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Connecting mutations of the RNA polymerase II C-terminal domain to complex phenotypic changes using combined gene expression and network analyses.

ABSTRACT: The C-terminal domain (CTD) of the largest subunit in DNA-dependent RNA polymerase II (RNAP II) is essential for mRNA synthesis and processing, through coordination of an astounding array of protein-protein interactions. Not surprisingly, CTD mutations can have complex, pleiotropic impacts on phenotype. For example, insertions of five alanine residues between CTD diheptads in yeast, which alter the CTD's overall tandem structure and physically separate core functional units, dramatically reduce growth rate and result in abnormally large cells that accumulate increased DNA content over time. Patterns by which specific CTD-protein interactions are disrupted by changes in CTD structure, as well as how downstream metabolic pathways are impacted, are difficult to target for direct experimental analyses. In an effort to connect an altered CTD to complex but quantifiable phenotypic changes, we applied network analyses of genes that are differentially expressed in our five alanine CTD mutant, combined with established genetic interactions from the Saccharomyces cerevisiae Genome Database (SGD). We were able to identify candidate genetic pathways, and several key genes, that could explain how this change in CTD structure leads to the specific phenotypic changes observed. These hypothetical networks identify links between CTD-associated proteins and mitotic function, control of cell cycle checkpoint mechanisms, and expression of cell wall and membrane components. Such results can help to direct future genetic and biochemical investigations that tie together the complex impacts of the CTD on global cellular metabolism.

SUBMITTER: Rogers C

PROVIDER: S-EPMC2894937 | biostudies-literature | 2010

REPOSITORIES: biostudies-literature

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Connecting mutations of the RNA polymerase II C-terminal domain to complex phenotypic changes using combined gene expression and network analyses.

Rogers Carlyle C Guo Zhenhua Z Stiller John W JW

PloS one 20100630 6

The C-terminal domain (CTD) of the largest subunit in DNA-dependent RNA polymerase II (RNAP II) is essential for mRNA synthesis and processing, through coordination of an astounding array of protein-protein interactions. Not surprisingly, CTD mutations can have complex, pleiotropic impacts on phenotype. For example, insertions of five alanine residues between CTD diheptads in yeast, which alter the CTD's overall tandem structure and physically separate core functional units, dramatically reduce ...[more]

PMID: 20613981

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Project description: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 ÂºC 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.

Project description:BackgroundRPB1, the largest subunit of RNA polymerase II, contains a highly modifiable C-terminal domain (CTD) that consists of variations of a consensus heptad repeat sequence (Y1S2P3T4S5P6S7). The consensus CTD repeat motif and tandem organization represent the ancestral state of eukaryotic RPB1, but across eukaryotes CTDs show considerable diversity in repeat organization and sequence content. These differences may reflect lineage-specific CTD functions mediated by protein interactions. Mammalian CTDs contain eight non-consensus repeats with a lysine in the seventh position (K7). Posttranslational acetylation of these sites was recently shown to be required for proper polymerase pausing and regulation of two growth factor-regulated genes.ResultsTo investigate the origins and function of RPB1 CTD acetylation (acRPB1), we computationally reconstructed the evolution of the CTD repeat sequence across eukaryotes and analyzed the evolution and function of genes dysregulated when acRPB1 is disrupted. Modeling the evolutionary dynamics of CTD repeat count and sequence content across diverse eukaryotes revealed an expansion of the CTD in the ancestors of Metazoa. The new CTD repeats introduced the potential for acRPB1 due to the appearance of distal repeats with lysine at position seven. This was followed by a further increase in the number of lysine-containing repeats in developmentally complex clades like Deuterostomia. Mouse genes enriched for acRPB1 occupancy at their promoters and genes with significant expression changes when acRPB1 is disrupted are enriched for several functions, such as growth factor response, gene regulation, cellular adhesion, and vascular development. Genes occupied and regulated by acRPB1 show significant enrichment for evolutionary origins in the early history of eukaryotes through early vertebrates.ConclusionsOur combined functional and evolutionary analyses show that RPB1 CTD acetylation was possible in the early history of animals, and that the K7 content of the CTD expanded in specific developmentally complex metazoan lineages. The functional analysis of genes regulated by acRPB1 highlight functions involved in the origin of and diversification of complex Metazoa. This suggests that acRPB1 may have played a role in the success of animals.

Dataset Information

Connecting mutations of the RNA polymerase II C-terminal domain to complex phenotypic changes using combined gene expression and network analyses.

Publications

Connecting mutations of the RNA polymerase II C-terminal domain to complex phenotypic changes using combined gene expression and network analyses.

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