Action Of Ribosome-inactivating Proteins Research Articles

Effect of heritable symbionts on maternally-derived embryo transcripts

Maternally-transmitted endosymbiotic bacteria are ubiquitous in insects. Among other influential phenotypes, many heritable symbionts of arthropods are notorious for manipulating host reproduction through one of four reproductive syndromes, which are generally exerted during early developmental stages of the host: male feminization; parthenogenesis induction; male killing; and cytoplasmic incompatibility (CI). Major advances have been achieved in understanding mechanisms and identifying symbiont factors involved in reproductive manipulation, particularly male killing and cytoplasmic incompatibility. Nonetheless, whether cytoplasmically-transmitted bacteria influence the maternally-loaded components of the egg or early embryo has not been examined. In the present study, we investigated whether heritable endosymbionts that cause different reproductive phenotypes in Drosophila melanogaster influence the mRNA transcriptome of early embryos. We used mRNA-seq to evaluate differential expression in Drosophila embryos lacking endosymbionts (control) to those harbouring the male-killing Spiroplasma poulsonii strain MSRO-Br, the CI-inducing Wolbachia strain wMel, or Spiroplasma poulsonii strain Hyd1; a strain that lacks a reproductive phenotype and is naturally associated with Drosophila hydei. We found no consistent evidence of influence of symbiont on mRNA composition of early embryos, suggesting that the reproductive manipulation mechanism does not involve alteration of maternally-loaded transcripts. In addition, we capitalized on several available mRNA-seq datasets derived from Spiroplasma-infected Drosophila melanogaster embryos, to search for signals of depurination of rRNA, consistent with the activity of Ribosome Inactivating Proteins (RIPs) encoded by Spiroplasma poulsonii. We found small but statistically significant signals of depurination of Drosophila rRNA in the Spiroplasma treatments (both strains), but not in the symbiont-free control or Wolbachia treatment, consistent with the action of RIPs. The depurination signal was slightly stronger in the treatment with the male-killing strain. This result supports a recent report that RIP-induced damage contributes to male embryo death.

Topological requirements for recognition and cleavage of DNA by ribosome-inactivating proteins.

Ribosome-inactivating proteins (RIPs) were demonstrated to exhibit a unique enzymatic activity on cleaving supercoiled double-stranded DNA into the nicked or linear form. Although there is an interaction between supercoiled DNA and RIP, no sequence-specific recognition was involved. Instead, RIPs recognize supercoiled DNA by conformational specificity. Negatively supercoiled DNA is the preferential conformation in the action of RIPs. When double-stranded DNA occurs in the supercoiled form, even if with lower linking number, RIPs can still convert it into nicked or linear form. Terminal-labelling experiments indicated that radioactivity was incorporated into putative 5'-ends of nicked or linear DNA generated by RIPs. We conclude that RIPs act as a novel supercoil-dependent endonuclease when they cleavage supercoiled DNA. The impossibility that contaminating enzymes in the RIP preparations cleaved the supercoiled DNA is briefly discussed.