The RNA polymerase II CTD kinase Ctk1 functions in translation elongation

Abstract

Translation is a highly complex process that is regulated by a multitude of factors. Here, we show that the conserved kinase Ctk1 functions in translation by enhancing decoding fidelity. Ctk1 associates with translating ribosomes in vivo and is needed for efficient translation. Ctk1 phosphorylates Rps2, a protein of the small ribosomal subunit, on Ser 238. Importantly, Ctk1-depleted as well as rps2-S238A mutant cells show a defect in translation elongation through an increase in the frequency of miscoding. The role of Ctk1 in translation may be conserved as the mammalian homolog of Ctk1, CDK9, also associates with polysomes. Since Ctk1 interacts with the TREX (transcription and mRNA export) complex, which couples transcription to mRNA export, Ctk1/CDK9 might bind to correctly processed mRNPs during transcription and accompany the mRNP to the ribosomes in the cytoplasm, where Ctk1 enhances efficient and accurate translation of the mRNA.

Figure 1.

Ctk1 associates with polysomes. (A) Sedimentation behavior of Ctk1-TAP on sucrose density gradients. (Top panel) Ribosomal fractions (40S, 60S, 80S, and polysomes) were determined by OD_{254 nm} measurement of the gradient fractions. (Bottom panel) The presence of Ctk1-TAP in each fraction was visualized by Western blotting using an anti-protein A antibody. (Bottom panel) For comparison, the sedimentation behavior of a ribosomal protein of the large ribosomal subunit (Rpl6) and of the small ribosomal subunit (Rps8) is shown. (B, top panel) Sedimentation behavior of Ctk1-TAP on sucrose density gradients containing EDTA, which disrupts mono- and polysomes. (Bottom panel) Ctk1 shifts to the fractions containing ribosomal subunits. (C) The distribution of Ctk1 shifts to the 80S fraction when cycloheximide is omitted. The sucrose density gradient is as in A, but lacking cycloheximide. Without cycloheximide the ribosomes run off the mRNA, and the amount of polysomes is specifically reduced. Compared with A, Ctk1, Rpl6, and Rps8 shift to the fractions of the 80S peak. (D) Ctk1 is lost from the high-density fractions in gradients with puromycin. The sucrose density gradient is as in C, but containing puromycin, which leads to a complete loss of polysomes. Ctk1 as well as Rpl6 and Rps8 are lost from the high-density fractions. (E) Ctk1 binds to ribosomes in vivo. Cells were cross-linked with formaldehyde prior to lysis of the cells in buffer containing 500 mM KCl and separation of the lysate on a 500 mM KCl sucrose density gradient. Ctk1, Rpl6, and Rps8 were visualized by Western blotting of each fraction.

Figure 2.

Ctk1 is necessary for efficient translation. (A) Translation-active extracts were prepared from wild-type (WT) and GAL1∷CTK1-TAP cells grown in galactose-containing medium (Ctk1 expressed), and equal amounts of each extract were tested in an in vitro translation assay with endogenous mRNA (left panel), exogenous total RNA (middle panel), and in vitro transcribed mRNA coding for luciferase (right panel). Both extracts have the same translational activity. (B) Depletion of Ctk1 for 8 h leads to a translation defect of ∼30%. Wild-type and GAL1∷CTK1-TAP cells were shifted to glucose-containing medium for 8 h before translation-active extracts were prepared. Assays were performed as in A. (C) After 18 h of depletion of Ctk1, translation activity drops to ∼30%. Experiments were performed as in A after an 18-h shift to glucose-containing medium. For each panel in A–C, the activity of the wild-type extracts was set to 100%. Values and error bars represent the results of two independent experiments of three independent extracts each. (D) Growth curves of wild-type (WT, filled circle/square) and GAL1∷CTK1-TAP (open circle/square) cultures in galactose-containing medium (YPG, squares) and after shift to glucose-containing medium (YPD, circles) by measuring their optical density at 600 nm. (E) Depletion of Ctk1 after shift to glucose-containing medium. The same amount of whole-cell extracts from GAL1∷CTK1-TAP cells grown in galactose-containing medium (G: YPG) or shifted to glucose-containing medium (D: YPD) for the indicated time points was analyzed for the amount of Ctk1-TAP by SDS-PAGE and Western blotting with PAP antibody. As loading control, the amount of actin was assessed. (Lane 1) For comparison with the physiological Ctk1 expression, an extract from a CTK1-TAP strain, which expresses Ctk1 under its endogenous promoter, is shown. (F) Growth of CTK1-TAP, wild-type (WT), GAL1∷CTK1-TAP, and Δctk1 cells on plates. Tenfold dilutions of cells were spotted on glucose-containing medium (YPD) or galactose-containing medium (YPG) and grown at 30°C for 2 and 3 d, respectively.

