Abstrakt
Background And Aims
Mutations in the splicing factor gene SF3B1 are frequently found in myelodysplastic syndromes (MDS). They result in aberrant splicing of multiple genes, such as the genes involved in mitochondrial metabolism. Furthermore, the aberrant splicing may cause changes in circular RNA (circRNA) production. Here, we investigated the impact of SF3B1 mutations on gene expression, circRNA production, and cellular bioenergetics.
Methods
Expression profiles were analyzed by RNA sequencing in MDS patient primary cells. siRNA knockdown of SF3B1 and circRNAs was performed on NALM6 cell lines with or without SF3B1 mutations (mut or wt, respectively). Energetic metabolism was investigated by Seahorse analysis and targeted metabolomics.
Results
We detected 1,001 differentially expressed genes between K700E and K666N/E SF3B1 mutated patient samples, suggesting that particular mutations have specific effect on RNA expression. In patients with SF3B1 mutations, we further detected substantial upregulation of circRNAs processed from ZEB1 gene, encoding a transcription factor, and we demonstrated that these circRNAs are related to mitochondrial activity. In NALM-6 cell lines, we observed that mut-cells have significantly lower glycolysis and glycolytic capacity, and more than four-times higher ATP/ADP ratio, suggesting increased respiratory metabolism and inhibition of glycolysis. Lower glycolysis further indicated decreased proliferation, which was confirmed with AlamarBlue assay.
Conclusions
K700E and K666N/E mutations have different effect on RNA expression, possibly leading to different cellular phenotypes. Mutated SF3B1 has impact on cellular bioenergetics and leads to deregulation of ZEB1-circRNAs, which may contribute to defects in mitochondrial metabolism.