Two Paenibacillus spp. strains promote grapevine wood degradation by the fungus Fomitiporia mediterranea: from degradation experiments to genome analyses.

Ascomycetes, basidiomycetes and deuteromycetes can degrade wood, but less attention has been paid to basidiomycetes involved in Esca, a major Grapevine Trunk Disease. Using a wood sawdust microcosm system, we compared the wood degradation of three grapevine cultivars inoculated with Fomitiporia mediterranea M. Fisch, a basidiomycete responsible for white-rot development and involved in Esca disease. The grapevine cultivar Ugni blanc was more susceptible to wood degradation caused by F. mediterranea than the cultivars Cabernet Sauvignon and Merlot. Solid-state Nuclear Magnetic Resonance (NMR) spectroscopy showed that F. mediterranea preferentially degrades lignin and hemicellulose over cellulose (preferential, successive or sequential white-rot). In addition, co-inoculation of sawdust with two cellulolytic and xylanolytic bacterial strains of Paenibacillus (Nakamura) Ash (Paenibacillus sp. (S231-2) and P. amylolyticus (S293)), enhanced F. mediterranea ability to degrade Ugni blanc. The NMR data further showed that the increase in Ugni blanc sawdust degradation products was greater when bacteria and fungi were inoculated together. We also demonstrated that these two bacterial strains could degrade the wood components of Ugni blanc sawdust. Genome analysis of these bacterial strains revealed numerous genes predicted to be involved in cellulose, hemicellulose, and lignin degradation, as well as several other genes related to bacteria-fungi interactions and endophytism inside the plant. The occurrence of this type of bacteria-fungus interaction could explain, at least in part, why necrosis develops extensively in certain grapevine varieties such as Ugni blanc.

The Promoter Region of the Proto-Oncogene MST1R Contains the Main Features of G-Quadruplexes Formation.

MST1R (RON) is a receptor of the MET tyrosine kinase receptor family involved in several cancers such as pancreas, breast, ovary, colon, and stomach. Some studies have shown that overexpression of MST1R increases the migratory and invasive properties of cancer cells. The promoter region of the oncogene MST1R is enriched in guanine residues that can potentially form G-quadruplexes (G4s), as it was observed in other oncogenic promoters such as KRAS and c-MYC. There is abundant literature that links the presence of G4s in promoter regions of oncogenes to diverse gene regulation processes that are not well understood. In this work, we have studied the reverse and forward sequence of MST1R promoter region using the G4Hunter software and performed biophysical studies to characterize the best scored sequences.

hnRNPA1/UP1 Unfolds KRAS G-Quadruplexes and Feeds a Regulatory Axis Controlling Gene Expression.

Recent studies have proven that the genetic landscape of pancreatic cancer is dominated by the KRAS oncogene. Its transcription is controlled by a G-rich motif (called 32R) located immediately upstream of the TSS. 32R may fold into a G-quadruplex (G4) in equilibrium between two G4 conformers: G9T (T M = 61.2 °C) and G25T (T M = 54.7 °C). We found that both G4s bind to hnRNPA1 and its proteolytic fragment UP1, promoting several contacts with the RRM protein domains. 1D NMR analysis of DNA imino protons shows that, upon binding to UP1, G25T is readily unfolded at both 5' and 3' tetrads, while G9T is only partially unfolded. The impact of hnRNPA1 on KRAS expression was determined by comparing Panc-1 cells with two Panc-1 knockout cell lines in which hnRNPA1 was deleted by the CRISPR/Cas9 technology. The results showed that the expression of KRAS is inhibited in the knockout cell lines, indicating that hnRNPA1 is essential for the transcription of KRAS. In addition, the knockout cell lines, compared to normal Panc-1 cells, show a dramatic decrease in cell growth and capacity of colony formation. Pull-down and Western blot experiments indicate that conformer G25T is a better platform than conformer G9T for the assembly of the transcription preinitiation complex with PARP1, Ku70, MAZ, and hnRNPA1. Together, our data prove that hnRNPA1, being a key transcription factor for the activation of KRAS, can be a new therapeutic target for the rational design of anticancer strategies.

Structure of two G-quadruplexes in equilibrium in the KRAS promoter.

KRAS is one of the most mutated oncogenes and still considered an undruggable target. An alternative strategy would consist in targeting its gene rather than the protein, specifically the formation of G-quadruplexes (G4) in its promoter. G4 are secondary structures implicated in biological processes, which can be formed among G-rich DNA (or RNA) sequences. Here we have studied the major conformations of the commonly known KRAS 32R, or simply 32R, a 32 residue sequence within the KRAS Nuclease Hypersensitive Element (NHE) region. We have determined the structure of the two major stable conformers that 32R can adopt and which display slow equilibrium (>ms) with each other. By using different biophysical methods, we found that the nucleotides G9, G25, G28 and G32 are particularly implicated in the exchange between these two conformations. We also showed that a triad at the 3' end further stabilizes one of the G4 conformations, while the second conformer remains more flexible and less stable.