Figure 3.

Ctk1 plays a role in decoding fidelity. (A) Ctk1-depleted cells are sensitive to translation inhibitors. GAL1∷CTK1-TAP and wild-type (WT) cells were grown for 18 h in YPD and then plated on YPD plates, on which a filter containing paromomycin, hygromycin B, geneticin, cycloheximide, or anisomycin was placed. The size of the halo indicates the sensitivity of the strain toward the drug. (B) Schematic showing the poly(U) assay. The rate of translation elongation was determined by measuring the incorporation of radioactively labeled phenylalanine (F*; left arrow), and the rate of miscoding was measured by the incorporation of radioactively labeled leucine (L*; right arrow) when poly(U) RNA was used as a template in the translation assay. (C) Depletion of Ctk1 leads to a decrease in the efficiency of translation elongation. The incorporation of [¹⁴C]-labeled phenylalanine into the polypeptide chain of nuclease-treated extracts prepared from wild-type (WT) and GAL1∷CTK1-TAP cells after 18 h of depletion in glucose-containing medium was measured. The activity of wild-type cells was set to 100%. (D) Loss of Ctk1 function leads to an increase in the frequency of miscoding. Cells depleted for Ctk1 by growth in glucose-containing medium (YPD) for 18 h show an increase in the frequency of miscoding as measured by the incorporation of [¹⁴C]-labeled leucine. (E,F) The defect of Ctk1-depleted cells in translation elongation (E) and translational accuracy (F) can be rescued by reconstitution with purified CTDK-I complex in a dose-dependent manner. Purified CTDK-I complex was added to extracts depleted for Ctk1 for 18 h, and the efficiency of translation elongation and the miscoding frequency was measured as in C and D, respectively. As negative control, an eluate from a mock purification of a nontagged wild-type strain was added to the translation assays, and the activities of the CTDK-I-treated extracts were calculated relative to the mock-treated ones. One representative experiment of three independent experiments is shown.

Figure 4.

Ctk1 plays a role in decoding fidelity in vivo. (A) Schematic showing the dual reporter system used for determination of miscoding frequency in B. (B) Loss of Ctk1 function leads to an increase in the frequency of miscoding. Cells depleted for Ctk1 by growth in glucose-containing medium (YPD) show an increase in the frequency of miscoding with all three stop codons (UAA, UAG, and UGA) compared with cells expressing Ctk1 (YPG), whereas an increased rate of frameshift events does not occur (−1 FS and +1 FS). (C) Depletion of Ctk1 leads to a slightly faster polysome run-off after inhibition of translation initiation. Wild-type (WT) and GAL1∷CTK1-TAP cells grown in YPD were shifted to glucose-lacking medium (YP) to inhibit translation initiation for the indicated time points and polysome profiles analyzed.

Figure 5.

