CTD kinase I is involved in RNA polymerase I transcription

Abstract

RNA polymerase II carboxy terminal domain (CTD) kinases are key elements in the control of mRNA synthesis. Yeast CTD kinase I (CTDK-I), is a non-essential complex involved in the regulation of mRNA synthesis at the level of transcription elongation, pre-mRNA 3' formation and nuclear export. Here, we report that CTDK-I is also involved in ribosomal RNA synthesis. We show that CTDK-I is localized in part in the nucleolus. In its absence, nucleolar structure and RNA polymerase I transcription are affected. In vitro experiments show an impairment of the Pol I transcription machinery. Remarkably, RNA polymerase I co-precipitates from cellular extracts with Ctk1, the kinase subunit of the CTDK-I complex. In vitro analysis further demonstrates a direct interaction between RNA polymerase I and Ctk1. The results suggest that CTDK-I might participate in the regulation of distinct nuclear transcriptional machineries, thus playing a role in the adaptation of the global transcriptional response to growth signalling.

Figure 1

Ctk1 interacts with Pol I in vivo and in vitro. (A and B) Ctk1–Myc purification. (A) Fractionation scheme used to purify Ctk1–Myc from yeast exponential cells by ion exchange chromatographies. (B) The different fractions recovered were analysed by western blot with anti-Myc antibodies (upper panel), and polyclonal antibodies raised against Pol I (lower panel). I and FT indicate input and flow through samples, respectively. (C) Ctk1–HA immunopurification. After the second chromatography, fractions that contained the minor elution peak of Ctk1–HA from CTK1–HA cell extract, and identical fractions from wild-type (Ctk1) cell extract, were pooled and subjected to HA immuno-affinity purification. Eluates were analysed by western blot with anti-HA (left panel), anti-Pol I (right upper panel) and anti-Rrn3 (right lower panel) antibodies. (D and E) In vitro co-immunoprecipitation experiments. (D) Pol I, Pol IΔ and Pol I^* purified from yeast cell were analysed by silver staining. A43^* corresponds to a degraded form of the A43 subunit. (E) Pol I was incubated with different combinations of recombinant Ctk proteins each tagged with 2 HA epitopes (HA-rCtk). Immunoprecipitation was performed with polyclonal antibodies against the largest subunit of Pol I, A190. Western blot analysis with anti-Pol I (upper panel) and anti-HA (lower panel) antibodies. Asterisk indicates a band corresponding to immunoglobulin heavy chains.

Figure 2

Immunolocalization of the Ctk proteins. Indirect immunofluorescence was performed with: (A and B) CTK1–Myc, (C) CTK2–Myc and (D) RPB3-HA early exponential cells. DNA was stained with DAPI (A), while Ctk1 (A and B), Ctk2 (C) and Rpb3 (D) were detected with mouse monoclonal anti-Myc (A, B and C) or anti-HA (D) antibodies, and A190 (B, C and D) was detected using a rabbit antibody. Three distinct representative nuclei are shown in each case.

Figure 3

Immunofluorescence microscopy analysis of the nucleolus. Wild-type (WT), Δctk1, Δctk3 and rpb3-2 early exponential cells were analysed with either anti-A190 (A) or anti-Nop1 (B) antibodies, and DAPI staining. Three distinct representative nuclei are shown in each case.

Figure 4

(A, B and C) In vivo transcription analyses. (A) Wild-type (WT), Δctk1, Δctk3 and ctk1-mut isogenic cells, were cultivated in SD-casa to early log phase, prior to incubation with ³H-Uracil for 30 min. After extraction, total RNA was separated by electrophoresis on urea–polyacrylamide gels prior to auto-radiography (upper panel) or ethidium bromide staining (lower panel). (B and C) Wild-type (WT), Δpdr13 and Δctk1 isogenic cells, were cultivated in SD-casa to an OD of either 1 or 2. After extraction, total RNA was analysed by primer extension with two oligonucleotides specific for the 35 and 25S rRNA species, respectively. Products were separated by electrophoresis on urea–polyacrylamide gels prior to auto-radiography (B), and 35S products analysed with a PhosphorImager (C). Grey, black and white boxes correspond to 35S amounts in WT, Δpdr13 and Δctk1 cells, respectively. Results correspond to three independent experiments. (D and E) rDNA chromatin immunoprecipitation. After cross-link, cell lysis and sonication, wild-type (WT) and Δctk1 extracts from cells grown in SD-casa to an OD of 2 were immunoprecipitated with anti-A190 antibodies. (D) Recovered proteins were analysed by western immunoblotting with anti-A190 antibodies. (E) Total and immuno-precipitated DNA were analysed by real-time PCR. Fragment corresponding to the 35S rDNA promoter (left panel), and enrichment of the 35S rDNA promoter compared to a non-specific 5S fragment (right panel). Grey and white boxes correspond to WT and Δctk1 extracts, respectively. Results correspond to five independent experiments.

Figure 5

In vitro analyses. (A, B and C) Transcription assay. (A) Pol I from wild-type (WT) and Δctk1 PA600 fractions were analysed by western blot with anti-Pol I antibodies. (B) Specific in vitro transcription of a mini rDNA transcription unit was carried out with PA600 fractions purified from wild-type (WT), Δpdr13 and Δctk1 strains. (C) Specific in vitro transcription with different combinations of wild-type (WT) and Δctk1 PA600 fractions. (D and E) In vitro binding of Pol I to the 35S rDNA promoter. (D) After incubation with immobilized templates containing (pSIRT), or not (Δprom), the 35S rDNA promoter, bound proteins were eluted from released templates and analysed by western immunoblot with anti-Pol I antibodies. (E) Quantification of three independent experiments. Grey boxes, Δprom template; white boxes, pSIRT template.