Probing the integration of steps in the gene expression pathway through analysis of the Spt4-Spt5 transcription elongation complex in Saccharomyces cerevisiae

Eukaryotic gene expression involves a series of complex, dynamic events, requiring the coordinated activities of numerous macromolecular machines. Gene expression begins with transcription, the process by which RNA is synthesized from a DNA template, resulting in the transfer of genetic information from genome to RNA. Transcription can both directly and indirectly affect numerous other gene expression events such as chromatin remodeling and RNA processing and export, though the exact nature of these connections is still not well understood. In order to better understand the mechanism of transcription and its role in RNA metabolism, we have chosen to study the Spt4-Spt5 complex in the budding yeast Saccharomyces cerevisiae. Functionally conserved from yeast through humans, Spt4-Spt5 regulates transcription through its association with elongating RNA Polymerase II.; We first asked if Spt4-Spt5 plays a role in pre-mRNA processing. We find that it physically and genetically interacts with the mRNA capping machinery in yeast, and that spt4 and spt5 mutants accumulate intron-containing transcripts. Using splicing-sensitive DNA microarrays, we find that proper splicing of specific subsets of intron-containing genes requires functional Spt4-Spt5. The characteristics of these genes suggest that Spt4-Spt5 plays a role in coordinating splicing with transcription under kinetically challenging conditions in vivo. Preliminary in vitro analysis suggests a possible direct participation of Spt5 in splicing reactions.; We next extended our genome-wide analysis to include mutations in components of all steps of the gene expression pathway, from transcription through translation and decay of mRNA. Using hierarchical clustering and machine-learning techniques to recognize gene expression phenotypes caused by these mutations provides insight into how the major machineries of the gene expression pathway communicate with one another. Additionally, this analysis reveals functional roles for specific factors at multiple steps in the gene expression pathway. Similar gene expression phenotypes for transcription elongation and mRNA export factor mutants suggest a role for Spt4-Spt5 in nuclear mRNA export. Genetic and mRNA visualization analysis confirms this prediction. Taken together these data provide evidence for an important role for Spt4-Spt5 in the temporal and functional coupling between molecules required for proper gene expression.