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Human RNA polymerase III transcriptomes and phosphatase regulation of yeast MAF1.

机译：人RNA聚合酶III转录组和酵母MAF1的磷酸酶调节。

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摘要

RNA Polymerase (Pol) III transcribes small noncoding RNAs (e.g., tRNAs) important for translational capacity. Maf1 is a repressor of Pol III conserved from yeast to human, required for repression of Pol III in response to multiple environmental stresses, such as nutrient deprivation. Interestingly, Maf1 is a phosphoprotein, being phosphorylated in good growth conditions and dephosphorylated in poor growing conditions. The phosphatase acting on Maf1 has not been determined. I investigated the identity of the phosphatase in yeast Saccharomyces cerevisiae using a genetic Maf1-Pol III fusion construct in combination with molecular and biochemical assays. I queried members of Protein Phosphatase 2A and 4 complexes (PP2A and PP4, respectively) for their role in dephosphorylation of Maf1 and determined that PP4---containing Pph3 and Psy2, together with accessory factors Rrd1 and Tip41---is the major Maf1 phosphatase, acting in response to multiple stresses. Maf1 interacts with Pph3 in vivo, and biochemical purification of TAP-tagged Pph3-bound complex shows activity on purified, endogenously phosphorylated Maf1 in vitro, suggesting that PP4 is a direct phosphatase of Maf1.;In human cells, regulation of Pol III also involves chromatin regulation, not apparent in studies of Pol III in yeast. The full repertoire of Pol III-transcribed genes in human cells has not been defined. I determined the full Pol III transcriptome in human (HeLa) cells by chromatin immunoprecipitation (ChIP), coupled with microarray (ChIP-array) or high throughput sequencing (ChIP-seq), for Pol III subunit Rpc32, and Pol III transcription factors Brf1, Brf2 and TFIIIC63. I also determined the Pol III transcriptome in other cell types to address the possibility of cell type-specific activation of Pol III genes. Although many active Pol III genes were shared between all cell types assayed, a large number of genes were differentially enriched with Pol III. I compared enrichment of Pol III with chromatin modifications, transcription factors, and Pol II ChIP-seq profiles and found a significant correlation for active Pol III genes with active chromatin modifications and occupancy of transcription factors and Pol II. These results suggest that active chromatin gates Pol III accessibility to the human genome.
机译：RNA聚合酶（Pol）III转录对翻译能力很重要的小型非编码RNA（例如tRNA）。 Maf1是从酵母到人保守的Pol III阻遏物，是对多种环境胁迫（例如营养缺乏）做出反应而抑制Pol III所必需的。有趣的是，Maf1是一种磷蛋白，在良好的生长条件下会被磷酸化，而在不良的生长条件下会去磷酸化。尚未确定作用于Maf1的磷酸酶。我使用基因Maf1-Pol III融合构建体与分子和生化分析相结合，研究了酵母酿酒酵母中磷酸酶的身份。我查询了蛋白磷酸酶2A和4个复合物（分别为PP2A和PP4）在Maf1脱磷酸中的作用，并确定包含PP4-的Pph3和Psy2以及辅助因子Rrd1和Tip41-是主要的Maf1。磷酸酶，响应多种压力而起作用。 Maf1在体内与Pph3相互作用，对带有TAP标签的Pph3结合的复合物进行生化纯化显示其对体外纯化的内源性磷酸化Maf1具有活性，这表明PP4是Maf1的直接磷酸酶。染色质调节，在酵母中的Pol III研究中不明显。尚未确定人类细胞中Pol III转录基因的全部组成。我通过染色质免疫沉淀（ChIP）结合微阵列（ChIP-array）或高通量测序（ChIP-seq），确定了人类（HeLa）细胞中的完整Pol III转录组，用于Pol III亚基Rpc32和Pol III转录因子Brf1 ，Brf2和TFIIIC63。我还确定了其他细胞类型中的Pol III转录组，以解决细胞类型特异性激活Pol III基因的可能性。尽管在所有检测的细胞类型之间共享许多活跃的Pol III基因，但大量的基因差异富集了Pol III。我比较了具有染色质修饰，转录因子和Pol II ChIP-seq图谱的Pol III富集，发现具有活跃染色质修饰和转录因子和Pol II占有率的活性Pol III基因具有显着相关性。这些结果表明活性染色质门Pol III对人类基因组的可及性。

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  • 作者

    Oler, Andrew Joseph.;

  • 作者单位

    The University of Utah.;

  • 授予单位 The University of Utah.;
  • 学科 Biology Molecular.;Biology Bioinformatics.;Biology Cell.
  • 学位 Ph.D.
  • 年度 2011
  • 页码 129 p.
  • 总页数 129
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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