Cellular localisation and stress induced clustering of RNase Y

Abstract

Rapid mRNA degradation facilitates adaptation to changes in environment. In E. coli, mRNA degradation is predominantly carried out by a complex known as the RNA degradosome, composed of helicases, ribonucleases and glycolytic enzymes, scaffolded by the endoribonuclease RNase E. In bacteria lacking RNase E, a non-homologous endoribonuclease; RNase Y, often carries out analogous functions. The components of the RNase E degradosome are recruited to the membrane in an RNase Edependent manner. However, the interaction partners of RNase Y in B. subtilis are not normally localised to the membrane, suggesting that the degradosome is not stably associated. Using fluorescence microscopy, I have found that RNase Y and some of its interaction partners cluster upon dissipation of the proton motive force with CCCP. This clustering appears to be driven by RNase Y, as it takes place both in B. subtilis and when RNase Y is heterologously expressed in E. coli in the absence of its native interaction partners. However, RNase Y interaction partners form CCCP induced foci in an RNase Y deficient strain, and foci formed by several interaction partners do not colocalise with RNase Y clusters. Additionally, the formation of multimers by RNase Y in vitro, and colocalisation of RNase Y with YocM, a reporter for protein aggregation, suggests that clustering is not induced degradosome formation, but rather a novel, non-specific aggregation triggered by a decrease in cytoplasmic pH. RNases E and Y are both postulated to be membrane-associated in almost all organisms where they occur, but the reason for this localisation is not well understood. I have found that release of RNase Y to the cytoplasm is toxic in B. subtilis and slows growth when heterologously expressed in E. coli. This aberrant localisation interferes with vital cellular processes including ribosome maturation, nucleoid condensation and bacterial cell division.