The DLK1-DIO3 C/D box snoRNA SNORD113-6/AF357425 plays a dual role in pre-mRNA processing and 2'O-ribose methylation in integrin signalling and arterial fibroblast function

We have previously shown that C/D box snoRNAs transcribed from the DLK1-DIO3 locus on human chromosome 14 (14q32) are associated with cardiovascular disease. DLK1-DIO3 snoRNAs are 'orphan snoRNAs' that have no known targets. We aimed to identify RNA targets and elucidate the mechanism-of-action of human SNORD113-6 (AF357425 in mice). As AF357425-knockout cells were non-viable, we induced overexpression or inhibition of AF357425 in primary murine fibroblasts and performed RNA-Seq. We identified several premRNAs with conserved AF357425/SNORD113-6 D'-seed binding sites in the last exon/3'UTR, which directed pre-mRNA processing and splice-variant specific protein expression. We also pulled down the snoRNA-associated methyltransferase fibrillarin from AF357425-High vs. AF357425-Low fibroblast lysates, followed by RNA isolation, rRNA depletion and RNA-Seq. Identifying mostly mRNAs, we subjected these to PANTHER pathway analysis and observed enrichment for genes in the integrin pathway. We confirmed 2'O-ribose methylation in 6 integrin pathway mRNAs (MAP2K1, ITGB3, ITGA7, PARVB, NTN4 and FLNB). Methylation and mRNA expression were decreased while mRNA degradation was increased under AF357425/SNORD113-6 inhibition in both murine and human primary fibroblasts, but effects on protein expression were more ambiguous. Integrin signalling is crucial for cell-cell and cell-matrix interactions, and correspondingly, we observed altered human primary arterial fibroblast barrier function upon SNORD113-6 inhibition. Overall design: RNA-seq on whole cell lysates of primary murine ear cells with AF357425 overexpression (AF357425-High) or inhibition (AF357425-Low), and on RNA from fibrillarin pulldown of AF357425-High and AF357425-Low fibroblast lysates.