Selfish conflict underlies RNA-mediated parent-of-origin effects.

Genomic imprinting-the non-equivalence of maternal and paternal genomes-is a critical process that has evolved independently in many plant and mammalian species1,2. According to kinship theory, imprinting is the inevitable consequence of conflictive selective forces acting on differentially expressed parental alleles3,4. Yet, how these epigenetic differences evolve in the first place is poorly understood3,5,6. Here we report the identification and molecular dissection of a parent-of-origin effect on gene expression that might help to clarify this fundamental question. Toxin-antidote elements (TAs) are selfish elements that spread in populations by poisoning non-carrier individuals7-9. In reciprocal crosses between two Caenorhabditis tropicalis wild isolates, we found that the slow-1/grow-1 TA is specifically inactive when paternally inherited. This parent-of-origin effect stems from transcriptional repression of the slow-1 toxin by the PIWI-interacting RNA (piRNA) host defence pathway. The repression requires PIWI Argonaute and SET-32 histone methyltransferase activities and is transgenerationally inherited via small RNAs. Remarkably, when slow-1/grow-1 is maternally inherited, slow-1 repression is halted by a translation-independent role of its maternal mRNA. That is, slow-1 transcripts loaded into eggs-but not SLOW-1 protein-are necessary and sufficient to counteract piRNA-mediated repression. Our findings show that parent-of-origin effects can evolve by co-option of the piRNA pathway and hinder the spread of selfish genes that require sex for their propagation.

Structure and heme-binding properties of HemQ (chlorite dismutase-like protein) from Listeria monocytogenes.

Chlorite dismutase-like proteins are structurally closely related to functional chlorite dismutases which are heme b-dependent oxidoreductases capable of reducing chlorite to chloride with simultaneous production of dioxygen. Chlorite dismutase-like proteins are incapable of performing this reaction and their biological role is still under discussion. Recently, members of this large protein family were shown to be involved in heme biosynthesis in Gram-positive bacteria, and thus the protein was renamed HemQ in these organisms. In the present work the structural and heme binding properties of the chlorite dismutase-like protein from the Gram-positive pathogen Listeria monocytogenes (LmCld) were analyzed in order to evaluate its potential role as a regulatory heme sensing protein. The homopentameric crystal structure (2.0Å) shows high similarity to chlorite-degrading chlorite dismutases with an important difference in the structure of the putative substrate and heme entrance channel. In solution LmCld is a stable hexamer able to bind the low-spin ligand cyanide. Heme binding is reversible with KD-values determined to be 7.2µM (circular dichroism spectroscopy) and 16.8µM (isothermal titration calorimetry) at pH 7.0. Both acidic and alkaline conditions promote heme release. Presented biochemical and structural data reveal that the chlorite dismutase-like protein from L. monocytogenes could act as a potential regulatory heme sensing and storage protein within heme biosynthesis.

Manipulating conserved heme cavity residues of chlorite dismutase: effect on structure, redox chemistry, and reactivity.

Chlorite dismutases (Clds) are heme b containing oxidoreductases that convert chlorite to chloride and molecular oxygen. In order to elucidate the role of conserved heme cavity residues in the catalysis of this reaction comprehensive mutational and biochemical analyses of Cld from "Candidatus Nitrospira defluvii" (NdCld) were performed. Particularly, point mutations of the cavity-forming residues R173, K141, W145, W146, and E210 were performed. The effect of manipulation in 12 single and double mutants was probed by UV-vis spectroscopy, spectroelectrochemistry, pre-steady-state and steady-state kinetics, and X-ray crystallography. Resulting biochemical data are discussed with respect to the known crystal structure of wild-type NdCld and the variants R173A and R173K as well as the structures of R173E, W145V, W145F, and the R173Q/W146Y solved in this work. The findings allow a critical analysis of the role of these heme cavity residues in the reaction mechanism of chlorite degradation that is proposed to involve hypohalous acid as transient intermediate and formation of an O═O bond. The distal R173 is shown to be important (but not fully essential) for the reaction with chlorite, and, upon addition of cyanide, it acts as a proton acceptor in the formation of the resulting low-spin complex. The proximal H-bonding network including K141-E210-H160 keeps the enzyme in its ferric (E°' = -113 mV) and mainly five-coordinated high-spin state and is very susceptible to perturbation.

Impact of subunit and oligomeric structure on the thermal and conformational stability of chlorite dismutases.

Chlorite dismutases (Cld) are unique heme b containing oxidoreductases that convert chlorite to chloride and dioxygen. Recent phylogenetic and structural analyses demonstrated that these metalloproteins significantly differ in oligomeric and subunit structure. Here we have analyzed two representatives of two phylogenetically separated lineages, namely pentameric Cld from Candidatus "Nitrospira defluvii" and dimeric Cld from Nitrobacter winogradskyi having a similar enzymatic activity at room temperature. By application of a broad set of techniques including differential scanning calorimetry, electronic circular dichroism, UV-vis and fluorescence spectroscopy the temperature-mediated and chemical unfolding of both recombinant proteins were analyzed. Significant differences in thermal and conformational stability are reported. The pentameric enzyme is very stable between pH 3 and 10 (T(m)=92°C at pH 7.0) and active at high temperatures thus being an interesting candidate for bioremediation of chlorite. By contrast the dimeric protein starts to unfold already at 53°C. The observed unfolding pathways are discussed with respect to the known subunit structure and subunit interaction.

Redox thermodynamics of high-spin and low-spin forms of chlorite dismutases with diverse subunit and oligomeric structures.

Chlorite dismutases (Clds) are heme b-containing oxidoreductases that convert chlorite to chloride and dioxygen. In this work, the thermodynamics of the one-electron reduction of the ferric high-spin forms and of the six-coordinate low-spin cyanide adducts of the enzymes from Nitrobacter winogradskyi (NwCld) and Candidatus "Nitrospira defluvii" (NdCld) were determined through spectroelectrochemical experiments. These proteins belong to two phylogenetically separated lineages that differ in subunit (21.5 and 26 kDa, respectively) and oligomeric (dimeric and pentameric, respectively) structure but exhibit similar chlorite degradation activity. The E°' values for free and cyanide-bound proteins were determined to be -119 and -397 mV for NwCld and -113 and -404 mV for NdCld, respectively (pH 7.0, 25 °C). Variable-temperature spectroelectrochemical experiments revealed that the oxidized state of both proteins is enthalpically stabilized. Molecular dynamics simulations suggest that changes in the protein structure are negligible, whereas solvent reorganization is mainly responsible for the increase in entropy during the redox reaction. Obtained data are discussed with respect to the known structures of the two Clds and the proposed reaction mechanism.