Mammalian Rrn3 is required for the formation of a transcription competent preinitiation complex containing RNA polymerase I

Abstract

Mammalian Rrn3, an essential, polymerase-associated protein, is inactivated when cells are treated with cycloheximide, resulting in the inhibition of transcription by RNA polymerase I. Although Rrn3 is essential for transcription, its function in rDNA transcription has not been determined. For example, it is unclear whether Rrn3 is required for initiation or elongation by RNA polymerase I. Rrn3 has been shown to interact with the 43-kDa subunit of RNA polymerase I and with two of the subunits of SL1. In the current model for transcription, Rrn3 functions to recruit RNA polymerase I to the committed complex formed by SL1 and the rDNA promoter. To examine the question as to whether Rrn3 is required for the recruitment of RNA polymerase I to the template, we developed a novel assay similar to chromatin immunoprecipitation assays. We found that RNA polymerase I can be recruited to a template in the absence of active Rrn3. However, that complex will not initiate transcription, even after Rrn3 is added to the reaction. Interestingly, the complex that forms in the presence of active Rrn3 is biochemically distinguishable from that which forms in the absence of active Rrn3. For example, the functional complex is fivefold more resistant to heparin than that which forms in the absence of Rrn3. Our data demonstrate that Rrn3 must be present when the committed template complex is forming for transcription to occur.

Figure 1

Rrn3 is required for “productive” recruitment. (A, B) In each reaction, the template indicated (immobilized on magnetic beads) was preincubated with an S100 extract from cells treated with cycloheximide (CHX S100) in the absence or presence of recombinant Rrn3 as indicated. Subsequently, the templates were washed in transcription buffer (gap in the horizontal line), resuspended, and incubated for an additional 15 min at 30°C (Preincubation 2). Following the second wash, the templates were again resuspended and combined as indicated. NTPs including [α-³²P]UTP were then added, and transcription was allowed to proceed for 10 min. Where indicated, Rrn3 was added at the beginning of the transcription reaction. The transcripts were then analyzed by denaturing urea-PAGE and autoradiography. (C) A transcription reaction was carried out after the first wash in the presence of unlabeled NTPs. The templates were then washed and pooled, and transcription was allowed to proceed in the presence of NTPs including [α-³²P]UTP. All templates were generated by PCR and immobilized on avidin-magnetic beads through a biotin incorporated in a common 5′ primer and were distinguished by the 3′ PCR primer, resulting in transcripts (Trans.) of either 480 (Template A) or 303 nt (Template B).

Figure 2

Coimmunoprecipation of rDNA with RNA Pol I using anti-FLAG antibody. (A) Schematic illustrating the steps in the PCR assay to detect protein–DNA interactions. (B) Five microliters of S100 from N1S1C3 (lane 2) or NISI cells (lane 3) was incubated with 60 ng of template containing the rDNA promoter. C-20 buffer was added to bring the volume to 30 μl. The incubation was carried out at room temperature for 20 min. Twenty microliters of a 50% suspension of anti-FLAG agarose beads was added and the mixture tumbled at 4°C for 1 h. The beads were washed and bound DNA isolated as described in Materials and Methods. DNA was amplified and electrophoresed on a 1% agarose gel and stained with ethidium bromide. Lane 1 depicts a positive control for the first PCR reaction, and lane 4 a positive control for the second PCR reaction. Lane 5 contains pGEM (Promega) molecular weight markers. (C) S100 extracts from N1S1C3 cells were incubated with either 60 ng of wild-type (lane 3) or pUC 19 DNA (lane 4) as described in (B). After washing the beads, DNA was isolated and amplified with primers specific for either the rDNA template or pUC 19 template. Lane 1 contains a positive control PCR reaction for the wild-type template and lane 2 contains a positive control PCR reaction for the pUC template. (D) S100 extracts from N1S1C3 cells were incubated with 60 ng of both the rDNA and pUC 19 templates as indicated in (B). The DNA immunoprecipatated from the reaction was amplified with primers for both the rDNA and pUC 19 templates. Lane 1 indicates the template input and lane 2 indicates the DNA amplified from this reaction. Primers are described in Materials and Methods. PCR products were electrophoresed on a 1% agarose gel and stained with ethidium bromide. Wild-type template amplifies as a 690-bp fragment and pUC as a 1300-bp fragment.

