Poster abstracts

Poster number 9 submitted by Stella Kahn

Probing the role of aconitase in coupling metabolic flux to RNA decay within bacterial ribonucleoprotein bodies in Caulobacter crescentus

Stella Kahn (Chemistry; University of Pittsburgh), Hadi Yassine (Biology; Indiana University), Mary English (Chemistry; University of Pittsburgh), Ian Gushurst (Chemistry; University of Pittsburgh), Jared Schrader (Biology; Indiana University), Seth Childers (Chemistry; University of Pittsburgh)

Abstract:
Bacterial ribonucleoprotein bodies (BR-bodies) organize the RNA degradosome through RNA-mediated phase separation of the endoribonuclease RNase E. Past proteomic studies have identified 111 client proteins of BR-bodies; roughly 30% of these proteins are related to RNA breakdown, but a substantial fraction diverges in function (e.g. metabolism (34%), redox homeostasis (4%)). This raises fundamental questions about the overall biochemistry orchestrated in BR-bodies and if there is cross-regulation that occurs between RNA decay and metabolism.  Here we investigate aconitase, a TCA cycle enzyme and major RNase E client within BR-bodies. Past work has demonstrated that aconitase moonlights as an RNA-binding protein capable of protecting its own mRNA 5’ UTR from degradation when intracellular iron is scarce. We hypothesize that cross-regulation may be more substantial, and that enzymatic flux of aconitase is functionally coupled to the RNA degradosome, impacting the activity or localization of other core clients such as the exoribonuclease PNPase. Initial aconitase in vivo assays demonstrated a 4.6-fold increase in aconitase activity in mid-log phase over stationary phase. Through reconstitution of RNase E and aconitase in vitro, we have observed partitioning of apo-aconitase into RNase E droplets and found that citrate, the substrate of aconitase, can reduce RNase E phase separation in the presence of aconitase. From these, we are testing our initial model that aconitase serves as a metabolic sensor that relays cellular physiological state to the RNA degradosome via BR-body function and material property changes.  To test our working model, we’ll present our current finding from a combination of in vivo and in vitro aconitase activity and RNA-binding assays.

References:
Al-Husini, Nadra, Tomares, Dylan T. et al. “a-Proteobacterial RNA degradosomes assemble liquid-liquid phase-separated RNP bodies.” J. Molecular Cell vol. 71,6 (2018): 1027-1039
Benjamin, Julie-Anna M., Masse´, Eric. “The iron-sensing aconitase B binds its own mRNA to prevent sRNA-induced mRNA cleavage.” Nucleic Acids Res., vol. 42,15 (2014): 10023-10026.
Nandana, Vidhyadhar et al. “The BR-body proteome contains a complex network of protein-protein and protein-RNA interactions.” Cell Rep. vol. 42,10 (2023)

Keywords: Aconitase, bacterial ribonucleoprotein bodies, Metabolsim