Molecular mechanisms of oxidation-mediated RNA-protein cross-links – OxiXlink

Proteins play multiple roles in RNA metabolism, regulating almost all aspects of the RNA functions, and form tight non-covalent complexes, mostly mediated by electrostatic charges and hydrogen bonds. In those complexes, oxidative-stress mediated, and highly reactive, chemical groups of RNA and protein partners may also form covalent links, forming stable cross-linked (X-linked) adducts. Such covalent X-linked RNA-protein adducts are reported in the ribosome structure, and appear under intracellular high-level oxidative stress. However, the underlying molecular mechanisms of X-links formation under these conditions still remain largely unexplored.
To address these questions we will use rather simple architecture of the enteric F-specific RNA phages combining ssRNA(+) and capsid proteins. Preliminary data obtained on native Qbeta and MS2 virions show that oxidation abrogates virus infectivity, by likely preventing the vRNA injection into the host cell or even impeding the post-infection translation step. We developed simplified in vitro models to gain more insights on location and nature of potential RNA-proteins X-links and to decipher the molecular mechanism(s) leading to formation of RNA-protein X-links upon exposure of model RNA-protein complexes to mild (and also physiological) oxidative agents, like hypochlorous acid (HOCl), hydrogen peroxide (H2O2) and peroxinitrite (ONOO-).
Characterization of RNA-protein X-links in such simplified systems can be performed by the very powerful LC-ESI-Q-TOF MS/MS methodology. Regarding the formation of covalent X-links upon exposure to HOCl, this analytical strategy was already validated for the model combining CP dimers and short RNA sequence (approx. 20 nucleotides) of the so-called operator of the phage replicase gene. structure-function relationships at the CP/MP and RNA levels will be investigated to define the molecular basis responsible for the formation of such X-links. By combining available structural information on the RNA-CP or RNA-MP binding modes, AbOxiSeq data and “X-links experiments” performed on mutated CP/MP and RNA sequences, we aim at defining the minimal pattern of RNA-CP/MP interactions and/or structural elements that allow X-links formation in the MS2 phage model. To gain molecular insights on the location of such reactive sites at the nucleotide-resolution, we will employ a specific variant of previously published AlkAnilineSeq protocol. This variant protocol (termed here as AbOxiSeq), preferentially detects RNA abasic sites, due to modified conditions of RNA cleavage, reducing RNA cleavage at m7G and other sensitive RNA nucleotides. Application of AbOxiSeq will allow precise mapping of the oxidation-sensitive nucleotide residues in model RNAs (our preliminary data show that guanosine (G) residues are particularly exposed, most likely due to oxidation to 8-oxoG). Since this technology is not limited by RNA size, mapping can be efficiently done using both model RNAs (of 40 or more nucleotides), as well as complete phage/viral RNAs extracted from intact VLPs or phage/viral particles. Sensitivity of the method is very high and formation of abasic sites was detected for MS2 phage particles and naked RNA exposed to HOCl.
In our project we will use a combination of highly sensitive MS characterization with high throughput analysis of RNA AP sites by deep sequencing. Oxidation-driven RNA-protein adducts are probably a common feature of the oxidative stress conditions in vivo, but little is known on their formation, stability and potential function (or loss of function) in the cell.