Superoxide and other derivative reactive oxygen species (ROS) promote atherosclerosis (athero) as well as vascular smooth muscle cell (SMC) and macrophage inflammatory signaling. Anti-atherogenic strategies targeting O2--producing NADPH oxidases, however, increase susceptibility to infection. This project’s goal is to discern novel mechanisms for constraining ROS-promoted atherogenesis while minimizing adverse effects on immunity. One such mechanism may involve the ubiquitously expressed noncoding small nucleolar (sno) RNAs from the ribosomal protein L13a (Rpl13a) locus: SNORD32A, SNORD33, SNORD34, and SNORD35A. We found that these snoRNAs augment ROS levels and oxidative stress in vitro and in vivo. Our Preliminary Studies with Rpl13a-snoRNA-/- (snoKO) mice and SMCs derived from them show: (1) snoKO SMCs have lower levels of ROS, cell proliferation and migration than congenic WT SMCs. (2) Compared with WT SMCs, snoKO SMCs express 5.7-fold more cytochrome C oxidase subunit 4 isoform 2 (COX4I2), which reduces mitochondrial O2- production. (3) SnoKO carotid arteries develop less athero than WT carotids when transplanted orthotopically into Apoe-/- mice. (4) Compared with Apoe-/- mice, snoKO/Apoe-/- mice develop 40% less brachiocephalic athero. (5) Compared with snoRNA+/+ brachiocephalic arteries or carotid grafts, snoKO arteries demonstrate less SMC-to-foam-cell transdifferentiation, a process potentiated by ROS. SnoRNAs bind to their target RNAs via an antisense domain, then recruit the enzyme fibrillarin, which effects RNA 2’-O-methylation. SnoRNAs canonically modify ribosomal RNA; however, we discovered that at least one of the Rpl13a snoRNAs can target mRNA for 2’-O-methylation—a process that alters mRNA abundance and translation. Nonetheless, specific mRNAs that constitute targets for pro-oxidant effects of Rpl13a-snoRNAs remain obscure. This project will therefore test the hypotheses that Rpl13a snoRNAs promote athero, particularly by potentiating SMC-to-foam cell transdifferentiation, and that that Rpl13a-snoRNA-guided mRNA 2’-O-methylation affects protein expression of key ROS-regulating enzyme(s) in SMCs and Mφs, including COX4I2. To do so, this project will compare athero in Rpl13a-snoRNA-/-/Apoe-/- versus Apoe-/- mice, and use bone marrow transplantation to discern the roles of Rpl13a-snoRNAs in bone marrow-derived cells versus arterial wall-derived cells. We will investigate how Rpl13a-snoRNAs affect foam cell formation in macrophages and SMCs, and determine whether COX4I2 engenders lower ROS levels and inflammation in snoKO SMCs. Finally, we will identify mRNA targets of Rpl13a-snoRNAs in SMCs and macrophages, by performing transcriptome-wide mapping of 2’-O-methylation sites on mRNA from WT and Rpl13a-snoRNA-/- SMCs and macrophages, by using the RibOxi-seq and crosslinking, ligation, and sequencing of hybrids (CLASH) approach. By elucidating mechanisms by which snoRNAs regulate ROS in SMCs and macrophages, this project should identify...