C/D box snoRNA SNORD113-6 guides 2′-O-methylation and protects against site-specific fragmentation of tRNALeu(TAA) in vascular remodeling

C/D box small nucleolar RNAs (snoRNAs) of the DLK1-DIO3 locus are associated with vascular remodeling and cardiovascu-lar disease. None of these snoRNAs has any known targets yet except for one, AF357425/SNORD113-6. We previously showed that this snoRNA targets mRNAs of the integrin signaling pathway and affects arterial fibroblast function. Here, we aimed to identify whether AF357425/SNORD113-6 can also target small RNAs. We overexpressed or inhibited AF357425 in murine fibroblasts and performed small RNA sequencing. Expression of transfer (t)RNA fragments (tRFs) was predominantly regulated. Compared with overexpression, AF357425 knockdown led to an overall decrease in tRFs but with an enrichment in smaller tRFs (<30 nucleotides). We focused on tRNA leucine anti-codon TAA (tRNALeu(TAA)), which has a conserved predicted binding site for AF357425/ SNORD113-6. Adjacent to this site, the tRNA is cleaved to form tRFLeu 47-64 in both primary murine and human fibro-blasts and in intact human arteries. We show that AF357425/ SNORD113-6 methylates tRNALeu(TAA) and thereby prevents the formation of tRFLeu 47-64. Exposing fibroblasts to oxidative or hypoxic stress increased AF357425/SNORD113-6 and tRNALeu(TAA) expression, but AF357425/SNORD113-6 knockdown did not increase tRFLeu 47-64 formation under stress even further. Thus, independent of cellular stress, AF357425/SNORD113-6 protects against site-specific frag-mentation of tRNALeu(TAA) via 2'O-ribose-methylation.