ABSTRACT: The C-terminal domain (CTD) of the largest subunit in DNA-dependent RNA polymerase II (RNAP II) is essential for mRNA syntheses, coordinating an astounding array of protein-protein interactions. In yeast, mutations that separate established functional units of the CTD result in pleiotrophic effects, producing slow growth phenotypes and the accumulation of abnormally large cells. In general, the downstream pathways for such CTD related phenotypes are uncharacterized and probably highly complex. We also observed that a sizeable fraction of CTD mutants retain multiple buds on parent cells and are elongated relative to wild-type yeast. FACScan analyses revealed a trend toward increased DNA content over multiple rounds of growth, indicating that insertions into the normal tandemly repeated CTD result in aneuploidy. To begin to address the role of the CTD in such complex changes in cell structure and behavior, we applied an integrated approach using the Saccharomyces cerevisiae Genome Database (SGD) and microarray data. We were able to identify candidate genetic networks that could explain chromosome missegregation and related phenotypic traits in CTD mutants. These networks provide links between CTD-associated proteins and kinetochore function, control of cell cycle checkpoint mechanisms, and expression of cell wall and membrane components. The essential elements required for CTD function have been determined in yeast though substitution and insertion mutations (West and Corden 1995; Stiller and Cook 2004; Liu et al. 2008); the core CTD functional unit lies within tandem heptapeptides or â??diheptadsâ??. In addition, CTD mutants with progressively longer polyalanine insertions between diheptads show a continuous decline in growth rates, and the induction of conditional phenotypes; this has been demonstrated out to five alanine (5A) insertions (Stiller and Cook, 2004). Attempts to restore the global amino acid register via extending insertions to seven alanines between diheptad units proved to be lethal, leading to the conclusion that too great a separation between functional units puts undo stress on at least some essential CTD-protein interactions. (Liu et al, 2008). During these prior investigations, we observed that mutants containing 5A insertions are, on average, twice as large as normal yeast, prompting us to investigate these cells in more detail. Yeast cells were transformed with mutated CTD sequences containing 5 alanine insertions between diheptad units via the plasmid shuffle, as described in detail previously (West and Corden 1995; Stiller and Cook 2004). For comparison yeast cells were transformed with YSPTSPS sequences via the plasmid shuffle, as described in detail previously (West and Corden 1995; Stiller and Cook 2004). RNA was extracted from fresh pellets of yeast cultures at log phase, grown to an optical density of 0.8 at 600nm in 100 ml of YPD medium (1% yeast extract, 2% peptone, and 2% dextrose) at 30 ºC in a shaker at 225 rpm. Total RNA was extracted using Qiagenâ??s RNeasy Kit (Valencia, CA). Four replicates for each sample yeast strain (5A and wild-type CTD control) were prepared, and 10ug total RNA for every replicates were sent to Duke university DNA microarray center for further analyses. Array ID YO06N from Operon Yeast Genome Oligo Set version 1.1.2 was used for the hybridization. The direct labeling protocol was performed for sample RNA, which included steps of first strand synthesis, slide preparation, hydrolysis, cDNA purification, hybridization and array washing. Cy3 and Cy5 were used for labeling the samples. Maui hybridization and TIGR washing system were used in this protocol for array hybridization and washing respectively. The protocol can be found in Duke University microarray center website as http://www.genome.duke.edu/cores/microarray/services/spotted-arrays/protocols/. Fluorescent DNA bound to the microarray was detected with a GenePix 4000B scanner (Axon Instruments, Foster city, CA), using the GenePix 4000 software package to locate signal from spots. Data processing was performed using Duke Univerisity BASE web server (https://base-server.duhs.duke.edu/). GO annotation was used for gene ontology.
