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Kaplan2013: High-Resolution, Quantitative Genetic Analysis of RNA Polymerase II

ABSTRACT: Dynamic control of gene expression is crucial for most aspects of cell physiology and at its core is RNA polymerase II (RNAPII). Using 53 RNAPII point mutants, we generated a point mutant epistatic miniarray profile (pE-MAP) comprising ~60,000 quantitative genetic interactions in Saccharomyces cerevisiae. This enabled functional assignment of RNAPII sub-domains, including connections to protein complexes. Using splicing microarrays and point mutants altering elongation rates in vitro, we found an inverse relationship between RNAPII speed and in vivo splicing efficiency. Furthermore, the pE-MAP classified groups of fast and slow mutants that favor upstream and downstream start site selection, respectively. Finally, the pE-MAP identified Sub1 as a positive transcription factor regulating start site selection and splicing. These data reveal striking coordination of polymerization rate with transcription initiation and splicing, suggesting transcription rate is tuned to coordinate multiple gene expression steps. The pE-MAP approach provides a powerful strategy to understand other multi-functional machines at amino acid resolution.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE47429 | GEO | 2013/08/06

SECONDARY ACCESSION(S): PRJNA205623

REPOSITORIES: GEO

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Project description:Dynamic control of gene expression is crucial for most aspects of cell physiology and at its core is RNA polymerase II (RNAPII). Using 53 RNAPII point mutants, we generated a point mutant epistatic miniarray profile (pE-MAP) comprising ~60,000 quantitative genetic interactions in Saccharomyces cerevisiae. This enabled functional assignment of RNAPII sub-domains, including connections to protein complexes. Using splicing microarrays and point mutants altering elongation rates in vitro, we found an inverse relationship between RNAPII speed and in vivo splicing efficiency. Furthermore, the pE-MAP classified groups of fast and slow mutants that favor upstream and downstream start site selection, respectively. Finally, the pE-MAP identified Sub1 as a positive transcription factor regulating start site selection and splicing. These data reveal striking coordination of polymerization rate with transcription initiation and splicing, suggesting transcription rate is tuned to coordinate multiple gene expression steps. The pE-MAP approach provides a powerful strategy to understand other multi-functional machines at amino acid resolution. Two channel microarrays were used. RNA isolated from a large amount of wt yeast from a single culture was used as a common reference. This common reference was used in one of the channels for each hybridization and used in the statistical analysis to obtain an average expression-profile for each deletion mutant relative to the wt. Two independent cultures were hybridized on two separate microarrays. For the first hybridization the Cy5 (red) labeled cRNA from the deletion mutant is hybridized together with the Cy3 (green) labeled cRNA from the common reference. For the replicate hybridization, the labels are swapped. Each gene is represented twice on the microarray, resulting in four measurements per mutant. Using the Erlenmeyer growth protocol up to five deletion strains were grown on a single day. In the tecan platereader, up to eleven deletion strains could be grown on a single day. Wt cultures were grown parallel to the deletion mutants to assess day-to-day variance. Strains with point mutations in either rpb1, rpb2 or rpb7 subunits of RNA polymerase II (RNAPII) examined under normal growth conditions. The mutated RNAPII subunit is present on URA3 or LEU2 marked CEN plasmid, and the corresponding genomic RNAPII subunit deleted. Strains labeled as wildtype also have the relevant genomic RNAPII subunit deleted, but complemented with wildtype RNAPII subunit on CEN plasmid.

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Kaplan2013: High-Resolution, Quantitative Genetic Analysis of RNA Polymerase II

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