Protein Degradation of RNA Polymerase II-Association Factor 1(PAF1) Is Controlled by CNOT4 and 26S Proteasome

Abstract

The PAF complex (PAFc) participates in various steps of the transcriptional process, from initiation to termination, by interacting with and recruiting various proteins to the proper locus for each step. PAFc is an evolutionarily conserved, multi-protein complex comprising PAF1, CDC73, CTR9, LEO1, yRTF1 and, in humans, hSKI8. These components of PAFc work together, and their protein levels are closely interrelated. In the present study, we investigated the mechanism of PAF1 protein degradation. We found that PAF1 protein levels are negatively regulated by the expression of CNOT4, an ortholog of yNOT4 and a member of the CCR4-NOT complex. CNOT4 specifically controls PAF1 but not other components of PAFc at the protein level by regulating the polyubiquitination of PAF1 and its subsequent degradation by the 26S proteasome. The degradation of PAF1 was found to require nuclear localization, as no PAF1 degradation by CNOT4 and the 26S proteasome was observed with NLS (nucleus localization signal)-deficient PAF1 mutants. However, chromatin binding by PAF1 was not necessary for 26S proteasome- or CNOT4-mediated degradation. Our results suggest that CNOT4 controls the degradation of chromatin-unbound PAF1 via the 26S proteasome.

Fig 1. PAF1 is regulated by CNOT4 at the protein level.

(A, B) The levels of endogenous PAF1 protein in CNOT4-overexpressing or silenced whole-cell lysates were analyzed by immunoblot. The silencing efficiency of CNOT4 was measured by RT-PCR (B). (C) The PAF1 and CNOT4 protein levels were measured in cells transfected with different siRNAs. (D) HEK293 cells were transfected with control or CNOT4 siRNA specific to the UTR, along with CNOT4 expression vectors. The protein levels of endogenous PAF1, endogenous CNOT4 and overexpressed V5-CNOT4 were measured by immunoblot assay. (E) After CNOT4 siRNA was transfected into HEK293 cells, PAF1 and CNOT4 mRNA levels were measured by conventional PCR (left) and quantitative real-time PCR (right). The protein levels of endogenous PAF1 were detected by immunoblot assay (left, bottom).

Fig 2. CNOT4 regulates PAF1 but not other PAFc components.

(A, B) Endogenous PAFc protein levels were measured in CNOT4-overexpressing or silenced HEK293 cells via Western blot. The silencing efficiency of CNOT4 was measured by RT-PCR (B, bottom). (C) Cells were transfected with the indicated plasmids, and cell lysates were prepared and immunoprecipitated with Anti-Myc. The interaction between mPAF1 and mLEO1 was determined by Western blotting. (D) Myc-mPAF1 WT or 231–535 was transfected along with sicontrol or siCNOT4 in HEK293 cells. Myc-tagged mPAF1 proteins levels were analyzed by immunoblotting, and CNOT4 depletion was measured by RT-PCR (bottom).The arrows indicate the Myc-mPAF1 of WT or mutant.

Fig 3. CNOT4 controls the degradation of PAF1 via the 26S proteasome.

(A) Pulse-chase experiment. V5-mPAF1-transfected cells were metabolically labeled with ³⁵S-Met for 60 min and chased for the indicated time periods. V5-mPAF1 was immunoprecipitated with anti-V5, and the remaining V5-mPAF1 protein was analyzed by autoradiography (left, bottom) and quantified (right). (B, C). Myc-mPAF1-transfected (B) or non-transfected (C) HEK293 cells were treated with the indicated inhibitors for 6 hr: MG132 (10 μM), chloroquine (CQ, 50 μM), NH₄Cl (20 mM), PS341 (10 μM). Exogenous Myc-mPAF1 (B) or endogenous PAF1 (C) proteins in whole-cell lysates were detected with anti-Myc or anti-PAF1 antibodies, respectively. (D) Empty or CNOT4-V5-transfected cells were treated with MG132 (10 μM) for 6 hr. Total cellular extracts were prepared, and each protein was detected by immunoblot assay.

Fig 4. CNOT4 regulates the ubiquitination status of the PAF1 protein.

(A) Physical interaction between PAF1 and CNOT4. Myc-mPAF1 was overexpressed in HEK293 cells. Myc-mPAF1 was immunoprecipitated from cell lysates, and the physical interaction between PAF1 and CNOT4 was assessed via immunoblotting. CTR9 was used as the positive control. (B) The indicated plasmids were transfected into HEK293 cells. After 48 hr, cells were harvested, and cell extracts were subjected to immunoprecipitation with an anti-Myc antibody. Ubiquitination of mPAF1 was detected by immunoblot assay with anti-HA. (C) Myc-mPAF1-transfected cells were treated with MG132 (10 μM) for 6 hr before harvest. Myc-mPAF1 was immunoprecipitated using an anti-Myc antibody and blotted with anti-Myc or anti-K48UB antibodies. (D) HEK293 cells were transfected with the indicated plasmids. Myc-mPAF1 was immunoprecipitated with anti-Myc from whole-cell lysates and blotted with anti- HA or anti-Myc antibodies. (E) HEK293 cells were transfected with Myc-mPAF1 along with control or CNOT4 siRNA. After treatment with MG132 (10 μM) for 6 hr, whole-cell extracts were immunoprecipitated with an anti-Myc antibody. Immunoprecipitates were subjected to Western blot analysis using anti-Myc or anti-HA antibodies. As the input control, 5% of the total lysates used for immunoprecipitation was loaded. The knockdown efficiency of siCNOT4 was measured by RT-PCR.

Fig 5. The 255–275 amino acid on PAF1 is required for its degradation.

(A) Schematic diagram of WT and mutant PAF1. “K” designates lysine residues. (B, C) Cells were transfected with Myc-tagged WT or mutant PAF1, and the sub-cellular localization of these proteins was analyzed by immunofluorescence staining with an anti-Myc antibody (green) and DAPI (blue) (B). The percentages of cells containing WT or mutant PAF1 in the “nucleus only” or in the “nucleus and cytoplasm, cytoplasm only” are shown (C). (D) HEK293 cells were transfected with the indicated plasmids for 48 hours. Cells were treated with MG132 (10 μM) for 6 hr, followed by immunoblot analysis. (E) WT PAF1- or mPAF1△255-275-transfected cells were treated with MG132 (10 μM) for 6 hr, and the levels of each PAF1 protein were detected by immunoblot against anti-PAF1. (F) Cells were transfected with WT or mutant PAF1 along with control or CNOT4 siRNA. Top, WCLs were prepared from half of the cells and subjected to Western blot analysis. Bottom, Total RNA was prepared from the remaining cells and analyzed by RT-PCR.

Fig 6. CNOT4 affects PAF1 independent of chromatin binding.

(A) HEK293 cells were fractionated into chromatin-unbound (U) and bound (B) fractions, and the chromatin association of the proteins was determined via Western blotting. GAPHD and H3 were used as markers of the chromatin-unbound and bound fractions, respectively. (B, C) Chromatin-unbound and bound PAF1 was determined by immunoblotting in CNOT4-overexpressed (B) and silenced (C) cells. (D) WT or Myc-mPAF1 Δ285-355-transfected HEK293 cells were treated with MG132 (10 μM) for 6 hr, and Myc-tagged PAF1 protein levels in whole-cell lysates were detected. (E) WT or Myc-mPAF1 Δ285–355 was transfected along with control or CNOT4 siRNA. Myc-tagged PAF1 protein levels in whole-cell lysates were determined using an antibody against Myc. The pGFP plasmid was included as a transfection control. CNOT4 depletion was measured by RT-PCR.