Figure 3

RNA Pol I from inactive extracts interacts with rDNA. S100 extracts from control N1S1C3 cells (lanes 1–4) or CHX-treated cells (lanes 7–10) were incubated with either wild-type template or pUC 19 template for 20 min at room temperature. Samples were processed and DNA amplified as described in Materials and Methods and the legend to Figure 2. Rrn3 (300 ng) was added as indicated (lanes 4 and 9). The products shown in lanes 5 and 6 are positive PCR controls for the wild-type and pUC templates, respectively.

Figure 4

Transcription causes RNA Pol I to dissociate from the template. (A) S100 extracts from N1S1C3 control cells were incubated with 60 ng of wild-type template for 20 min at room temperature. NTPs (lanes 2 and 4) or NTPs plus 8 μg/ml of heparin were added (lane 6) and the reaction continued for 15 min at either 4°C ((lanes 2 and 3) or 30°C (lanes 1, 4, 5, and 6). Samples were processed and DNA amplified as described in Materials and Methods and the legend to Figure 2. (B) In vitro transcription of S100 extracts from control N1S1C3 cells in the absence (lane 1) or presence of heparin (lanes 2 and 3). Transcription conditions have been previously reported and the products analyzed by urea/PAGE and autoradiography (19). In reactions containing heparin, the NTP mixture contained unlabeled ATP, GTP, and CTP with 1 μCi of [³²P]UTP. (C) S100 extracts from either control N1S1C3 cells (lanes 1–3) or CHX-treated cells (lanes 4–6) were incubated with wild-type template to allow formation of the initiation complex. NTPs were added (lanes 2 and 5) or NTPs plus 8 μg/ml of heparin (lanes 3 and 6) and the reaction incubated at 30°C for 15 min. Samples were processed and the DNA amplified as described in Materials and Methods and the legend to Figure 2.

Figure 5

Preinitiation complexes formed in the presence or absence of Rrn3 differ in their sensitivity to heparin. (A) S100 extracts from either control (lanes 1–5) or CHX-treated (lanes 6–9) N1S1C3 cells were incubated with 60 ng of wild-type template for 15 min at room temperature. Heparin was added at the concentration indicated in the total volume of 20 μl and the incubation continued for an additional 15 min at 30°C. Samples were processed and DNA amplified as described in Materials and Methods and the legend to Figure 2. (B) S100 extracts from CHX-treated N1S1C3 cells, some of which had been preincubated with 300 ng Rrn3 for 20 min (lanes 5-8), were incubated with wild-type template (lanes 1–8) for 20 min at room temperature. Heparin or buffer was then added to the final concentrations indicated in a final volume of 20 μl and the incubation continued for 15 min at 30°C. Samples were processed and DNA amplified as described in Materials and Methods and the legend to Figure 2. (C) RT-PCR fluorescence curves for S100 extracts from control or CHX-treated N1S1C3 cells incubated in the presence or absence of varying amounts of heparin. The experiment was carried out as indicated in (A). Conditions for RT-PCR are as stated in Materials and Methods. The figure shows a representative experiment. Crossover values for a typical experiment were control, 27.68; control plus 1.6 μg/ml heparin, 29.14; CHX plus 1.6 μg/ml heparin, 30.46; control plus 8 μl/ml heparin, 30.75. (D) Graphic representation of fractional change in crossover value of S100 extracts from control and CHX-treated N1S1C3 cells incubated with or without heparin as indicated. The fractional change in crossover values was calculated relative to control S100 value. Values represent the average of three independent experiments ± SD. (E) S100 extracts from CHX-treated N1S1C3 were incubated with heparin or heparin and Rrn3 as described in (B). The fractional change in crossover values was calculated relative to the value with no heparin. Values represent the average of four independent experiments ± SD.

