Complexity of living organisms increases as a function of evolution. According to the central dogma, proteins represent the primary form of genetic output in living organisms and therefore all biological functions and increases in complexity must have arisen from proteins. Yet, in direct contradiction to this fundamental dogma , the proportion of protein-coding genes actually decreases as a function of organismal complexity. How can this paradigm be reconciled? How else can genetic information be stored and transmitted in higher organisms without involving proteins? Interestingly, the increase in organismal complexity does in fact coincide with a vast expansion of the noncoding genome, suggesting the regulatory RNAs encoded by this portion of the genome have the potential to act as potent regulators in diverse biological processes. Regulatory RNA may therefore represent the missing piece in our understanding of how genetic information is stored and transmitted in higher eukaryotes and how these organisms are able to bypass the upper limits of biological complexity that would otherwise be imposed by a regulatory network operating solely on proteins. Thus, a better understanding of the physiological roles and mechanisms of regulatory RNAs in health and disease is likely to have significant therapeutic implications and will undoubtedly shed new light on what the basic notion of a gene truly represents.

The work presented here will focus on two types of regulatory RNA: m6A-modified RNAs and lncRNAs. In chapter 2, we demonstrate that the m6A RNA modification is essential for proper regulation of hepatic lipid metabolism. In-vivo studies showed that both diet and sex alter the global landscape of m6A. Under control diet feeding, m6A is dynamically enriched at 3’ UTR regions on lipogenic mRNAs but enrichment at these sites decreases dramatically when mice are fed a lipid-rich diet. Furthermore, deletion of the m6A installing enzyme Mettl14 increases hepatic lipogenesis and triglyceride accumulation while also diminishing hepatic sexual dimorphism. Our study outlines a protective role for RNA modifications against the development of fatty liver disease and also fills a gap in our understanding of molecular mechanisms underlying sex-specific differences in hepatic lipid traits.

In chapter 3, we report findings from a preclinical investigation of Mexis therapy in atherosclerosis. We previously demonstrated the macrophage-specific lncRNA MeXis enhances Abca1 expression thereby boosting cholesterol efflux capacity of macrophages. To test the therapeutic effects of MeXis overexpression within lesions, we used a novel genetic model that allows spatial control of Mexis expression from the endogenous locus. We show that Mexis overexpression is associated with increased Abca1 mRNA and protein levels, enhanced cholesterol efflux to an ApoA1 acceptor, and reduced foam cell formation in vitro and in vivo. However, when we performed a bone-marrow transplant (BMT) and infused LDLR-/- mice with Cre+ and Cre- marrow, we did not observe major differences in plaque burden between groups. Surprisingly, Mexis overexpression in the atherosclerosis model did however lead to a substantial elevation in systemic inflammatory markers. Our findings have important implications for proposed strategies that aim to enhance lncRNA expression for chronic disease treatment and highlights the importance of carefully interrogating lncRNA effects in multiple contexts.

In chapter 4, we demonstrate the macrophage-specific lncRNA AK165607 encodes a novel micropeptide termed ORF5. We further demonstrate ORF5 interacts with aggregation-prone proteins while they are being transported to the plasma membrane in secretory lysosomes for extracellular release. Loss of ORF5 triggers widespread rupturing of lysosome and results in the intracellular accumulation of toxic protein aggregates. In-vivo experiments revealed loss of ORF5 suppresses macrophage inflammatory response and impairs extracellular secretion of S100A8