Mapping the interaction site of Rpb4 and Rpb7 subunits of RNA polymerase II in Saccharomyces cerevisiae

Rpb4 and Rpb7, the fourth and the seventh largest subunits of RNA polymerase II, form a heterodimer in Saccharomyces cerevisiae. To identify the site of interaction between these subunits, we constructed truncation mutants of both these proteins and carried out yeast two hybrid analysis. Deletions in the amino and carboxyl terminal domains of Rpb7 abolished its interaction with Rpb4. In comparison, deletion of up to 49 N-terminal amino acids of Rpb4 reduced its interaction with Rpb7. Complete abolishment of interaction between Rpb4 and Rpb7 occurred by truncation of 1-106, 1-142, 108-221, 172-221 or 198-221 amino acids of Rpb4. Use of the yeast two-hybrid analysis in conjunction with computational analysis of the recently reported crystal structure of Rpb4/Rpb7 sub-complex allowed us to identify regions previously not suspected to be involved in the functional interaction of these proteins. Taken together, our results have identified the regions that are involved in interaction between the Rpb4 and Rpb7 subunits of S. cerevisiae RNA polymerase II in vivo.     

The RNA polymerase II Rpb4/7 subcomplex regulates cellular lifespan through an mRNA decay process

In budding yeast, a highly conserved heterodimeric protein complex that is composed of the Rpb4 and Rpb7 proteins within RNA polymerase II shuttles between the nucleus and cytoplasm where it coordinates various steps of gene expression by associating with mRNAs. Although distinct stages of gene expression potentially contribute to the regulation of cellular lifespan, little is known about the underlying mechanisms. Here, we addressed the role of the dissociable Rpb4/7 heterodimeric protein complex in the regulation of replicative lifespan during various stages of gene expression in the yeast Saccharomyces cerevisiae. We observed that the loss of Rpb4 resulted in a shortened lifespan. In contrast, we found that defects in the dissociation of Rpb4/7 from the RNA polymerase core complex and in translation initiation steps affected by Rpb4/7 did not impact lifespan. Tandem affinity purification experiments demonstrated that Rpb7 physically associates with Tpk2 and Pat1, which are both implicated in mRNA degradation. Consistent with this data, the loss of the mRNA decay regulators Pat1 and Dhh1 reduced the cellular lifespan. In summary, our findings further reinforce the pivotal role of Rpb4/7 in the coordination of distinct steps of gene expression and suggest that among the many stages of gene expression, mRNA decay is a critical process that is required for normal replicative lifespan.     

酵母RNA聚合酶ⅡRpb2和Rpb3两亚基间相互作用位点的定位

为研究S .pombeRNApolⅡ各亚基间体内装配成复合体的机制 ,本文首次用酵母双杂交系统鉴定了Rpb2和Rpb3两亚基间体内相互作用的位点。首先将Rpb2的 4个片段克隆至Gal4BD表达载体pAS2上 ,构建BD Rpb2片段融合蛋白重组质粒 ;同时将Rpb3克隆至Gal4AD表达载体pGADGH上 ,构建AD Rpb3融合蛋白重组质粒。其次 ,将pGADGHRpb3分别与pAS2Rpb2各片段重组质粒共转化到受体酵母菌Y1 90感受态细胞内 ,筛选并鉴定β gal活性阳性 (β gal+)的共转化子。最后 ,将β gal+共转化子中的Rpb2片段进行序列分析并进行同源序列比较确定其在Rpb2中的位置。结果表明 ,Rpb2与Rpb3相互作用的位点位于Rpb2的 90 2～ 989aa肽段内     

RAM pathway contributes to Rpb4 dependent pseudohyphal differentiation in Saccharomyces cerevisiae

Rpb4, a subunit of RNA Polymerase II plays an important role in various stress responses in budding yeast, Saccharomyces cerevisiae. In response to nitrogen starvation, diploid yeast undergoes a dimorphic transition to filamentous pseudohyphal growth, which is regulated through cAMP-PKA and MAP kinase pathway. In the present study, we show that disruption of Rpb4 leads to enhanced pseudohyphal growth, which is independent of nutritional status. We observed that the rpb4Delta/rpb4Delta cells exhibit pseudohyphae even in the absence of functional MAP kinase and cAMP-PKA pathways. Genome-wide expression profiling showed that in the absence of Rpb4 several genes controlling mother daughter cell separation are down regulated. Our genetic studies also provide evidence for involvement of RNA Pol II subunit Rpb4 in the expression of genes downstream of the RAM pathway. Finally, we show that this effect on expression of RAM pathway may at least be partially responsible for the pseudohyphal phenotype of rpb4Delta/rpb4Delta cells.     

Sequence divergence of the RNA polymerase shared subunit ABC14.5 (Rpb8) selectively affects RNA polymerase III assembly in Saccharomyces cerevisiae.

ABC14.5 (Rpb8) is a eukaryotic subunit common to all three nuclear RNA polymerases. In Saccharomyces cerevisiae, ABC14.5 (Rpb8) is essential for cell viability, however its function remains unknown. We have cloned and characterised the Schizosaccharomyces pombe rpb8(+) cDNA. We found that S.pombe rpb8, unlike the similarly diverged human orthologue, cannot substitute for S.cerevisiae ABC14. 5 in vivo. To obtain information on the function of this RNA polymerase shared subunit we have used S.pombe rpb8 as a naturally altered molecule in heterologous expression assays in S.cerevisiae. Amino acid residue differences within the 67 N-terminal residues contribute to the functional distinction of the two yeast orthologues in S.cerevisiae. Overexpression of the S.cerevisiae largest subunit of RNA polymerase III C160 (Rpc1) allows S.pombe rpb8 to functionally replace ABC14.5 in S.cerevisiae, suggesting a specific genetic interaction between the S.cerevisiae ABC14.5 (Rpb8) and C160 subunits. We provide further molecular and biochemical evidence showing that the heterologously expressed S.pombe rpb8 molecule selectively affects RNApolymerase III but not RNA polymerase I complex assembly. We also report the identification of a S.cerevisiae ABC14.5-G120D mutant which affects RNA polymerase III.