Constrained G4 structures unveil topology specificity of known and new G4 binding proteins.

G-quadruplexes (G4) are non-canonical secondary structures consisting in stacked tetrads of hydrogen-bonded guanines bases. An essential feature of G4 is their intrinsic polymorphic nature, which is characterized by the equilibrium between several conformations (also called topologies) and the presence of different types of loops with variable lengths. In cells, G4 functions rely on protein or enzymatic factors that recognize and promote or resolve these structures. In order to characterize new G4-dependent mechanisms, extensive researches aimed at identifying new G4 binding proteins. Using G-rich single-stranded oligonucleotides that adopt non-controlled G4 conformations, a large number of G4-binding proteins have been identified in vitro, but their specificity towards G4 topology remained unknown. Constrained G4 structures are biomolecular objects based on the use of a rigid cyclic peptide scaffold as a template for directing the intramolecular assembly of the anchored oligonucleotides into a single and stabilized G4 topology. Here, using various constrained RNA or DNA G4 as baits in human cell extracts, we establish the topology preference of several well-known G4-interacting factors. Moreover, we identify new G4-interacting proteins such as the NELF complex involved in the RNA-Pol II pausing mechanism, and we show that it impacts the clastogenic effect of the G4-ligand pyridostatin.

V-erbA generates ribosomes devoid of RPL11 and regulates translational activity in avian erythroid progenitors.

The v-erbA oncogene transforms chicken erythrocytic progenitors (T2EC) by blocking their differentiation and freezing them in a state of self-renewal. Transcriptomes of T2EC, expressing either v-erbA or a non-transforming form of v-erbA (S61G), were compared using serial analysis of gene expression and some, but not all, mRNA-encoding ribosomal proteins were seen to be affected by v-erbA. These results suggest that this oncogene could modulate the composition of ribosomes. In the present study, we demonstrate, using two-dimensional difference in gel electrophoresis, that v-erbA-expressing cells have a lower amount of RPL11 associated with the ribosomes. The presence of ribosomes devoid of RPL11 in v-erbA-expressing cells was further confirmed by immunoprecipitation. In order to assess the possible impact of these specialized ribosomes on the translational activity, we analyzed proteomes of either v-erbA or S61G-expressing cells using 2D/mass spectrometry, and identified nine proteins present in differing amounts within these cells. Among these proteins, we focused on HSP70 because of its involvement in erythroid differentiation. Our results indicate that, in v-erbA-expressing cells, hsp70 is not only transcribed but also translated more efficiently, as shown by polyribosome fractionation experiments. We demonstrate here, for the first time, the existence of ribosomes with different protein components, notably ribosomes devoid of RPL11, and a regulation of mRNA translation depending on v-erbA oncogene expression.

Unbalanced expression of CK2 kinase subunits is sufficient to drive epithelial-to-mesenchymal transition by Snail1 induction.

Epithelial-to-mesenchymal transition (EMT) is closely linked to conversion of early-stage tumours into invasive malignancies. Many signalling pathways are involved in EMT, but the key regulatory kinases in this important process have not been clearly identified. Protein kinase CK2 is a multi-subunit protein kinase, which, when overexpressed, has been linked to disease progression and poor prognosis in various cancers. Specifically, overexpression of CK2α in human breast cancers is correlated with metastatic risk. In this article, we show that an imbalance of CK2 subunits reflected by a decrease in the CK2ß regulatory subunit in a subset of breast tumour samples is correlated with induction of EMT-related markers. CK2ß-depleted epithelial cells displayed EMT-like morphological changes, enhanced migration, and anchorage-independent growth, all of which require Snail1 induction. In epithelial cells, Snail1 stability is negatively regulated by CK2 and GSK3ß through synergistic hierarchal phosphorylation. This process depends strongly on CK2ß, thus confirming that CK2 functions upstream of Snail1. In primary breast tumours, CK2ß underexpression also correlates strongly with expression of EMT markers, emphasizing the link between asymmetric expression of CK2 subunits and EMT in vivo. Our results therefore highlight the importance of CK2ß in controlling epithelial cell plasticity. They show that CK2 holoenzyme activity is essential to suppress EMT, and that it contributes to maintaining a normal epithelial morphology. This study also suggests that unbalanced expression of CK2 subunits may drive EMT, thereby contributing to tumour progression.

The protein ICP0 of herpes simplex virus type 1 is targeted to nucleoli of infected cells. Brief report.

This study describes the nucleolar localization of the viral protein ICP0 of herpes simplex virus type 1. We show that the RING finger domain of ICP0 is essential for ICP0 to localize in nucleoli of transfected and 4 hour-infected cells. ICP0 forms particular intranucleolar domains that do not correspond to any known nucleolar domains. This distribution was confirmed by immunoblots performed on fractionated infected cells. Quantitative RT-PCR experiments indicated that ICP0 did not increase the transcription from the RNA polymerase I (Pol I) promoter in transfected cells, an effect opposite to that observed on viral and cellular Pol II promoters. Nucleoli are thus, after PML bodies and centromeres, a novel nuclear structure targeted by ICP0.

Characterization by two-dimensional gel electrophoresis of host proteins whose synthesis is sustained or stimulated during the course of herpes simplex virus type 1 infection.

Herpes simplex virus type 1 (HSV-1) gene expression is concomitant with a selective shutoff of host protein synthesis. While the synthesis of the vast majority of cellular proteins is inhibited immediately after infection, several cellular proteins continue to be synthesized, even during the late phase of infection. Because these cellular proteins may intervene in the life cycle of the virus, we undertook two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) analyses to evaluate the proportion of cellular proteins that is represented by these particular proteins. Human cells were infected with HSV-1. At different times after infection, proteins were labeled with 35S just prior to harvesting. The rate of synthesis of a set of 183 acidic host proteins, as well as that of ribosomal proteins, was measured during the course of infection, after separation by 2-D PAGE. As expected, HSV-1 induces a strong inhibition of host protein synthesis immediately after infection. However, the synthesis of basic ribosomal proteins and that of an unexpected high proportion of the sub-set of cellular proteins analyzed is sustained or stimulated during HSV-1 infection. A 2-D PAGE analysis outlining the expression patterns of these proteins at different times of infection is presented.