RESUMO
The pre-integration steps of the HIV-1 viral cycle are some of the most valuable targets of recent therapeutic innovations. HIV-1 integrase (IN) displays multiple functions, thanks to its considerable conformational flexibility. Recently, such flexible proteins have been characterized by their ability to form biomolecular condensates as a result of Liquid-Liquid-Phase-Separation (LLPS), allowing them to evolve in a restricted microenvironment within cells called membrane-less organelles (MLO). The LLPS context constitutes a more physiological approach to study the integration of molecular mechanisms performed by intasomes (complexes containing viral DNA, IN, and its cellular cofactor LEDGF/p75). We investigated here if such complexes can form LLPS in vitro and if IN enzymatic activities were affected by this LLPS environment. We observed that the LLPS formed by IN-LEDGF/p75 functional complexes modulate the in vitro IN activities. While the 3'-processing of viral DNA ends was drastically reduced inside LLPS, viral DNA strand transfer was strongly enhanced. These two catalytic IN activities appear thus tightly regulated by the environment encountered by intasomes.
Assuntos
Integrase de HIV , HIV-1 , Integração Viral , Integrase de HIV/metabolismo , Integrase de HIV/química , Integrase de HIV/genética , HIV-1/metabolismo , HIV-1/fisiologia , Humanos , DNA Viral/metabolismo , DNA Viral/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/químicaRESUMO
As in many other organisms, tRNA-derived RNAs (tDRs) exist in plants and likely have multiple functions. We previously showed that tDRs are present in Arabidopsis under normal growth conditions, and that the ones originating from alanine tRNAs are the most abundant in leaves. We also showed that tDRs Ala of 20 nt produced from mature tRNAAla (AGC) can block in vitro protein translation. Here, we report that first, these tDRs Ala (AGC) can be found within peculiar foci in the cell that are neither P-bodies nor stress granules and, second, that they assemble into intermolecular RNA G-quadruplex (rG4) structures. Such tDR Ala rG4 structures can specifically interact with an Arabidopsis DEA(D/H) RNA helicase, the DExH1 protein, and unwind them. The rG4-DExH1 protein interaction relies on a glycine-arginine domain with RGG/RG/GR/GRR motifs present at the N-terminal extremity of the protein. Mutations on the four guanine residues located at the 5' extremity of the tDR Ala abolish its rG4 structure assembly, association with the DExH1 protein, and foci formation, but they do not prevent protein translation inhibition in vitro. Our data suggest that the sequestration of tDRs Ala into rG4 complexes might represent a way to modulate accessible and functional tDRs for translation inhibition within the plant cell via the activity of a specific RNA helicase, DExH1.
Assuntos
Arabidopsis , Quadruplex G , Arabidopsis/genética , RNA Helicases/genética , RNA , RNA de TransferênciaRESUMO
Fluorogenic dimers with polarity-sensitive folding are powerful probes for live-cell bioimaging. They switch on their fluorescence only after interacting with their targets, thus leading to a high signal-to-noise ratio in wash-free bioimaging. We previously reported the first near-infrared fluorogenic dimers derived from cyanine 5.5 dyes for the optical detection of G protein-coupled receptors. Owing to their hydrophobic character, these dimers are prone to form nonspecific interactions with proteins such as albumin and with the lipid bilayer of the cell membrane resulting in a residual background fluorescence in complex biological media. Herein, we report the rational design of new fluorogenic dimers derived from cyanine 5. By modulating the chemical structure of the cyanine units, we discovered that the two asymmetric cyanine 5.25 dyes were able to form intramolecular H-aggregates and self-quenched in aqueous media. Moreover, the resulting original dimeric probes enabled a significant reduction of the nonspecific interactions with bovine serum albumin and lipid bilayers compared with the first generation of cyanine 5.5 dimers. Finally, the optimized asymmetric fluorogenic dimer was grafted to carbetocin for the specific imaging of the oxytocin receptor under no-wash conditions directly in cell culture media, notably improving the signal-to-background ratio compared with the previous generation of cyanine 5.5 dimers.