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. 2014 Jun 20;289(25):17446-52.
doi: 10.1074/jbc.M114.568014. Epub 2014 May 5.

Rpb4 subunit functions mainly in mRNA synthesis by RNA polymerase II

Daniel Schulz  1 , Nicole Pirkl  1 , Elisabeth Lehmann  1 , Patrick Cramer  2
Affiliations

Rpb4 subunit functions mainly in mRNA synthesis by RNA polymerase II

Daniel Schulz et al. J Biol Chem. .

Abstract

RNA polymerase II (Pol II) is the central enzyme that carries out eukaryotic mRNA transcription and consists of a 10-subunit catalytic core and a subcomplex of subunits Rpb4 and Rpb7 (Rpb4/7). Rpb4/7 has been proposed to dissociate from Pol II, enter the cytoplasm, and function there in mRNA translation and degradation. Here we provide evidence that Rpb4 mainly functions in nuclear mRNA synthesis by Pol II, as well as evidence arguing against an important cytoplasmic role in mRNA degradation. We used metabolic RNA labeling and comparative Dynamic Transcriptome Analysis to show that Rpb4 deletion in Saccharomyces cerevisiae causes a drastic defect in mRNA synthesis that is compensated by down-regulation of mRNA degradation, resulting in mRNA level buffering. Deletion of Rpb4 can be rescued by covalent fusion of Rpb4 to the Pol II core subunit Rpb2, which largely restores mRNA synthesis and degradation defects caused by Rpb4 deletion. Thus, Rpb4 is a bona fide Pol II core subunit that functions mainly in mRNA synthesis.

Keywords: RNA Metabolism; RNA Polymerase II; RNA Turnover; Transcription; mRNA Decay; mRNA Degradation.

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Figures

FIGURE 1.
FIGURE 1.
Rpb4 deletion causes severe defects in mRNA metabolism. A, labeled RNA expression -fold changes in Δrpb4a and the Δrpb4 of genes plotted against their position in chromosomes. Black lines represent the median expression of all genes of the respective chromosome. B, colony growth on YPD plates for the freshly prepared Δrpb4 and the Euroscarf Δrpb4a strain. Cells were plated from cryo-cultures and incubated for 4 days at 30 °C. C, -fold changes in DRs (log folds, x axis) and SRs (log folds, y axis) for the Euroscarf Δrpb4a mutant against the isogenic wild type. Each point corresponds to one mRNA, and the density of points is encoded by their brightness. Red contour lines define regions of equal density. The center of the distribution is shifted from the origin, indicating a global shift in median SRs and DRs (SR shift is reflected by red line relative to dashed x axis line; DR shift is reflected by red line relative to dashed y axis line). D, DR and SR -fold changes for the freshly prepared Δrpb4 mutant as in C. E, median -fold changes of total RNA levels (TE), mRNA DRs, and mRNA SRs for the Δrpb4 (black bars) and the Δrpb4a (gray bars) mutants over wild type determined from cDTA. The dashed gray line reflects the respective wild-type value, which is normalized to one. Error bars indicate S.D.
FIGURE 2.
FIGURE 2.
Covalent fusion of Rpb2 and Rpb4 compensates growth defects of Rpb4 deletion. A, the C terminus of Rpb2 (cyan) lies in close proximity to the N terminus of Rpb4 (yellow) and suggested the possibility of direct fusion of the two proteins with a linker containing three glycine residues. B, Western blot against Rpb4 for the wild type, the Rpb2-Rpb4 fusion mutant with free Rpb4, and the fusion mutant after genomic deletion of Rpb4. An asterisk marks low amounts of various N-terminal degradation products of the fusion protein that do not lead to significant amounts of free Rpb4. C, growth curves of wild-type, fusion mutant, and Δrpb4 strains in culture at 30 °C. Doubling times were determined from the slope during logarithmic growth. OD, optical density. D, cultures of wild-type, Rpb2–4 fusion, and Δrpb4 cells were grown to the logarithmic phase at 30 °C. For each strain, 1 A600 was spotted on plates in 10-fold serial dilutions on YPD plates. Plates were incubated either at 30 or 37 °C for 2–3 days. E, immunostaining of Rpb4 (green signal) in wild-type cells. F, colocalization of the nucleus stained with DAPI (blue signal) and Rpb4 (green signal) in wild-type cells. G, nuclei stained with DAPI in wild-type cells. H–J, immunostaining as in E–G, for the Rpb2-Rpb4 fusion mutant reveals a major nuclear localization.
FIGURE 3.
FIGURE 3.
Rpb2-Rpb4 fusion rescues SR and DR defects of Rpb4 deletion. A, -fold changes in DRs and SRs for the Rpb2-Rpb4 fusion mutant as in Fig. 1C. The gray ellipse reflects the 95% confidence region of the median SR and DR of the wild type (1). B, median -fold changes of total RNA expression (TE), mRNA DRs, and mRNA SRs for the Rpb2-Rpb4 fusion mutant (gray bars). For comparison the values for Δrpb4 were plotted again (black bars). The dashed gray line reflects the respective wild-type value, which is normalized to one. Error bars indicate S.D. C, Western blot of Rpb1, Rpb3, Rpb4, and tubulin (loading control) in wild-type and Rpb2-Rpb4 fusion mutant cells.
FIGURE 4.
FIGURE 4.
Rpb2-Rpb4 fusion mutant cells are fully capable of adapting to 37 °C. A, growth curves of wild-type and Rpb2-Rpb4 fusion cells at 37 °C in culture. Doubling times were calculated from the slope during logarithmic growth. OD, optical density. B, median -fold changes of total RNA expression (TE), mRNA DRs, and mRNA SRs between the Rpb2-Rpb4 fusion mutant at 30 and 37 °C (gray bars) and between the wild type at 30 and 37 °C (black bars). The dashed gray line reflects the respective wild-type value that is normalized to one. Error bars indicate S.D. C, -fold changes in DRs and SRs for the Rpb2-Rpb4 fusion at 37 °C as compared with the wild type at 37 °C. D, heat map comparing DRs between Rpb2-Rpb4 fusion and wild type at 30 and 37 °C. Transcripts colored in blue show lower DRs, and transcripts colored in red show higher DRs. Differences are shown as variation from the median global -fold change after median centering.
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