ABSTRACT
Synthesis of tRNA and 5S rRNA by RNA polymerase (pol) III is regulated by the mTOR pathway in mammalian cells. The mTOR kinase localizes to tRNA and 5S rRNA genes, providing an opportunity for direct control. Its presence at these sites can be explained by interaction with TFIIIC, a DNA-binding factor that recognizes the promoters of these genes. TFIIIC contains a TOR signaling motif that facilitates its association with mTOR. Maf1, a repressor that binds and inhibits pol III, is phosphorylated in a mTOR-dependent manner both in vitro and in vivo at serine 75, a site that contributes to its function as a transcriptional inhibitor. Proximity ligation assays confirm the interaction of mTOR with Maf1 and TFIIIC in nuclei. In contrast to Maf1 regulation in yeast, no evidence is found for nuclear export of Maf1 in response to mTOR signaling in HeLa cells. We conclude that mTOR associates with TFIIIC, is recruited to pol III-transcribed genes, and relieves their repression by Maf1.
Subject(s)
Intracellular Signaling Peptides and Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , RNA, Ribosomal, 5S/genetics , RNA, Ribosomal, 5S/metabolism , RNA, Transfer/genetics , RNA, Transfer/metabolism , Repressor Proteins/metabolism , Transcription Factors, TFIII/metabolism , Base Sequence , Binding Sites , Chromatin Immunoprecipitation , HeLa Cells , Humans , Mutagenesis, Site-Directed , Phosphorylation , RNA Polymerase III/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Repressor Proteins/chemistry , Repressor Proteins/genetics , Serine/chemistry , TOR Serine-Threonine KinasesABSTRACT
High-content cell-based screens provide a powerful tool to identify new chemicals that interfere with complex biological processes. Here, we describe the identification of a new inhibitor of microtubule dynamics (micropolyin) using a high-content screen. Integrated high-resolution imaging allowed for fast selection of hits and progression to target identification. Treatment of cells with micropolyin efficiently causes a pro-metaphase arrest, with abnormal spindle morphology and with the spindle assembly checkpoint activated. The arrest appears to result from interference of micropolyin with microtubule dynamics. We show in vitro that tubulin is indeed the target of micropolyin and that micropolyin inhibits microtubule polymerization. Our results demonstrate the power of high-content image- and cell-based screening approaches to identify potential new drug candidates. As our approach is unbiased, it should allow for discovery of new targets that may otherwise be overlooked.
