Suppressed SF3B1 Expression Lowers METTL3 Transcription and m6A RNA Expression

Abstract

Splicing factor 3b1 (SF3B1), a component of U2 small nuclear ribonucleoprotein (U2 snRNP), has been known for its essential roles in pre-mRNA splicing and alternative splicing. Here we show that knocking down (KD) of SF3B1 broadly induced a significant reduction in mRNA expression in the genome. One of the genes whose expression is reduced by SF3B1 KD is methyl-transferase-like 3 (METTL3), a writer of N6-methyladenosine (m(6)A). We demonstrate that expression of both METTL3 mRNA and protein is affected by SF3B1 KD, which further decreases the m(6)A RNA expression level. m(6)A-seq indicates that SF3B1 KD affects m(6)A distribution within multiple genes in the genome. In addition, a high proportion of hypo-methylation events by SF3B1 KD (similar to 70%) are overlapped in METTL3 KD cells, and a conserved m(6)A motif is observed in the hypo-methylated regions as in SF3B1 KD cells, suggesting the m(6)A decrease by SF3B1 is a direct effect of the reduced METTL3 expression. Furthermore, RT-qPCR using unlabeled RNA and 5-Bromouridine (BrU)-labeled nascent RNA and actinomycin D treatment demonstrates that transcription of METTL3 is significantly reduced but the mRNA decay rate is not altered, suggesting that METTL3 expression is altered at the transcription level. We further show that SF3B1 interacts with RNA polymerase (Pol) II in the RNA independent manner, further indicating the involvement of SF3B1 in transcription. Lastly, we demonstrate that the transcription inactive H3K27me3 on the METTL3 promoter was significantly increased whereas transcription active H3K4me3 was not changed by SF3B1 KD. Taken together, we conclude that reduced SF3B1 expression suppresses the transcription of METTL3 and inhibits m(6)A RNA expression.