Figure 5

Preinitiation complexes formed in the presence or absence of Rrn3 differ in their sensitivity to heparin. (A) S100 extracts from either control (lanes 1–5) or CHX-treated (lanes 6–9) N1S1C3 cells were incubated with 60 ng of wild-type template for 15 min at room temperature. Heparin was added at the concentration indicated in the total volume of 20 μl and the incubation continued for an additional 15 min at 30°C. Samples were processed and DNA amplified as described in Materials and Methods and the legend to Figure 2. (B) S100 extracts from CHX-treated N1S1C3 cells, some of which had been preincubated with 300 ng Rrn3 for 20 min (lanes 5-8), were incubated with wild-type template (lanes 1–8) for 20 min at room temperature. Heparin or buffer was then added to the final concentrations indicated in a final volume of 20 μl and the incubation continued for 15 min at 30°C. Samples were processed and DNA amplified as described in Materials and Methods and the legend to Figure 2. (C) RT-PCR fluorescence curves for S100 extracts from control or CHX-treated N1S1C3 cells incubated in the presence or absence of varying amounts of heparin. The experiment was carried out as indicated in (A). Conditions for RT-PCR are as stated in Materials and Methods. The figure shows a representative experiment. Crossover values for a typical experiment were control, 27.68; control plus 1.6 μg/ml heparin, 29.14; CHX plus 1.6 μg/ml heparin, 30.46; control plus 8 μl/ml heparin, 30.75. (D) Graphic representation of fractional change in crossover value of S100 extracts from control and CHX-treated N1S1C3 cells incubated with or without heparin as indicated. The fractional change in crossover values was calculated relative to control S100 value. Values represent the average of three independent experiments ± SD. (E) S100 extracts from CHX-treated N1S1C3 were incubated with heparin or heparin and Rrn3 as described in (B). The fractional change in crossover values was calculated relative to the value with no heparin. Values represent the average of four independent experiments ± SD.

Figure 6

Preinitiation complexes, formed in the presence or absence of Rrn3, differ in their sensitivity to sarkosyl. Reactions containing S100 extracts from control (lanes 1 and 2) or CHX-treated cells (lanes 3–6) were supplemented with sarkosyl (as indicated) to a final concentration of 0.025%. As indicated, the S100 extracts from CHX-treated N1S1C3 cells were preincubated with either 150 ng (lane 5) or 300 ng of Rrn3 (lane 6) at room temperature for 20 min. Sixty nanograms of wild-type template was then added to the reaction and the incubation continued for an additional 20 min at room temperature. Samples were processed and DNA amplified as described in Materials and Methods and the legend to Figure 2.

Figure 7

Rrn3 does not bind to RNA Pol I that has been recruited in the absence of active Rrn3. (A) Schematic of the reactions shown in (B) and (C). Each preincubation was done for 20 min at RT followed by immunoprecipitation with anti-FLAG agarose beads. (B) S100 (120 μl) from CHX-treated NISI cells was incubated with either 1.5 μg FLAG-tagged Rrn3 (lanes 2 and 3) or 500 ng of DNA (lanes 4 and 5) for 20 min at RT (preincubation 1). DNA (lane 3) or FLAG-tagged Rrn3 (lane 5) was then added to the reactions, and the incubation continued for another 20 min (preincubation 2). Twenty-five microliters of packed anti-FLAG agarose beads was added and the incubation continued for 1 h at 4°C. The beads were eluted with 50 μl of FLAG peptide (500 μg/ml) and the entire eluate fractionated by SDS-PAGE, transferred to PVDP, and probed with an antibody to rpa43. (C) S100 (5 μl) from CHX-treated NISI cells was incubated with either 0.3 μg FLAG-tagged Rrn3 (lanes 2 and 3) or 50 ng of template DNA (lanes 4 and 5) as indicated (preincubation 1). After a second incubation supplemented with either DNA (lane 3) or Rrn3 (lane 5) as indicated in the schematic, 10 μl of packed anti-FLAG agarose beads was added, and the incubation continued for 1 h at 4°C. Following that incubation, the DNA was isolated and amplified as described in Materials and Methods and the legend to Figure 2.