Transfer RNA (tRNA) biosynthesis is essential for processes like protein synthesis, cell cycle progression, mitochondrial function, and cell proliferation. Dysregulated tRNA biosynthesis contributes to diseases such as hepatocellular carcinoma (HCC). Maf1 is a key inhibitor of RNA polymerase III (Pol III), regulating tRNA biosynthesis by blocking the interaction between Pol III and transcription factors TFIIIB and TFIIIC. Maf1 also suppresses tumor growth and the AKT/mTOR pathway by enhancing PTEN expression. mTOR phosphorylates Maf1 at Ser-75, leading to its export to the cytosol and subsequent ubiquitination, promoting protein synthesis and cell cycle progression in HCC. Aurora-A, a serine/threonine kinase, is crucial for cell cycle regulation, and its overexpression is linked to cancer progression. However, the relationship between Maf1 and Aurora-A remains unclear.

HCC cells were transfected with pGFP-Aurora-A and pHA-Maf1 to investigate their correlation. Alisertib was added to inhibit Aurora-A activity. RT-qPCR was performed to determine the RNA expression of Aurora-A, Maf1, pre-tRNALeu and pre-tRNAiMet. Western blot was performed to analyze expression levels of Maf1 and Aurora-A. Translational inhibitor cycloheximide was added to observe the protein stability of Maf1. The protein interaction between Aurora-A and Maf1 was determined by co-immunoprecipitation and proximity ligation assay. The subcellular localization of Maf1 was observed by cell fraction assay and immunofluorescence. The cell proliferation was analyzed by CCK-8. The glycolytic activity was analyzed by Extracellular Acidification Rate (ECAR) assay.

Aurora-A interacts with the Maf1 C domain to promote its cytosolic localization and enhance its protein stability in a kinase-dependent manner. Mutation of one potential Aurora-A phosphorylation site on Maf1 results in the nucleolus localization and decreased the protein stability. In the cytosol, Maf1 may regulate mitochondrial function by reducing OXPHOS levels. Notably, overexpression of the Maf1 mutant (Maf1-MT) failed to suppress tRNA biosynthesis and restore mitochondrial function. Additionally, HCC cells with Maf1 overexpression were more sensitive to Aurora-A inhibitor.

Aurora-A promotes the protein stability and cytosolic localization of Maf1 via interacting with its C domain in a kinase-dependent manner in HCC cells. Aurora-A phosphorylation site mutant of Maf1 can promote nucleolar localization and lost the RNA polymerase III suppressor function and regulation of mitochondria. In Maf1 overexpressed HCC cells, Aurora-A inhibitor has a higher efficacy in inhibiting cell growth. In HCC patients with high Aurora-A and Maf1 expression, treatment with Aurora-A inhibitors can be a promising strategy in cancer therapy.