Rpb7 can interact with RNA polymerase II and support transcription during some stresses independently of Rpb4

Abstract

Rpb4 and Rpb7 are two yeast RNA polymerase II (Pol II) subunits whose mechanistic roles have recently started to be deciphered. Although previous data suggest that Rpb7 can stably interact with Pol II only as a heterodimer with Rpb4, RPB7 is essential for viability, whereas RPB4 is essential only during some stress conditions. To resolve this discrepancy and to gain a better understanding of the mode of action of Rpb4, we took advantage of the inability of cells lacking RPB4 (rpb4Delta, containing Pol IIDelta4) to grow above 30 degrees C and screened for genes whose overexpression could suppress this defect. We thus discovered that overexpression of RPB7 could suppress the inability of rpb4Delta cells to grow at 34 degrees C (a relatively mild temperature stress) but not at higher temperatures. Overexpression of RPB7 could also partially suppress the cold sensitivity of rpb4Delta strains and fully suppress their inability to survive a long starvation period (stationary phase). Notably, however, overexpression of RPB4 could not override the requirement for RPB7. Consistent with the growth phenotype, overexpression of RPB7 could suppress the transcriptional defect characteristic of rpb4Delta cells during the mild, but not during a more severe, heat shock. We also demonstrated, through two reciprocal coimmunoprecipitation experiments, a stable interaction of the overproduced Rpb7 with Pol IIDelta4. Nevertheless, fewer Rpb7 molecules interacted with Pol IIDelta4 than with wild-type Pol II. Thus, a major role of Rpb4 is to augment the interaction of Rpb7 with Pol II. We suggest that Pol IIDelta4 contains a small amount of Rpb7 that is sufficient to support transcription only under nonstress conditions. When RPB7 is overexpressed, more Rpb7 assembles with Pol IIDelta4, enough to permit appropriate transcription also under some stress conditions.

FIG. 1

Overexpression of RPB7 suppresses the growth defect of rpb4Δ cells at 34°C independently of the genetic background. A high-copy-number suppression approach was taken, with a yeast cDNA library under GAL1 promoter in a URA3 plasmid, to select for genes which could rescue rpb4Δ growth defect at 34°C (see Results). RPB7 was thus selected. pGAL1-RPB7 from one of the positive transformants was recovered, propagated in Escherichia coli, and introduced into MC11-1 or MC12-1 (see genotypes in Table 1) by the lithium acetate transformation method (see Materials and Methods). URA3 colonies were selected at 25°C. Transformants were then allowed to grow at 34°C on either YPG (inducing condition, left plate) or YPD (repressing condition, right plate).

FIG. 2

Overexpression of RPB7 suppresses the growth defect of rpb4Δ cells at both high and low temperatures. SUB62 (WT), MC11-1 (rpb4Δ), and AS33 (rpb4Δ strain carrying pMC116 [tetp-RPB7 URA3 plasmid]) were streaked onto YPD plates lacking tetracycline (designated “−tet” at the left of the figure), or containing 20 μg of tetracycline per ml (designated “+tet” at the left). Plates were incubated at the various temperatures indicated at the top. Pictures were obtained either after 3 days of incubation (at 24, 34, and 37°C) or after 7 days (at 9 and 13°C). After the 9°C picture was taken, the wild-type colonies continued to grow, whereas those of the other strains did not. The inset (lower left) shows a Western analysis (see Materials and Methods) of 50-μg extracts probed with anti-Rpb7 antibodies. Lanes: 1, extract from AS33 which had been grown on YPD lacking tetracycline; 2, extract from AS33 which had been grown on YPD containing 20 μg of tetracycline per ml; 3, extract from SUB62 cells which had been grown on YPD lacking tetracycline. The band seen above Rpb7 and marked by an arrowhead is nonspecific. It is shown to demonstrate equal loading.

FIG. 3

Overexpression of RPB7 prevents the accelerated death of rpb4Δ cells during starvation in stationary phase. AS1 (rpb4Δ strain overexpressing RPB7) cells were grown at 28°C on SC medium lacking uracyl and containing 2% galactose. SUB62 (wild-type) and MC11-1 (rpb4Δ) cells were grown in the same medium supplemented with uracyl. The viability was determined, at various time points after entry into stationary phase, by evaluating the plating efficiency on YPG at 25°C. Symbols: ■, SUB62; ⧫, MC11-1; ▴, AS1. The graph represents an average of three different experiments. The deviation was less than 25%.