Phosphorylation of Rps2 on Ser 238 by Ctk1 is needed for translational accuracy. (A) Complexes purified for the in vitro kinase assay shown in B. The CTDK-I complex was purified by a TAP tag on Ctk1 or Ctk3, the Bur1–Bur2 complex via TAP-tagged Bur1, and ribosomes were purified via TAP-tagged Rpl11a (L11a) or Rps2 (S2). A nontagged wild-type (WT) strain served as negative control. Proteins were separated by SDS-PAGE and stained with Coomassie. The left lane shows a molecular weight marker. (B) Ctk1 phosphorylates Rps2 in vitro. The protein complexes indicated by the component that was TAP-tagged for the purification were used in in vitro kinase assays. After incubation with radioactively labeled ATP, proteins were separated by SDS-PAGE, and phosphorylated proteins were visualized by autoradiography. An eluate of a nontagged wild-type strain served as negative control. Incubation of the control eluate alone (WT; lane 1) or of Rpl11a (L11) or Rps2 (S2) containing ribosomes with control eluate (lanes 5,6) did not give a radioactive signal. Incubation of Bur1, Ctk1, and Ctk3 with the control eluate (WT; lanes 2–4) gave signals for (auto)phosphorylated Bur1, Ctk1, and Ctk3. (Lane 7) Incubation of Ckt1 with Rpl11a-purified ribosomes yields an additional phosphorylated protein (indicated by a star). (Lane 8) This band shifts up when CBP-tagged Rps2-containing ribosomes (S2) are incubated with Ctk1, identifying Rps2 as the phosphorylated product (Rps2-TAP indicated by a star). (Lane 9) Bur1, used as a control kinase, does not phosphorylate Rps2. (C) Coomassie staining of purified ribosomes containing Rps2 or Rps2–S238A and CTDK-I complex used for the in vitro kinase assays shown in D. (D) Ctk1 phosphorylates Rps2 on S238. (Lane 4) Ctk1 phosphorylates itself, Ctk3, and Rps2. (Lane 5) In contrast, when ribosomes containing Rps2–S238A–CBP are used as substrate, no phosphorylation of Rps2 can be observed. (E,F) rps2-S238A extracts show a minor decrease in translation elongation and an increase in miscoding events. Experiments were performed as described in Figure 3B. (G) rps2-S238A cells are sensitive to drugs that impair translation elongation.

Figure 6.

The function of Ctk1 in translation might be conserved. (A) CDK9, the human homolog of Ctk1, associates with polysomes. Extracts of 293T cells were separated by sucrose density centrifugation. (Top panel) The ribosomal fractions were visualized by OD_{254 nm} measurement of the gradient fractions. (Bottom panel) The presence of CDK9 in each fraction was analyzed by Western blotting with an antibody recognizing CDK9. (B) The association of CDK9 with polysomes is specific. OD_{254 nm} measurement (top panel) and CDK9 Western blot (bottom panel) of a sucrose gradient containing EDTA, which dissociates mono- and polysomes.

Figure 7.

(A, 1) Model of Ctk1’s function in translation. Phosphorylation of Rps2 on S238 by Ctk1 is needed for a higher accuracy of mRNA decoding. (2) Since Ctk1 is implicated in transcription of RNA Pol II and interacts with the TREX complex, it could exit the nucleus bound to correctly processed mRNPs. (3) Alternatively, Ctk1 could travel to the cytoplasm with preribosomes, as Ctk1 has been implicated in RNA Pol I transcription. After phosphorylation of Rps2 (1), Ctk1 most likely rapidly reenters the nucleus (4). See Discussion for details. (B–D) Cryo-EM model of an elongating 80S ribosome with messenger RNA, A, P, and E site tRNA, and Rps2 modeled in according to Spahn et al. (2001). (B) Cartoon of a cross-section allowing a view of the elongation process. Rps2 is positioned at the beginning of the mRNA entry tunnel. (C) View of the 40S subunit from the solvent site. The transparency was reduced to visualize the path of the mRNA, the tRNAs, and Rps2. Yeast Rps2 is present with reduced electron density in the 80S EM structure and as crystal structure of E. coli S5. (D) Closeup of the localization of Rps2 on the small ribosomal subunit at the entry tunnel of the mRNA. The large ribosomal subunit is shown in blue, the small ribosomal subunit is in yellow, mRNA is in orange (single spheres show the likely path of the mRNA, which is not seen in the electron density), A, P, and E site tRNAs are in green, and Rps2 is in red.