mTORC1 is a central regulator of growth in response to nutrient availability, but few direct targets have been identified. RNA polymerase (pol) III produces a number of essential RNA molecules involved in protein synthesis, RNA maturation, and other processes. Its activity is highly regulated, and deregulation can lead to cell transformation. The human phosphoprotein MAF1 becomes dephosphorylated and represses pol III transcription after various stresses, but neither the significance of the phosphorylations nor the kinase involved is known. We find that human MAF1 is absolutely required for pol III repression in response to serum starvation or TORC1 inhibition by rapamycin or Torin1. The protein is phosphorylated mainly on residues S60, S68, and S75, and this inhibits its pol III repression function. The responsible kinase is mTORC1, which phosphorylates MAF1 directly. Our results describe molecular mechanisms by which mTORC1 controls human MAF1, a key repressor of RNA polymerase III transcription, and add a new branch to the signal transduction cascade immediately downstream of TORC1.The PIKK family conserved TOR (target of rapamycin) kinase, originally discovered in Saccharomyces cerevisiae (9), is a central regulator of cell growth in response to nutrient availability and other environmental cues (see references 4 and 44 for reviews). TOR is part of two complexes, TORC1 and TORC2, of which the first is inhibited by low concentrations of the macrolide rapamycin. Inhibition of TORC1 by nutrient deprivation or rapamycin has broad consequences, among them the inhibition of ribosome biogenesis and protein translation. This inhibition is mediated in part through transcriptional repression of genes required for these processes such as the RNA polymerase (pol) I-transcribed large rRNA genes, the pol II-transcribed ribosomal protein genes, and a number of pol III-transcribed genes, including, for example, tRNA genes (4, 44).In yeast, repression of pol III transcription in response to nutrient deprivation, rapamycin treatment, or other stresses such as DNA damage and secretory pathway defects requires the repressor Maf1 (38) (see 6 and 42 for reviews). The protein is regulated by phosphorylation/dephosphorylation events, which control nuclear/cytoplasmic transport as well as the pol III repression function of the protein. The two processes are, however, at least partially independent (20,23,28,36,40). Several kinases have been implicated, in particular PKA and Sch9, the second of which appears to be the main Maf1 kinase (11,17,20,41). Recently, TORC1 was also described as a kinase that weakly phosphorylates yeast Maf1 on unknown residues within the nucleus (40).Human MAF1, like yeast Maf1, is a phosphoprotein. It is unclear, however, whether human MAF1 is indispensable for repression of pol III transcription in response to various stresses, and neither the function of MAF1 phosphorylation nor the MAF1 kinases have been identified. Here we show that mammalian cells lacking the MAF1 gene do not repress pol III transcript...