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

Abstract

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 pre-mRNAs 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 function upon SNORD113–6 inhibition.

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

“…We investigated whether AF357425/SNORD113-6 could also target small RNAs besides the previously identified integrin signaling mRNA targets. 33 We found that tRNAs are the predominant small RNA target of AF357425 in primary fibroblasts. Inhibition of AF357425/ SNORD113-6 led to an overall decrease in tRFs, and, compared with overexpression of the snoRNA, less larger (>30 nucleotides) and more smaller (<30 nucleotides) tRFs were formed.…”

“…11 Indeed, knockdown of SNORD113-6 altered human arterial fibroblast function. 12 C/D box snoRNAs associate with four conserved ribonucleoproteins NHP2L1, NOP56, NOP58, and Fibrillarin (FBL). FBL is the methyltransferase that catalyzes 2 0 Ome.…”

Unraveling the epitranscriptome of small non-coding RNAs in vascular cells

“… 7 They also reported a role for AF357425/SNORD113, focusing on its mRNA targeting capacity resulting in regulation of the integrin signaling pathway. 8 This new report expands the knowledge considerably, showing that AF357425/SNORD113-6 targets tRNAs, protecting the tRNA from cleavage into small fragments. Specifically, they demonstrated that AF357425/SNORD113-6 induces 2-O-methylation (2′Ome) of the mature tRNALeu(TAA), thereby protecting against site-specific tRNA fragmentation.…”

Orphan C/D box snoRNAs modulate fear-related memory processes in mice

“…They also reported direct impairment of translation by the methylation of the Pxdn coding sequence. Recently, van Ingen et al found that the orphan snoRNA snord113-6 is required for fibroblast cell survival and methylates multiple mRNAs within the integrin pathway; inhibition of this snoRNA reduced mRNA methylation and increased degradation of the targeted mRNAs, with mixed effects on subsequent protein expression (van Ingen et al, 2022). Furthermore, several studies have reported widespread 2’-O-methylation of eukaryotic mRNAs (Dai et al, 2017; Hsu et al, 2019), although the origin and function of these methylation sites is largely unknown.…”