2'-19F labelling of ribose in RNAs: a tool to analyse RNA/protein interactions by NMR in physiological conditions.

Protein-RNA interactions are central to numerous cellular processes. In this work, we present an easy and straightforward NMR-based approach to determine the RNA binding site of RNA binding proteins and to evaluate the binding of pairs of proteins to a single-stranded RNA (ssRNA) under physiological conditions, in this case in nuclear extracts. By incorporation of a 19F atom on the ribose of different nucleotides along the ssRNA sequence, we show that, upon addition of an RNA binding protein, the intensity of the 19F NMR signal changes when the 19F atom is located near the protein binding site. Furthermore, we show that the addition of pairs of proteins to a ssRNA containing two 19F atoms at two different locations informs on their concurrent binding or competition. We demonstrate that such studies can be done in a nuclear extract that mimics the physiological environment in which these protein-ssRNA interactions occur. Finally, we demonstrate that a trifluoromethoxy group (-OCF3) incorporated in the 2'ribose position of ssRNA sequences increases the sensitivity of the NMR signal, leading to decreased measurement times, and reduces the issue of RNA degradation in cellular extracts.

Surface Passivation with a Perfluoroalkane Brush Improves the Precision of Single-Molecule Measurements.

Single-molecule imaging is invaluable for investigating the heterogeneous behavior and interactions of biological molecules. However, an impediment to precise sampling of single molecules is the irreversible adsorption of components onto the surfaces of cover glasses. This causes continuous changes in the concentrations of different molecules dissolved or suspended in the aqueous phase from the moment a sample is dispensed, which will shift, over time, the position of chemical equilibria between monomeric and multimeric components. Interferometric scattering microscopy (iSCAT) is a technique in the single-molecule toolkit that has the capability to detect unlabeled proteins and protein complexes both as they adsorb onto and desorb from a glass surface. Here, we examine the reversible and irreversible interactions between a number of different proteins and glass via analysis of the adsorption and desorption of protein at the single-molecule level. Furthermore, we present a method for surface passivation that virtually eliminates irreversible adsorption while still ensuring the residence time of molecules on surfaces is sufficient for detection of adsorption by iSCAT. By grafting high-density perfluoroalkane brushes on cover-glass surfaces, we observe approximately equal numbers of adsorption and desorption events for proteins at the measurement surface (±1%). The fluorous-aqueous interface also prevents the kinetic trapping of protein complexes and assists in establishing a thermodynamic equilibrium between monomeric and multimeric components. This surface passivation approach is valuable for in vitro single-molecule experiments using iSCAT microscopy because it allows for continuous monitoring of adsorption and desorption of protein without either a decline in detection events or a change in sample composition due to the irreversible binding of protein to surfaces.

Is Uracil-DNA Glycosylase UNG2 a New Cellular Weapon Against HIV-1?

Uracil-DNA glycosylase-2 (UNG2) is a DNA repair protein that removes uracil from single and double-stranded DNA through a basic excision repair process. UNG2 is packaged into new virions by interaction with integrase (IN) and is needed during the early stages of the replication cycle. UNG2 appears to play both a positive and negative role during HIV-1 replication; UNG2 improves the fidelity of reverse transcription but the nuclear isoform of UNG2 participates in the degradation of cDNA and the persistence of the cellular genome by repairing its uracil mismatches. In addition, UNG2 is neutralized by Vpr, which redirects it to the proteasome for degradation, suggesting that UNG2 may be a new cellular restriction factor. So far, we have not understood why HIV-1 imports UNG2 via its IN and why it causes degradation of endogenous UNG2 by redirecting it to the proteasome via Vpr. In this review, we propose to discuss the ambiguous role of UNG2 during the HIV-1 replication cycle.

Backbone resonance assignment of the human uracil DNA glycosylase-2.

The HIV-1 viral protein R (Vpr) is incorporated into virus particle during budding suggesting that its presence in the mature virion is required in the early steps of the virus life cycle in newly infected cells. Vpr is released into the host cell cytoplasm to participate to the translocation of the preintegration complex (PIC) into the nucleus for integration of the viral DNA into the host genome. Actually, Vpr plays a key role in the activation of the transcription of the HIV-1 long terminal repeat (LTR), mediates cell cycle arrest in G2 to M transition, facilitates apoptosis and controls the fidelity of reverse transcription. Moreover, Vpr drives the repair enzyme uracil DNA glycosylase (UNG2) towards degradation. UNG2 has a major role in "Base excision repair" (BER) whose main function is to maintain genome integrity by controlling DNA uracilation. The interaction of Vpr with the cellular protein UNG2 is a key event in various stages of retroviral replication and its role remains to be defined. We have performed the structural study of UNG2 by NMR and we report its (1HN, 15N, 13Cα, 13Cß and 13C') chemical shift backbone assignment and its secondary structure in solution as predicted by TALOS-N. We aim to determine with accuracy by NMR, the residues of UNG2 interacting with Vpr, characterize their interaction and use the local structure of UNG2 and its interface with Vpr to propose potential ligands disturbing this interaction.