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. 2002 Oct;1(5):830-42.
doi: 10.1128/EC.1.5.830-842.2002.

The yeast pafl-rNA polymerase II complex is required for full expression of a subset of cell cycle-regulated genes

Stephanie E Porter  1 , Taylor M Washburn, Meiping Chang, Judith A Jaehning
Affiliations

The yeast pafl-rNA polymerase II complex is required for full expression of a subset of cell cycle-regulated genes

Stephanie E Porter et al. Eukaryot Cell. 2002 Oct.

Abstract

We have previously described an alternative form of RNA polymerase II in yeast lacking the Srb and Med proteins but including Pafl, Cdc73, Hprl, and Ccr4. The Pafl-RNA polymerase II complex (Paf1 complex) acts in the same pathway as the Pkc1-mitogen-activated protein kinase cascade and is required for full expression of many cell wall biosynthetic genes. The expression of several of these cell integrity genes, as well as many other Paf1-requiring genes identified by differential display and microarray analyses, is regulated during the cell cycle. To determine whether the Paf1 complex is required for basal or cyclic expression of these genes, we assayed transcript abundance throughout the cell cycle. We found that transcript abundance for a subset of cell cycle-regulated genes, including CLN1, HO, RNR1, and FAR1, is reduced from 2- to 13-fold in a paf1delta strain, but that this reduction is not promoter dependent. Despite the decreased expression levels, cyclic expression is still observed. We also examined the possibility that the Paf1 complex acts in the same pathway as either SBF (Swi4/Swi6) or MBF (Mbp1/Swi6), the partially redundant cell cycle transcription factors. Consistent with the possibility that they have overlapping essential functions, we found that loss of Paf1 is lethal in combination with loss of Swi4 or Swi6. In addition, overexpression of either Swi4 or Mbp1 suppresses some paf1delta phenotypes. These data establish that the Paf1 complex plays an important role in the essential regulatory pathway controlled by SBF and MBF.

