Ithaca College

Maki Inada. Investigating the Role of Phosphorylation Sites of the RNA Polymerase II CTD Code on Global Gene Expression in Schizosaccharomyces pombe. RNA 2012 conference. Ann Arbor, MI. May 2012. Co-authored with Robert Nichols, Jeffrey Pleiss, Beate Schwer.

ABSTRACT:  

Regulation of gene expression is essential for all living organisms. In eukaryotes, genetic information stored in the form of DNA must first be transcribed to a transient messenger RNA molecule that is highly processed prior to translation to functional protein. The structure of the transcriptional enzyme RNA polymerase II is believed to play a critical role in modulating gene expression. Specifically, the dynamic phosphorylation pattern on the carboxy terminal domain (CTD) of RNA polymerase II has been demonstrated to aid in the recruitment of numerous nuclear factors involved in RNA processing. Conserved throughout evolution, the Y1S2P3T4S5P6S7 heptapeptide repeat can be phosphorylated at any of the serines at positions 2, 5, and 7. The modification of residues of the CTD repeats is thought to create a readable ‘code’ for controlling transcription initiation, elongation, termination, changes in chromatin structure, and regulation of capping, splicing and polyadenylation. In order to characterize how the absence of specific phosphorylation marks in the CTD affect gene expression, mutants of fission yeast Schizosacchromyces pombe were rendered defective for phosphorylation by substituting alanines for serines at either positions 2 and 7 individually or in combination, S2A, S7A, and S2A/S7A respectively. Previous studies demonstrated mutants lacking some of these phosphorylation events were deficient in the expression of genes required for sexual differentiation. To examine the genome-wide effects these mutations have on gene expression as well as changes in pre-mRNA splicing, we conducted microarray analyses. We used custom designed splicing sensitive microarrays to allow us to detect upregulation or downregulation of specific introns, exons, or splice junctions for the S. pombe genome. In agreement with previous results, the expression of ste11, which is required for mating in S. pombe, is low in the S2A mutant, whereas levels are restored in the double mutant S2A/S7A. In addition, our microarray analyses reveal numerous changes in expression of both individual genes, as well as positionally related clusters of genes. These results and their consequences will be discussed.