Project description:Loss of nutrient supply elicits alterations of the SUMO proteome and sumoylation is crucial to various cellular processes including transcription. However, the physiological significance of sumoylation of transcriptional regulators is unclear. To begin clarifying this, we mapped the SUMO proteome under nitrogen-limiting conditions in Saccharomyces cerevisiae. Interestingly, several RNA polymerase III (RNAPIII) components are major SUMO targets under normal growth conditions, including Rpc53, Rpc82, and Ret1, and nutrient starvation results in rapid desumoylation of these proteins. These findings are supported by ChIP-seq experiments that show that SUMO is highly enriched at tDNA genes. Furthermore, RNA-seq experiments revealed that preventing sumoylation results in significantly decreased tRNA transcription. TORC1 inhibition resulted in the same effect, and our data indicate that the SUMO and TORC1 pathways are both required for robust tDNA expression. Importantly, tRNA transcription was strongly reduced in cells expressing a non-sumoylatable Rpc82-4KR mutant, which correlated with a misassembled RNAPIII transcriptional complex. Our data suggest that in addition to TORC1 activity, sumoylation of RNAPIII is key to reaching full translational capacity under optimal growth conditions.
Project description:Loss of nutrient supply elicits alterations of the SUMO proteome and sumoylation is crucial to various cellular processes including transcription. However, the physiological significance of sumoylation of transcriptional regulators is unclear. To begin clarifying this, we mapped the SUMO proteome under nitrogen-limiting conditions in Saccharomyces cerevisiae. Interestingly, several RNA polymerase III (RNAPIII) components are major SUMO targets under normal growth conditions, including Rpc53, Rpc82, and Ret1, and nutrient starvation results in rapid desumoylation of these proteins. These findings are supported by ChIP-seq experiments that show that SUMO is highly enriched at tDNA genes. Furthermore, RNA-seq experiments revealed that preventing sumoylation results in significantly decreased tRNA transcription. TORC1 inhibition resulted in the same effect, and our data indicate that the SUMO and TORC1 pathways are both required for robust tDNA expression. Importantly, tRNA transcription was strongly reduced in cells expressing a non-sumoylatable Rpc82-4KR mutant, which correlated with a misassembled RNAPIII transcriptional complex. Our data suggest that in addition to TORC1 activity, sumoylation of RNAPIII is key to reaching full translational capacity under optimal growth conditions.
Project description:The Target Of Rapamycin (TOR) protein is a Ser/Thr kinase that functions in two distinct multiprotein complexes: TORC1 and TORC2. These conserved complexes regulate many different aspects of cell growth in response to intra- and extracellular cues. Here we report the first bona fide substrate of yeast TORC1: the AGC-kinase Sch9. Six amino acids in the c-terminus of Sch9 are directly phosphorylated by TORC1. Phosphorylation of these residues is lost upon rapamycin-treatment as well as carbon- or nitrogen-starvation and transiently reduced following application of osmotic, oxidative or thermal stress. TORC1-dependent phosphorylation is required for Sch9 activity and replacement of residues phosphorylated by TORC1 with Asp/Glu renders Sch9 activity TORC1-independent. Sch9 is required for TORC1 to properly regulate ribosome biogenesis, translation initiation and entry into G0 phase, but not expression of Gln3-dependent genes. Our results suggest that Sch9 functions analogously to the mammalian TORC1 substrate S6K1 rather than the mTORC2 substrate PKB/Akt. Keywords: time course, cell type.
Project description:The Target Of Rapamycin (TOR) protein is a Ser/Thr kinase that functions in two distinct multiprotein complexes: TORC1 and TORC2. These conserved complexes regulate many different aspects of cell growth in response to intra- and extracellular cues. Here we report the first bona fide substrate of yeast TORC1: the AGC-kinase Sch9. Six amino acids in the c-terminus of Sch9 are directly phosphorylated by TORC1. Phosphorylation of these residues is lost upon rapamycin-treatment as well as carbon- or nitrogen-starvation and transiently reduced following application of osmotic, oxidative or thermal stress. TORC1-dependent phosphorylation is required for Sch9 activity and replacement of residues phosphorylated by TORC1 with Asp/Glu renders Sch9 activity TORC1-independent. Sch9 is required for TORC1 to properly regulate ribosome biogenesis, translation initiation and entry into G0 phase, but not expression of Gln3-dependent genes. Our results suggest that Sch9 functions analogously to the mammalian TORC1 substrate S6K1 rather than the mTORC2 substrate PKB/Akt. Keywords: time course, cell type. Global transcriptional analysis of rapamycin response was conducted on cells expressing either a wild-type, Sch9(WT), or TOR-independent allele of Sch9, Sch9(2D3E). Reference samples used were cells collected immediately prior to rapamycin treatment for the respective cell genotypes. Test samples were collected 20, 30, 60, 90, 120, and 180min post rapamycin treatment.
Project description:Snf1 and TORC1 are two global regulators that sense the nutrient availability and regulate the cell growth in yeast Saccharomyces cerevisiae. Here we undertook a systems biology approach to study the effect of deletion of these genes and investigate the interaction between Snf1 and TORC1 in regulation of gene expression and cell metabolism.
Project description:Snf1 and TORC1 are two global regulators that sense the nutrient availability and regulate the cell growth in yeast Saccharomyces cerevisiae. Here we undertook a systems biology approach to study the effect of deletion of these genes and investigate the interaction between Snf1 and TORC1 in regulation of gene expression and cell metabolism. 3 mutant strains (snf1?, tor1?, snf1?tor1?) together with 1 reference strain grown under both glucose-limited or amonia-limited defined media with three biological replicates for each strain
Project description:Interplay between nuclear RNA polymerases is key to growth control. Here, we explored the ways in which mRNA transcription by polymerase II (Pol II) is influenced by a defect in the biogenesis of Pol III. We used the cold-sensitive yeast mutant rpc128-1007, which prevents assembly of the Pol III complex and consequently leads to low tRNA levels. mRNA upregulation in rpc128-1007 cells was generally stronger and involved more genes than downregulation. The observed induction of mRNA expression was mostly indirect and resulted from the de-repression of general control transcription factor Gcn4. mRNA genes that were downregulated by the reduction of Pol III assembly comprise the proteasome complex. We also investigated the ways in which the reprogramming of Pol II genes is influenced by the rpc128-1007 suppressors RBS1 and PRT1, which encode the Pol III assembly factor and the subunit of translation initiation factor eIF3, respectively. Both of the suppressor genes countered the effects of rpc128-1007 on the expression of Gcn4-dependent genes and the effects of PRT1 were stronger than the effects of RBS1. Additionally, Rbs1 modulates Gcn4 activity in a manner that depends on of the Pho85 cyclin Pcl5. We have shown that the downregulation of Pcl5 protein levels by Rbs1 overproduction leads to a Gcn4 response that is likely related to the stabilization of Gcn4 protein. Altogether, our data contribute to the regulatory network which links transcription of different RNA classes