Epitranscriptomic RNA modifications play a crucial role in RNA processing, stability, and function. Small non-coding RNAs (sRNAs), such as tRNA-derived fragments (tDRs), likely inherit modifications from parent transcripts. Evidence supports that cell-free sRNA modifications regulate gene expression, modulate immune responses, and are closely linked to the pathogenesis of immune related diseases. Given that high-density lipoproteins (HDL) transport sRNAs from parent transcripts with extensive modifications, we predicted that many HDL-sRNAs harbor immune regulatory modifications that contribute to HDL functionality.

To assess HDL-sRNA modifications in atherosclerotic cardiovascular disease (ASCVD), total RNAs were isolated from HDL of healthy subjects and those with advanced ASCVD. HDL-sRNA modifications were quantified using liquid chromatography-tandem mass-spectrometry (LC-MS/MS), AlkB-facilitated RNA (de)Methylation Sequencing (ARM-seq), and qPCR.

LC-MS/MS revealed a significant increase in the relative levels of modified nucleosides on HDL in ASCVD and healthy individuals. ARM-seq identified candidate tDR-ArgACG fragments harboring 1-methyladenosine (m1A) modifications that were enriched in ASCVD subjects compared to controls. Bulk mRNA sequencing showed significant macrophage gene expression changes in response to ASCVD-HDL-sRNA uptake, including TMEM123, a transmembrane protein linked to immune cell migration and adhesion. Reconstituted HDL loaded with m1A-tDR-ArgACG were also found to significantly increase TMEM123 expression in primary macrophages, and blocking m1A using an anti-m1A neutralizing antibody attenuated this effect.

Results support a model in which HDL-delivered m1A-HDL-tDRs regulate immune signaling, macrophage activation, and pro-inflammatory sub-phenotypes within the atherosclerotic lesion.