino-bisphosphonate alendronate is considered as a potential anticancer drug. In the case of A431 cells, which express high levels of VEGF, it had a two-step effect. At 24h, the antitumor properties of alendronate were counterbalanced by a survival process, which consisted of an enhancement of VEGF expression (mRNA and protein secretion) and TGF alpha secretion. It was only at 48 h that alendronate displayed the expected antiproliferative and antiangiogenic properties. The first step, in which the PI3K pathway was engaged, could be prevented by the use of a VEGF-antisense oligonucleotide. The combination of such an antisense with small concentrations of alendronate (approximately 2 microM), which is of the order of clinically used concentrations, was shown to have an antiangiogenic effect as soon as 12h.

Intracellular fate of oligodeoxynucleotides: A microspectro FRET study

Fluorescence resonance energy transfer (FRET) under confocal microscopy allows direct intracellular measurements of both localisation and interactions of tagged molecules to be performed. In the antigene and antisense strategies, short oligonucleotide sequences are used to inhibit RNA translation in cell. Numerous techniques (Crook, R.M. “In Vitro Cellular Uptake, Distribution, and Metabolism of Oligonucleotides” Handbook of Experimental Pharmacology, Vol. 131 Antisense Research and Application : 103-140 (1998) were used to follow oligonucleotide penetration and distribution in cultured cells, but no in situ study of the oligonucleotide stability was performed. Therefore, microspectroFRET was chosen as a non destructive method to study in differents cell lines the fate of short oligonucleotides, fluorescently labeled at both ends. When an oligonucleotide is intact, FRET occurs, so, in this study, intramolecular FRET permits us to know the ratio of intact oligonucleotides during time, i. e. the ratio of potentially active oligonucleotides ; single fluorescent labeling is not sufficient : the observed fluorescence may be due to fluorophores alone or still grafted on the oligonucleotide. The oligonucleotides, labeled with fluorescein at the 3’ end and rhodamine at the 5’ end, were added to the cells and subcellular microvolumes (-10-18 cubic meter) were analysed with the confocal microspectrofluorometer (Vigny, P. Amirand, C. Ballini, J.P. Chinsky, L. Duquesne, M. Laigle, A. Manfait, A. Sureau, F. Turpin, P.Y. “Microspectrofluorometry of Single Living Cells” Spectroscopy of Biological Molecules - State of the Art : 345-348 (1989) developed in the laboratory. Different wavelengths of an Argon LASER were used for excitation of the samples. The spectra were recorded on an optical multichannels analyser after separation of the different emission wavelengths by a monochromator. After FFT smoothing and background subtraction, comparisons of the spectra were achieved and analysed in term of : Stability of the oligonucleotides, which is directly related to the transfer efficiency, as a function of time Quantitative localisation of the oligonucleotides, as fluorescence intensity is proportional to concentration Surrounding of the oligonucleotides, as the quatum yield of fluorescein is pH dependent. As an example of granulocytic cell model, we used K562 cells. But these cells have a large nucleus, preventing to distinguish subcellular compartments. So we also used Hela cells as an example of epithelial-like cells with a real differentiation between cytoplasm and nucleus. 3e Colloque SFp, Paris-Sud ‘99 icole Polytechnique, Palaiseau, 28 juin-2 juillet 1999

Translation termination-dependent deadenylation of MYC mRNA in human cells

The earliest step in the mRNA degradation process is deadenylation, a progressive shortening of the mRNA poly(A) tail by deadenylases. The question of when deadenylation takes place remains open. MYC mRNA is one of the rare examples for which it was proposed a shortening of the poly(A) tail during ongoing translation. In this study, we analyzed the poly(A) tail length distribution of various mRNAs, including MYC mRNA. The mRNAs were isolated from the polysomal fractions of polysome profiling experiments and analyzed using ligase-mediated poly(A) test analysis. We show that, for all the mRNAs tested with the only exception of MYC, the poly(A) tail length distribution does not change in accordance with the number of ribosomes carried by the mRNA. Conversely, for MYC mRNA, we observed a poly(A) tail length decrease in the fractions containing the largest polysomes. Because the fractions with the highest number of ribosomes are also those for which translation termination is more frequent, we analyzed the poly(A) tail length distribution in polysomal fractions of cells depleted in translation termination factor eRF3. Our results show that the shortening of MYC mRNA poly(A) tail is alleviated by the silencing of translation termination factor eRF3. These findings suggest that MYC mRNA is co-translationally deadenylated and that the deadenylation process requires translation termination to proceed.