Analysis of a Splice Array Experiment Elucidates Roles of Chromatin Elongation Factor Spt4–5 in Splicing

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Abstract
Splicing is an important process for regulation of gene expression in eukaryotes, and it has important functional links to other steps of gene expression. Two examples of these linkages include Ceg1, a component of the mRNA capping enzyme, and the chromatin elongation factors Spt4–5, both of which have recently been shown to play a role in the normal splicing of several genes in the yeast Saccharomyces cerevisiae. Using a genomic approach to characterize the roles of Spt4–5 in splicing, we used splicing-sensitive DNA microarrays to identify specific sets of genes that are mis-spliced in ceg1, spt4, and spt5 mutants. In the context of a complex, nested, experimental design featuring 22 dye-swap array hybridizations, comprising both biological and technical replicates, we applied five appropriate statistical models for assessing differential expression between wild-type and the mutants. To refine selection of differential expression genes, we then used a robust model-synthesizing approach, Differential Expression via Distance Synthesis, to integrate all five models. The resultant list of differentially expressed genes was then further analyzed with regard to select attributes: we found that highly transcribed genes with long introns were most sensitive to spt mutations. QPCR confirmation of differential expression was established for the limited number of genes evaluated. In this paper, we showcase splicing array technology, as well as powerful, yet general, statistical methodology for assessing differential expression, in the context of a real, complex experimental design. Our results suggest that the Spt4–Spt5 complex may help coordinate splicing with transcription under conditions that present kinetic challenges to spliceosome assembly or function. Splicing is a key process for the regulation of gene expression in eukaryotes and is credited as being the main reason for the extraordinary complexity of the human proteome relative to the human genome. Accurate splicing is crucial for normal protein function; aberrant transcripts due to splicing mutations are known causes for 15% of genetic diseases. Therefore, elucidation of splicing mechanisms will not only help in understanding the complexity and diversity of higher organisms, but also potentially aid in new therapeutic strategies for treatments of splicing-related genetic disorders. It has been previously shown that splicing has important links to other steps involved with gene expression. In this study, the authors pursue a genome-wide approach, using yeast-based, splicing-sensitive, DNA microarrays in order to further characterize the roles of select splicing factors. They devise novel statistical and computational methods that enable identification of specific sets of genes that are mis-spliced in the chosen splicing factors. Follow-up investigation of known attributes of the genes so elicited indicates that these factors may help coordinate splicing and transcription in situations where additional energy is required to effect splicing.