PgmNr Y473: P bodies regulate the rewiring of a transcription network by controlling the expression of the YOX1 repressor during DNA replication stress.

Authors:
R. Loll-Krippleber; GW. Brown


Institutes
Department of Biochemistry, University of Toronto, Toronto, Ontario, CA.


Keyword: RNA Processing

Abstract:

P bodies are RNA-protein granules that form in the cytoplasm of eukaryotic cells in response to various stresses and are thought to serve as sites of degradation and/or storage of mRNAs. We recently discovered that P bodies form in yeast in response to replication stress induced by HU (hydroxyurea), an anti-cancer drug that inhibits dNTP synthesis and slows DNA replication. P body components are required for cell survival of replication stress as mutants lacking key P body components Lsm1, Pat1 and Dhh1 are strongly sensitive to HU. Here, we aimed to identify mRNAs that are processed by P bodies during replication stress. First, we performed a transcriptome study on lsm1∆ cells upon acute HU exposure to identify mRNAs that are stabilized in the absence of a functional P body-dependent mRNA degradation pathway. Second, we used an SGA-based suppressor screen to identify genes whose expression is toxic in the absence of Lsm1 and Pat1 during replication stress. We found that the transcriptome in lsm1∆ is altered both during normal growth and during replication stress, with more than 800 mRNAs being stabilized in lsm1∆ compared to wild type. Interestingly, we found that inactivation of the coding sequence of 6 of those 800 mRNAs was able to suppress HU sensitivity of lsm1∆ and pat1∆ strains suggesting that these genes encode mRNAs that need to be degraded in a P body dependent manner upon HU exposure. Among these we identified YOX1, a gene encoding a transcription repressor critical for the regulation of cell cycle and DNA replication genes. The suppressor effect of yox1∆ in lsm1∆ and pat1∆ cells is specific to HU and not to other drugs that induce DNA replication stress or to other environmental stresses. Consistent with P bodies regulating YOX1 mRNA abundance, we found that YOX1 mRNA localizes to P bodies and accumulates at P bodies in the absence of the mRNA exonuclease Xrn1. To gain insight into the role of Yox1 during replication stress, we identified 156 genes that are down-regulated upon YOX1 overexpression. Among this set of targets, we notably found genes important for ribonucleotides biosynthesis (RNR genes), DNA unwinding during replication (MCM genes), cell cycle regulation and cytokinesis. Work is underway to identify which targets play a critical role during replication stress. Together, our data suggest a model where YOX1 mRNA abundance is post-transcriptionally regulated by P bodies in order to reduce the level of the Yox1 transcription repressor and therefore prevent repression of genes necessary for survival of DNA replication stress.



Yeast Database Genetic Index
1. gene symbol: LSM1; systematic name: YJL124C
2. gene symbol: PAT1; systematic name: YCR077C
3. gene symbol: YOX1; systematic name: YML027W