Unknown,Transcriptomics,Genomics,Proteomics

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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. 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.

ORGANISM(S): Saccharomyces cerevisiae

SUBMITTER: Marian Groot Koerkamp

PROVIDER: E-GEOD-47429 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The Saccharomyces cerevisiae Slx5/8 complex is the founding member of a recently defined class of ubiquitin ligases (STUbLs) that target sumoylated substrates. Slx5/8 has been implicated in genome stability and transcription, but the precise contribution to these processes is not clear. To characterize Slx5/8 function, we determined genome-wide changes in gene expression upon loss of either subunit. The majority of mRNA changes are part of a general stress response, also exhibited by mutants of other genome integrity pathways and therefore indicative of an indirect effect on transcription. Genome-wide binding analysis reveals a uniquely centromeric location for Slx5. Detailed phenotype analyses of slx5D and slx8D mutants show severe mitotic defects that include aneuploidy, spindle mispositioning, fish hook spindles and aberrant spindle kinetics. This is associated with accumulation of the PP2A regulatory subunit Rts1 at centromeres prior to entry into anaphase. Knockdown of the human STUbL orthologue RNF4 also results in chromosome segregation defects due to formation of chromosome bridges. The study shows that STUbLs have a conserved role in maintenance of chromosomal stability and the results link SUMO-dependent ubiquitination to a centromere-specific function during mitosis. 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.

Project description:To understand the organisation of the glucose regulatory system, we analysed 91 deletion mutants of established glucose signalling and metabolic pathway members in Saccharomyces cerevisiae by DNA microarrays. These deletion mutants do not induce pathway-specific transcriptional responses reflecting the tight interconnection between pathways of the glucose regulatory system. Instead, one main transcriptional response is discerned, which varies in direction to mimic either a high or a low glucose response. The study reveals both known and unknown relationships within and between individual pathways and their members. Metabolic components of the glucose regulatory system are most frequently affected at the transcriptional level. A new network approach is applied that exposes the hierarchical organisation of the glucose regulatory system. Tps2 and Tsl1, two enzymes involved in trehalose biosynthesis, are predicted to be the most downstream transcriptional components. This prediction is further validated by epistasis analysis of Tps2 double mutants. Taken together, this suggests that changes in perceived glucose levels ultimately lead to a shift in trehalose biosynthesis. RNA isolated from a large amount of wt yeast from a single culture was used as a common reference. This common reference was used for each separate 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. Up to five deletion strains were grown on a single day. Wt cultures were grown parallel to the deletion mutants to assess day-to-day variance.

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

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