Caspase-mediated cleavage of miRNA processing proteins Drosha, DGCR8, Dicer, and TRBP2 in heat-shocked cells and its inhibition by HSP70 overexpression.

Cells respond to stress through adaptive mechanisms that limit cellular damage and prevent cell death. MicroRNAs act as regulators of stress responses and stress can impact the functioning of miRNA biogenesis pathways. We were interested in the effect that severe proteotoxic stress capable of inducing apoptosis may have on miRNA biogenesis and the impact of the molecular chaperone protein HSP70 under these conditions. We found that the miRNA processing enzymes Drosha and Dicer and their accessory proteins DGCR8 and TRBP2 are cleaved by caspases in apoptotic cells. Overexpression of HSP70 prevented caspase activation and the degradation of these processing proteins. Caspase cleavage of TRBP2 was mapped to amino acid 234 which separates the two dsRNA-binding domains from the C-terminal Dicer interacting domain. Overexpression of TRBP2 was found to increase miRNA maturation, while expression of either of the fragments generated by caspase cleavage impaired maturation. These results indicate that inactivation of miRNA biogenesis is a critical feature of apoptosis and that cleavage of TRBP2, rather than simply a loss of function, serves to create positive acting inhibitors of pre-miRNA maturation.

In vitro growth of microsporidia Anncaliia algerae in cell lines from warm water fish.

Anncaliia algerae is an aquatic microsporidium that most commonly infects mosquitoes but can be grown on the rabbit kidney cell line, RK-13. Spores were purified from RK-13 cultures and added to cell lines from warm water fish and from an insect. The cell lines were GFSK-S1 and GFB3C-W1 from goldfish skin and brain respectively, ZEB2J from zebrafish embryos, FHMT-W1 from fathead minnow testis, and Sf9 from ovaries of a fall armyworm moth. All cultures were maintained at 27°C. Infection was judged to have taken place by the appearance of sporonts and/or spores in cells and occurred in all cell lines. Spores were also isolated from ZEB2J cultures and used to successfully infect new cultures of ZEB2J, RK-13 and Sf9. These results suggest that cells of a wide range of vertebrates support A. algerae growth in vitro and fish cells can produce spores infectious to cells of mammals, fish, and insects.