Abstract 233: PANDORA-seq Reveals Hidden Regulatory Small RNA Landscape Associated With Atherosclerosis Development In Hypercholesterolemic LDL Receptor-deficient Mice

Abstract

Introduction: Small non-coding RNAs (sncRNAs) have been demonstrated to have diverse roles in regulating molecular processes. While the standard RNA sequencing (RNA-seq) method has advanced sncRNA discovery, many RNA modifications can interfere with library construction procedures, preventing the discovery of a subset of highly modified sncRNAs including transfer RNA-derived small RNAs (tsRNAs) and ribosomal RNA-derived small RNAs (rsRNAs) that may have important functions in disease development. To address this technical obstacle, we recently developed a novel PANDORA-seq method to overcome RNA modification-elicited sequence interferences ( Nat Cell Biol 2021:424-436). This study aims to identify novel sncRNAs that are associated with atherosclerosis development by using PANDOR-seq. Hypothesis: PANDORA-seq enables us to reveal unprecedented sncRNA landscape associated with atherosclerosis development in LDL receptor-deficient (LDLR -/- ) mice. Methods: To identify sncRNAs associated with atherosclerosis development, LDLR -/- mice were fed a low-cholesterol diet (LCD; 0.02% cholesterol) or high-cholesterol diet (HCD; 0.5% cholesterol) for 9 weeks. Total RNA isolated from intima was subjected to both traditional RNA-seq and PANDORA-seq, and the sequencing data were analyzed by sncRNA analyzing software SPORTS1.1 . Results and Conclusions: By overcoming RNA modification-elicited limitations, PANDORA-seq unveiled a rsRNA- and tsRNA-enriched sncRNA landscape in the atherosclerotic intima of LDLR -/- mice which was strikingly different from that detected by traditional RNA-seq. While micro-RNAs (miRNAs) were the dominant sncRNAs detected by traditional RNA-seq (55.9% of total sncRNAs), PANDORA-seq substantially increased the reads of rsRNAs and tsRNAs which account for 77.4% and 5% of total sncRNAs, respectively. PANDORA-seq also detected 1,383 differentially expressed sncRNAs induced by HCD feeding including 1160 rsRNAs and 195 tsRNAs but traditional RNA-seq only detected 16 differentially expressed rsRNAs and tsRNAs. Thus, PANDORA-seq revealed the hidden rsRNAs and tsRNAs associated with atherosclerosis development, and the functions of those previously underexplored sncRNAs warrant future investigations.