FIG. 4

Overexpressed RPB4 cannot replace RPB7. WY-73 (A), AS25 (B), and AS24 (C), whose genotypes are depicted in the left panel, were grown on appropriate selective media until mid-log phase. Similar numbers of cells were then streaked on either a selective plate lacking uracyl or on an SC plate containing 50 μg of 5-FOA as indicated at the top of the figure. Plates were incubated at 30°C for 3 days (SD-uracil plate) or for 5 days (5-FOA plate). Other experiments, similar to the one described here except that the plates were incubated at various temperatures, ranging from 25 to 34°C, gave similar results.

FIG. 5

Coimmunoprecipitation of the overproduced Rpb7 with Pol IIΔ4. (A) IP of Pol II with anti-Rpb1. Equal amounts of protein (200 μg), extracted from logarithmically growing SUB62 (wild-type) or AS1 (rpb4Δ GAL1p-RPB7) cells, were used for IP with the anti-C-terminal-domain antibody of Rpb1 (8WG16 MAb). These antibodies were chosen because they had already been demonstrated to be suitable for IP of the full complement of Pol II, including stoichiometric amounts of Rpb4 and Rpb7 (7, 9). IP followed by Western analysis was carried out as described in Materials and Methods. Subunits detected by specific antibodies (see Materials and Methods) are marked by arrows. Inclusion of nonspecific antibodies, instead of 8WG16 MAb, produced no detectable signals of Rpb1, Rpb4, or Rpb7 (results not shown). Lanes 1 (SUB62) and 2 (AS1) show the supernatant (S/N) left after the IP (due to technical limitations only 20 of 200 μg was loaded onto the gel). Lane 4 shows the IP of SUB62; lane 5 shows the IP of AS1. Lane 3 shows a 1:10 dilution of the material used in lane 4 in order to estimate the ratio between the Rpb7 band intensity in lane 5 to that in lane 4. (B) IP of Pol II with anti-myc antibodies that recognize the Rpb7-myc2. Equal amounts of protein (500 μg) extracted from logarithmically growing AS33 (lane 6), AS30 (lanes 7 and 9), and AS35 (lane 8) were taken for IP with 10 μg of the 9E10 MAb as described for panel A. Subunits, detected by the respective specific antibodies (see Materials and Methods), are marked by arrows. Lane 9 shows a 1:10 dilution of the material used in lane 7. This is shown to estimate the ratio between the Rpb1 and Rpb2 band intensities in lane 8 to those in lane 7. The band underneath Rpb1 is nonspecific. The absence of a gene or a plasmid (−), the presence of one copy of a gene (+), or the overexpression of RPB7 or RPB7-myc2 (+++) are indicated.

FIG. 6

Steady-state level of the global poly(A)⁺ mRNA during mild and severe HS: effect of RPB4 deletion and overexpression of RPB7. SUB62 (wild-type), MC11-1 (rpb4Δ), and AS1 (rpb4Δ GAL1p-RPB7) strains were grown at 22°C on galactose-containing medium as described in Materials and Methods. In mid-log phase (at 10⁷ cells/ml), cells were shifted to either 34 or 39°C and harvested at the indicated time points after the shift. RNA was extracted, and 1 μg of the RNA samples was dot blotted onto nitrocellulose filter in duplicate. One set of dots was hybridized with ³²P-labeled poly(dT) to detect the poly(A)-containing mRNA (designated mRNA at the left or right of the figure) and the other set was hybridized with ³²P-labeled rDNA to detect the rRNA (designated rRNA at the left or right of the figure). This procedure was described in detail previously (5). The absence of a gene (−), the presence of one copy of a gene (+), and the overexpression of a gene (+++) are indicated.

FIG. 7

Levels of specific mRNAs during mild or severe HS: effect of RPB4 deletion and overexpression of RPB7. Strains, cell growth, HS at 34°C (A) or at 39°C (B), and RNA extraction procedures were as described for Fig. 6. RNA samples (8 μg) were analyzed by Northern blot hybridization as described previously (5, 7). The application and transfer of equal amounts of RNA was verified by ethidium bromide staining (shown at the bottom). The same filter was used for sequential hybridization with the indicated DNA probes. The absence of a gene (−), the presence of one copy of a gene (+), and the overexpression of a gene (+++) are indicated.