Functional Analysis Of DdRpb4 And DdRpb7, Two Subunits Of Dictyostelium Discoideum RNA Polymerase II

Abstract

The process of eukaryotic transcription and its regulation has been an interesting area of research for decades. With more insights into the process of transcriptional regulation of genes, studies have revealed a transcriptional regulation at the level of RNA polymerase II in response to nutritional stress. Further studies in our laboratory and others’, using Saccharomyces cerevisiae as a model system, had shown that two subunits of core RNA polymerase II, RPB4 and RPB7 play a crucial role in response to nutritional starvation. Similarly, these proteins are also known to play important roles in stress response in higher eukaryotes. Additionally, altering levels of Rpb4 and Rpb7 can differentially affect starvation response in S. cerevisiae (Singh et al., 2007). Multiple tissue blot analyses had shown that both these subunits are differentially expressed in different human tissues more significantly in heart, kidney and brain (Khazak et al., 1995; Khazak et al., 1998; Schoen et al., 1997). These findings have led us to investigate in Dictyostelium discoideum, a cellular slime mold, the possible role of these subunits during starvation-induced development. D. discoideum cells exist as unicellular amoebae in soil. In this organism, growth and differentiation phases are distinctly separated, which is an advantage for investigating the functions of these subunits during growth and development. Cells respond to nutritional starvation by undergoing a series of morphological changes coordinated with transcriptional changes giving rise to a terminally differentiated structure referred to as fruiting body which has live spores suspended on top of stalk of dead cells. Though starvation-induced development is accompanied by differential expression of genes, few studies related to the transcription machinery, RNA polymerase II have been reported so far. Purification and presence of all three RNA polymerases from D. discoideum had been reported earlier but the details of their structures and regulation have not been explored in detail (Pong and Loomis, 1973; Renart et al., 1985). One interesting observation reported by Lam and colleagues (Lam et al., 1992) was that CTD of the largest subunit of RNA polymerase II, Rpb1, is highly conserved with 24 heptapeptide repeats and expression of RPB1 transcript was regulated during development. Thus, we carried out experiments to characterize Rpb4 and Rpb7, two subunits of D. discoideum RNA polymerase II to understand any role of these subunits during development. Identification of Rpb4 and Rpb7, two subunits of D. discoideum RNA polymerase II To identify the homologs of S. cerevisiae Rpb4 and Rpb7 in D. discoideum, we employed bioinformatics and genetic approaches. Firstly, we searched D. discoideum database for all protein sequences of S. cerevisiae RNA polymerase II subunits. We could obtain sequences homologous to all twelve subunits in D. discoideum. Among the 12 subunits of D. discoideum RNA polymerase II, we chose to characterize two subunits, DdRpb4 and DdRpb7. We cloned the open reading frames of these two genes from D. discoideum Ax2 cells and cloned them in yeast expression vectors for complementation studies. In S. cerevisiae, Rpb4 is a non-essential protein but rpb4∆ cells show abnormal phenotypes. Few phenotypes of rpb4∆ cells, such as temperature sensitivity, defective in response to nutritional starvation and defective in activated transcription, were employed to identify the D. discoideum homolog of ScRpb4 (Woychik and Young, 1989; Pillai et al., 2001: Pillai et al., 2003). We observed that DdRPB4 can rescue temperature sensitivity corroborated with its ability to activate transcription from HSE containing promoters and sporulation defects of Scrpb4Δ mutant to the wild type. However, DdRPB4 can rescue neither the defect in activated transcription of GAL10 and INO1 promoters nor the elongated morphology exhibited by Scrpb4Δ mutant. On the other hand, we observed that DdRPB7 can complement the lethality associated with ScRPB7 deletion and can partially rescue the phenotypes associated with Scrpb4∆ strain similar to ScRPB7 (Sharma and Sadhale, 1999; Singh et al., 2004). Taken together, we have identified D. discoideum Rpb4 and Rpb7 as bona fide homologs of S. cerevisiae Rpb4 and Rpb7, respectively. Analysis of Rpb4 and Rpb7 in D. discoideum Since yeast RNA polymerase II subunits, Rpb4 and Rpb7, play an important role in the regulation of genes responsive to starvation stress, we carried out experiments to characterize Rpb4 and Rpb7 during growth and starvation-induced development in D. discoideum. Temporal and spatial expression profiles show avaried but similar pattern of RPB4 and RPB7 transcripts during D. discoideum development. We observed similarity between ScRpb4 and DdRpb4 in its ability to interact with DdRpb7 and to localise in both nuclear and cytoplasmic compartments. Attempts to knock out or reduce the levels of DdRpb4 and DdRpb7 by homologous recombination and antisense approaches, respectively, failed. However, since altering levels of Rpb4 and Rpb7 in S. cerevisiae can affect different stress response pathways, we had used overexpression to alter the level of Rpb4 and analysed its effect on D. discoideum development. We overexpressed DdRpb4 as GFP fusion protein in Ax2 cells and observed that D. discoideum cells overexpressing DdRpb4 showed normal growth and development similar to the wild type protein. Interestingly, we observed that Ax2 cells overexpressing DdRpb4 have drastically reduced levels of the endogenous protein. Thus, we have identified a post-transcriptional control on the level of Rpb4 in D. discoideum. Role of S. cerevisiae Rpb4/Rpb7 subcomplex in stress In S. cerevisiae, Rpb4 and Rpb7 interact with each other and carry out important functions (Choder, 2003; Sampath and Sadhale, 2004). Employing the functional conservation of Rpb4 and Rpb7 across various model systems, we further investigated the role of the subcomplex in S. cerevisiae. Since Rpb7 is an essential gene, we have generated rpb7Δstrain in the presence of plasmids expressing Rpb7 or its homologs. We have generated a S. cerevisiae strain lacking both RPB4 and RPB7 and introduced Rpb4 and Rpb7 homologs from either D. discoideum or C. albicans. We analysed these strains under stresses such as high temperature and nutrient starvation. The results of these experiments have provided how the differences in Rpb4 and Rpb7 proteins and their ability to form a subcomplex could be reflected in differential stress responses. Besides the high functional conservation of these proteins, their interaction with other regulatory proteins might also be critical for a proper response to nutritional stress.