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Sabikunnahar, Bristy

Cellular, Molecular, and Biomedical Sciences Graduate Program

2023

Ph. D.

Long non-coding RNAs (lncRNAs) are one of the most commonly transcribed functional non-coding RNAs in mammalian genomes, yet majority of them remain understudied. Recent advancement in high throughput sequencing and computational knowledge have revealed the identity of thousands of lncRNAs and their association with numerous diseases and cellular processes. In the last two decades, flurries of studies showed experimentally that lncRNAs regulate a myriad of essential biological processes including immune system. Although a large number of lncRNAs have been identified in a variety of immune cells, only a handful of them have been characterized for biological function and mechanisms. In this dissertation, we functionally characterized a mouse lncRNA named U90926, which was previously identified in our lab from transcriptomic data in activated macrophages.First, we have investigated the role of U90926 in macrophage function. We generated a U90926-deficient mice (U9-KO) to study U90926 function. We showed that the expression of U90926 is highly induced in macrophages and dendritic cells by toll-like receptor activation, in a p38 MAP kinase- and MyD88-dependent manner. Then, we have investigated the role of U90926 in regulating macrophage function upon lipopolysaccharide (LPS) stimulation, such as cytokine secretion, expression of co- stimulatory molecules and gene expression. Surprisingly, we found minimal effects of U90926 deficiency in cultured macrophages. The lack of macrophage-intrinsic effect by U90926, led us to explore protein-coding potential aspect of this putative lncRNA. Interestingly, we found that U90926 RNA localizes to the cytosol, associates with ribosomes, and contains an open reading frame that encodes a novel glycosylated protein (termed U9-ORF), which is secreted from the cell. This led us to use an in vivo model of endotoxic shock where macrophages interact with many other cell types, for further characterization of U90926. Our data show that in comparison with WT mice, U9-KO mice exhibited increased sickness responses and mortality. Mechanistically, serum levels of IL- 6 were elevated in U9-KO mice, and IL-6 neutralization improved endotoxemia outcomes in U9-KO mice. Taken together, these results suggest that U90926 expression is protective during endotoxic shock, which is potentially mediated by the paracrine and/or endocrine actions of the novel U9-ORF protein secreted by activated myeloid cells. We have also examined the role of U90926 in adipose tissue homeostasis, using our U9-KO mouse model. U90926 has been characterized as a repressor of adipogenesis in 3T3-L1 preadipocytes by another group. This led us to hypothesize that U90926 deficiency in vivo will enhance adipogenesis or obesity-related phenotypes. However, we did not find any effect of U90926 in weight gain or other obesity-related phenotypes like fat mass, adipose tissue morphology, metabolic parameters, and adipose tissue gene expression, both in the context of a normal diet or high-fat diet. Taken together, these data suggest that U90926 may not have an essential role in adipocyte biology beyond its role in myeloid cell-mediated inflammation.