Post-transcriptional modifications on tRNA fragments confer functional changes to high-density lipoproteins in atherosclerosis

Epitranscriptomic modifications on RNA play critical roles in stability, processing, and function, partly by influencing interactions with RNA-binding proteins and receptors. The diverse role(s) of post-transcriptional RNA modifications on cell-free small non-coding RNAs (sRNA) remain poorly understood, particularly in disease contexts. Although HDL confer many beneficial properties to health, HDL particles lose these features during atherosclerotic cardiovascular disease (ASCVD). As HDL transport sRNA fragments from classes of RNA known to harbor extensive modifications, we predicted that changes to regulatory modifications on HDL-sRNAs may contribute to HDL functionality changes in ASCVD. To assess HDL-RNA modification levels in ASCVD, total RNA were isolated from HDL of healthy subjects (CAC=0) and those with advanced atherosclerosis, as determined by coronary artery calcium (CAC) scores >21 Agatston units. HDL-RNA were quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and AlkB-facilitated RNA (de)Methylation Sequencing (ARM-seq). LC-MS/MS revealed elevated levels of modified nucleosides in HDL-sRNAs from ASCVD subjects, while ARM-seq detected an enrichment of m1A-modified sRNAs-derived from parent tRNAs (tDR), particularly tDR-ArgACG-1. To assess the impact of disease HDL on immune cells, primary macrophages were treated with HDL isolated from ASCVD (CAC+) and control subjects, and results showed that ASCVD-HDL directly induced expression of genes associated with immune signaling and cell adhesion, including TMEM123, a transmembrane protein linked to immune cell migration, adhesion, and cytoskeletal organization. In addition, recombinant HDL particles loaded with m1A-tDR-ArgACG-1 were found to significantly increase TMEM123 expression in treated primary macrophages. Together, these findings propose a model in which HDL-delivered m1A-HDL-tDRs directly stimulate immune signaling, macrophage activation, and pro-inflammatory sub-phenotypes within the atherosclerotic lesion. This study highlights HDL-associated RNA modifications as previously unrecognized contributors to immune modulation, with direct implications for targeting modified HDL-tDRs as a new approach to therapy for ASCVD.