Current work reports the use of single-stranded RNA toeholds of different lengths to promote the reassociation of various RNA-DNA hybrids, which results in activation of multiple split functionalities inside human cells. The process of reassociation is analyzed and followed with a novel computational multistrand secondary structure prediction algorithm and various experiments. All of our previously designed RNA/DNA nanoparticles employed single-stranded DNA toeholds to initiate reassociation. The use of RNA toeholds is advantageous because of the simpler design rules, the shorter toeholds, and the smaller size of the resulting nanoparticles (by up to 120 nucleotides per particle) compared to the same hybrid nanoparticles with single-stranded DNA toeholds. Moreover, the cotranscriptional assemblies result in higher yields for hybrid nanoparticles with ssRNA toeholds.

Flavin-Containing Monooxygenases are conserved xenobiotic-detoxifying enzymes. Recent studies have revealed endogenous functions of FMOs in regulating longevity in Caenorhabditis elegans and in regulating aspects of metabolism in mice. To explore the cellular mechanisms of FMO’s endogenous function, here we demonstrate that all five functional mammalian FMOs may play similar endogenous roles to improve resistance to a wide range of toxic stresses in both kidney and liver cells. We further find that stress-activated c-Jun N-terminal kinase activity is enhanced in FMO-overexpressing cells, which may lead to increased survival under stress. Furthermore, FMO expression modulates cellular metabolic activity as measured by mitochondrial respiration, glycolysis, and metabolomics analyses. FMO expression augments mitochondrial respiration and significantly changes central carbon metabolism, including amino acid and energy metabolism pathways. Together, our findings demonstrate an important endogenous role for the FMO family in regulation of cellular stress resistance and major cellular metabolic activities including central carbon metabolism.

Flavin containing monooxygenases (FMOs) are promiscuous enzymes known for metabolizing a wide range of exogenous compounds. In C. elegans, fmo-2 expression increases lifespan and healthspan downstream of multiple longevity-promoting pathways through an unknown mechanism. Here, we report that, beyond its classification as a xenobiotic enzyme, fmo-2 expression leads to rewiring of endogenous metabolism principally through changes in one carbon metabolism (OCM). These changes are likely relevant, as we find that genetically modifying OCM enzyme expression leads to alterations in longevity that interact with fmo-2 expression. Using computer modeling, we identify decreased methylation as the major OCM flux modified by FMO-2 that is sufficient to recapitulate its longevity benefits. We further find that tryptophan is decreased in multiple mammalian FMO overexpression models and is a validated substrate for FMO-2. Our resulting model connects a single enzyme to two previously unconnected key metabolic pathways and provides a framework for the metabolic interconnectivity of longevity-promoting pathways such as dietary restriction. FMOs are well-conserved enzymes that are also induced by lifespan-extending interventions in mice, supporting a conserved and important role in promoting health and longevity through metabolic remodeling.

No abstract

An organism’s ability to perceive and respond to changes in its environment is crucial for its health and survival. Our approach to identify molecular mechanisms of aging is to focus on common mechanisms downstream of multiple pathways. This approach led to our discovery of a gene, flavin-containing monooxygenase (fmo)-2, that is both necessary and sufficient to increase lifespan and healthspan downstream of several longevity interventions, including dietary restriction and hypoxia. Surprisingly, we also find that in both hypoxia and dietary restriction models, fmo-2 is induced by cell non-autonomous signaling pathways, consistent with the worms’ perceiving the stress (e.g. low oxygen, lack of food) and changing physiology as a result. Our current work focuses on 1) the signaling networks that regulate stress perception and integrate multiple signals to change physiology, and 2) the mechanism of FMO-2-mediated longevity. Our new data suggest that these cell non autonomous networks pathways utilize both overlapping and distinct signaling mechanisms to converge on upregulation of the same gene. They also suggest that these pathways can be manipulated by small molecule drugs to increase lifespan by “tricking” the organism into activating stress response networks. We further find that FMO enzyme expression has a drastic effect on endogenous metabolism, primarily through tryptophan and one carbon metabolism. Ultimately, we aim to leverage our results in a translational framework to identify key signals, genes, and mechanisms where organisms respond to the perception of environmental stress to improve health and slow aging.