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Figures

FIG. 1.
FIG. 1.
Deletion of the PAF1 gene causes a decrease in mRNA levels for many cell cycle-regulated genes. Asynchronous cultures of wild type (YJJ662) and paf1Δ (YJJ664) cells were grown to a Klett density of 60 (2 × 106 cells/ml), total RNA was harvested, and 10 μg of RNA per lane was fractionated and used to detect mRNAs for specific cell cycle-regulated genes as described in Materials and Methods. The cycle peak designations are from the microarray analysis of Spellman et al. (; http://genome-www.stanford.edu/cellcycle/). The fold decrease of each mRNA in the paf1Δ strain compared to that in the wild type was calculated from an average of at least two experiments after normalization to 18S rRNA. RPS9A is an example of a gene that decreases in paf1Δ but is not cell cycle regulated. PIR3 is an example of a cell cycle-regulated gene that does not decrease in a paf1Δ strain. Results for two different strain backgrounds are shown for PIR3. The other mRNAs shown in this figure were from the D273-10b background. Similar results were obtained from both strains for the other mRNAs shown in this figure.
FIG. 2.
FIG. 2.
Synchronized wild-type, paf1Δ, swi4Δ, and mbp1Δ strains have similar budding profiles. Strains were synchronized with α-factor as described in Materials and Methods. Samples were taken at 15-min intervals, and the number of small budded cells in each sample was determined. Data are graphed as the percentage of cells with small buds versus doublings, rather than time, to directly compare the budding indices of the slow-growing paf1Δ strain to the other strains. Doublings for the wild type (YJJ755), swi4Δ (YJJ1173), and mbp1Δ (YJJ1239) were calculated by dividing the time, in hours, of each time point by 1.5 h. Doublings for paf1Δ (YJJ756) were calculated by dividing the time, in hours, of each time point by 3 h.
FIG. 3.
FIG. 3.
mRNA abundance for cycling genes is decreased throughout the cell cycle in the paf1Δ strain. Fractionated total RNA from synchronized wild-type (YJJ755) and paf1Δ (YJJ756) cells was analyzed with probes (see Table 2) that detect periodic genes that peak in the indicated parts of the cell cycle. PIR3 expression peaks in M/G1 but is not affected by lack of Paf1. Each graph shows the normalized amount of mRNA for that gene over a period of 1.5 doublings for wild-type and paf1Δ strains. The mRNA/18S rRNA units are arbitrary phosphorimager numbers and are not comparable between probes. However, each individual panel represents equal amounts of RNA from wild-type and paf1Δ cells fractionated on the same gel for direct comparison.
FIG. 4.
FIG. 4.
Cell cycle-regulated genes still cycle in paf1Δ. Representative data from Fig. 3 showing the cell cycle profiles from paf1Δ (YJJ756) cells of three G1-phase genes (CLN1, RNR1, and HO) normalized to 18S rRNA are shown here on an expanded scale. Profile of the non-cell-cycle-regulated gene RPS9A is shown for comparison.
FIG. 5.
FIG. 5.
Gene expression profiles of swi4Δ and mbp1Δ strains are not identical to that of a paf1Δ strain. Asynchronous cultures of wild type (YJJ662), paf1Δ (YJJ664), swi4Δ (YJJ1000), and mbp1Δ (YJJ1067) cells were grown to a Klett density of 60 (2 × 106 cells/ml). Total RNA was harvested, 10 μg of RNA per lane was fractionated, and specific mRNAs were detected using probes for cell cycle-regulated genes and normalized to 18S rRNA. The fold change of each mRNA in paf1Δ, swi4Δ, and mbp1Δ strains compared to that for the wild type is an average of at least two experiments. PIR3 is a cell cycle-regulated gene that does not decrease in a paf1Δ strain (see Fig. 1). Note that the asynchronous samples shown in this figure were from the D273-10b genetic background; similar results were obtained in the A364a background.
FIG. 6.
FIG. 6.
The gene expression profiles of swi4Δ and mbp1Δ strains do not mimic the cycling profiles seen in a paf1Δ strain. RNA from synchronized wild-type (YJJ755), paf1Δ (YJJ756), swi4Δ (YJJ1173), and mbp1Δ (YJJ1239) cells was fractionated and probed for the indicated transcripts. The graphs show the amount of normalized mRNA for the indicated genes over a period of 1.5 doublings for wild-type versus paf1Δ, swi4Δ, and mbp1Δ strains. The mRNA/18S rRNA units are arbitrary phosphorimager counts and are not comparable between probes. However, each individual panel represents equal amounts of RNA from wild type and the indicated mutant strains fractionated on the same gels for a direct comparison.
FIG. 7.
FIG. 7.
paf1Δ swi4Δ and paf1Δ swi6Δ, but not paf1Δ mbp1Δ, are synthetically lethal. Heterozygous diploids containing paf1Δ and swi4Δ, paf1Δ and swi6Δ, or paf1Δ and mbp1Δ mutations were sporulated, and tetrads were dissected onto YPD plates containing 1 M sorbitol and incubated at 30°C. Tetrads were also dissected onto YPD plates and onto YPD plates at low temperature (23°C) with similar results. Representative tetrads are shown. Spore genotypes are indicated below each tetrad: W, wild type; p, paf1Δ; s, swi4Δ or swi6Δ as appropriate; m, mbp1Δ; pm, paf1Δ mbp1Δ. Genotypes were scored as described in Materials and Methods.
FIG. 8.
FIG. 8.
Both Mbp1 and Swi4, when overexpressed, can partially suppress paf1Δ. Spot assays were performed as described in Materials and Methods. WT + v1, YJJ662 transformed with YEp24; paf1Δ + v1, YJJ664 transformed with YEp24; paf1Δ + Mbp1, YJJ664 transformed with BK72; paf1Δ + v2, YJJ664 transformed with YEp352; paf1Δ + Swi4, YJJ664 transformed with B327. Spots from left to right were made by applying 4 μl of a cell suspension containing 107, 106, 105, 104, and 103 cells/ml. Similar results were obtained with two independent sets of transformants.
FIG. 9.
FIG. 9.
Output of promoter/reporter constructs does not correlate with measurements of RNA abundance. Constructs containing the GLK1, FKS1, and CLN1 promoters driving expression of the luciferase gene were transformed into wild-type (YJJ662) and paf1Δ (YJJ664) strains. The FKS1 and CLN1 constructs were also transformed into swi4Δ (YJJ1233) and mbp1Δ (YJJ1067) strains. Extracts were made and luciferase activity was measured as described in Materials and Methods. The bars represent the average and standard error derived from two independent experiments, with the activity of each construct measured in quadruplicate in each assay. Data are presented as relative luciferase units per milligram of total protein from the extract.
FIG. 10.
FIG. 10.
The Paf1 complex is required for full expression of many SBF- and MBF-regulated genes. The model, described further in the Discussion, summarizes the genetic and molecular analyses in this work. Mutation of PAF1 results in reduced expression of Swi4 targets like CLN1 and Mbp1 targets like RNR1. The synthetic lethality of paf1Δ in combination with swi4Δ or swi6Δ indicates that the Paf1 complex has essential functions that are redundant with these factors. High-copy suppression of paf1Δ phenotypes by SWI4 and MBP1 is consistent with these factors acting downstream of the Paf1 complex, or at a different stage of transcription.
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