Regulation of RNA Polymerase III Transcription Involves SCH9-dependent and SCH9-independent Branches of the Target of Rapamycin (TOR) Pathway

Abstract

Maf1 is a conserved repressor of transcription that functions at the downstream end of multiple nutrient and stress signaling pathways. How these different signaling pathways converge on Maf1 is not known. Previous work in yeast indicates that protein kinase A (PKA) regulates RNA polymerase (pol) III transcription, in part, by phosphorylating multiple sites in Maf1. Here we present additional evidence for this view and show that a parallel nutrient and stress-sensing pathway involving Sch9, an homologous kinase to metazoan S6 kinase, targets Maf1 at a subset of PKA sites. Using ATP analog-sensitive alleles of PKA and Sch9, we find that these two kinases account for the bulk of the phosphorylation on consensus PKA sites in Maf1. Deletion of Sch9 reduces RNA pol III transcription in a Maf1-dependent manner, yet the cells remain susceptible to further repression by rapamycin and other treatments. Because the rapamycin-sensitive kinase activity of the TORC1 complex is necessary for Sch9 function in vivo and in vitro, our results show that transcriptional regulation of RNA pol III and the coordinate control of ribosomal protein genes can be achieved by Sch9-dependent and -independent branches of the target of rapamycin (TOR) signaling pathway.Maf1 is essential for repressing transcription by RNA polymerase (pol) 2 III in yeast, and this function is conserved in mammals (1-4). In addition, human Maf1 is known to directly repress RNA pol II transcription of certain protein coding genes such as the TATA box-binding protein, TBP, and to indirectly repress transcription by RNA pol I (3). Repression by Maf1 occurs in response to a wide variety of nutritional and stress conditions, but the nature of the signaling pathways mediating these responses and how they converge on Maf1 is largely unknown (5).In budding yeast, Maf1 is phosphorylated on consensus PKA sites under optimal growth conditions and is rapidly dephosphorylated under starvation or stress conditions that repress transcription (5, 6). Dephosphorylation of these PKA sites correlates with the relocation of Maf1 from the cytoplasm to the nucleus and is thought to regulate its inhibitory interaction with RNA pol III (5-9). Nuclear import of Maf1 is directed by two redundant nuclear localization sequences (NtNLS and CtNLS) that are differentially sensitive to PKA site phosphorylation. Specifically, the NtNLS is inhibited by phosphorylation as acidic substitutions at the PKA sites, which are normally fully functional, can prevent nuclear import and repression when the CtNLS is disabled (6). The localization of Maf1 in the cytoplasm serves to fine-tune its regulation but is not essential for preventing repression at inappropriate times. The accumulation of Maf1 in the nucleus, either by mutation of the protein or by deletion of the exportin of the protein, Msn5, does not cause repression in the absence of additional changes triggered by cellular signaling pathways (6, 10).Numerous studies have implicated the RAS/PKA pathway and the rapamycin-sensitive TOR pathway as po...

Characterization of the rapamycin-sensitive phosphoproteome reveals that Sch9 is a central coordinator of protein synthesis

“…While this manuscript was in preparation, Lee et al (2009) reported that Sch9 phosphorylates Maf1; but, in contrast to our results, their work suggested that Sch9 and PKA perform equally important roles in Maf1 regulation. We believe that this discrepancy is due to protocol differences in the handling of yeast cells prior to protein extract.…”

Covalent Small Ubiquitin-like Modifier (SUMO) Modification of Maf1 Protein Controls RNA Polymerase III-dependent Transcription Repression

“…In yeast, Maf1 is regulated through phosphorylation by PKA and the TORC1-regulated Sch9 kinase (21,22). Phosphorylation of Maf1 at these sites results in its inactivation by preventing its nuclear accumulation under non-stress conditions.…”

Life in the midst of scarcity: adaptations to nutrient availability in Saccharomyces cerevisiae

“…For instance, both kinases have an additive effect for the expression of proteins required for nucleotide metabolism but they exert opposite effects on the expression on proteins involved in detoxification or proteolysis . In addition, they independently determine the sensitivity of yeast cells for the ATP-analogue 1NM-PP1 as illustrated by the observation that a mutant with combined analogue-sensitive sch9 as tpk1 as tpk2 as tpk3 as alleles is more sensitive than the single sch9 as mutant or the tpk1 as tpk2 as tpk3 as strain (Yorimitsu et al 2007;Huber et al 2009;Lee et al 2009). …”

“…For RNA polymerase III-dependent transcription, the downstream target of Sch9 is Maf1, the repressor that is also regulated by PKA and TORC1. Here, Sch9 was first reported to phosphorylate Maf1 at a subset of the sites that are also recognized by PKA (Lee et al 2009). Most recently, it was shown that Sch9 phosphorylates Maf1 at seven distinct sites in vitro and that it thereby can not only partially inhibit nuclear accumulation of Maf1 after rapamycin treatment, but also completely block the repressive association between Maf1 and the RNA Pol III subunit Rpc82 (Huber et al 2009).…”