Sunday, October 27, 2024
10/27/2024
Project Summary and Abstract Acute lung injury (ALI) is a critical respiratory condition with high mortality. Although the etiology of ALI is diverse, its pathogenesis is commonly linked to an uncontrolled inflammatory response, driven by an overproduction of proinflammatory mediators such as cytokines. Despite the pressing need, no effective therapies exist, and current literature offers limited new information regarding the molecular components contributing to the inflammatory processes in ALI. In this context, short non-coding RNAs (sncRNAs) have been posited for their roles in inflammation. While many studies have delved into the roles of microRNAs in ALI, recent advances in RNA biology research have unveiled the existence of additional, and often more abundant, classes of functional sncRNAs such as tRNA halves, the most abundant subgroup of tRNA-derived sncRNAs, and rRNA fragments (rRFs). There is a growing recognition of the essential roles of these sncRNAs in a wide array of biological processes and human diseases. Yet, most of these non-miRNA-sncRNAs remain largely uncharacterized as standard RNA-seq methods cannot capture sncRNAs lacking 5′-phosphate and 3′-hydroxyl ends, representing a significant scientific knowledge gap. For instance, during tRNA half biogenesis, the tRNA cleavage by angiogenin, a predominant endoribonuclease, leaves a 2′,3′-cyclic phosphate (cP) at the 3′-end of 5′-tRNA half, preventing such sncRNAs from being captured using standard RNA-seq. Our lab has developed the “cP-RNA- seq” which can specifically sequence tRNA halves and other cP-containing RNAs. In this study, we aim to leverage our sequencing technique to uncover the dysregulation of these “hidden” sncRNAs, with a particular focus on tRNA halves and rRFs, in Lipopolysaccharide (LPS) induced ALI models in human cells and mice. Preliminary studies indicate a significant upregulation of specific tRNA halves and rRFs in human monocyte- derived macrophages (HMDMs) and murine lung tissues upon LPS exposure. Further, LPS-induced accumulation of these sncRNAs is observed in extracellular vesicles secreted from HMDMs and obtained from murine bronchoalveolar lavage. Additional findings suggest tRNA halves and rRFs strongly activate endosomal Toll-like receptors and induce cytokine production in HMDMs and murine lung slices. We hypothesize that the increased expression of tRNA halves and rRFs significantly enhances the inflammatory process in ALI by inducing cytokine production via the endosomal TLR axis and by promoting immune cell infiltration. We will comprehensively characterize LPS-induced tRNA halves and rRFs in ALI models of human cells and mice (Aim 1), investigate the proinflammatory effects of these sncRNAs by examining their endosomal TLR stimulation activity (Aim 2), and further establish their role in regulating immune cell migration (Aim 3). Our study will uncover a novel sncRNA pathway in the molecular pathogenesis of ALI and may have implications for the future exploration of biomarkers and efficacious therapeutic applications targeting these sncRNAs in ALI.
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