NF-kappa B p65 regulates snRNA U6 N6-methlyadenosine modification and pre-mRNA alternative splicing by binding to and shuttling METTL16 into nucleus

In the spliceosome, snRNA U6 can form the catalytic core with snRNA U2 to catalyze pre-mRNA alternative splicing. The ACA(43)GAGA box of snRNA U6 is involved in the first step of alternative splicing by base-pairing with the 5' splicing site of pre-mRNA. The A43 in U6 can be modified into m6A43 (m6A U6), however, its effect on pre-mRNA alternative splicing remains unclear. In this study, we first found that TAT increased the m6A U6 level and the splicing of minigene CD44V5 pre-mRNA. We then demonstrated that synthesized short methylated U6 increased the splicing of both minigene CD44V5 pre-mRNA in vivo and beta-globin pre-mRNA in vitro. Transcriptome-wide analysis using SPLICE-q revealed that TAT increased the splicing efficiency (SE) in both the whole transcriptome and single-spliced genes that was 16 times higher than multiple-spliced genes. We also found that NF-kappa B p65 (p65) increased the m6A U6 level and its sole methyltransferase METTL16 protein level in the nucleus while nuclear localization signal mutation of p65 decreased the m6A U6 level. Analysis of the existing transcriptome data derived from cells treated with a p65 activator TNF-alpha and cells transfected with p65-targeted miRNA miR520b confirmed a strong association between p65 expression and the SE at the transcriptome level. Lastly, we constructed a series of site-specific and domain-deletion mutants and demonstrated that p65 bound to METTL16 through amino acid resides L413 and P517 of p65 and the methyltransferase domain (aa 2-289) and the disordered region (aa 401-514) of METTL16. Taken together, these results demonstrated that p65 regulates the m6A modification of U6 and pre-mRNA splicing through binding to and shuttle METTL16 into nucleus and suggest a new mechanism to protect cells against harmful stimuli.