PAF1 is regulated by CNOT4 at the protein level.

<p>(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).</p

CNOT4 affects PAF1 independent of chromatin binding.

<p>(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.</p

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

<p><b>(</b>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>(</b>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. <b>(</b>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.</p

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

<p>(A) Schematic diagram of WT and mutant PAF1. “K” designates lysine residues. <b>(</b>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. <b>(</b>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. <b>(</b>F) Cells were transfected with WT or mutant PAF1 along with control or CNOT4 siRNA. <i>Top</i>, WCLs were prepared from half of the cells and subjected to Western blot analysis. <i>Bottom</i>, Total RNA was prepared from the remaining cells and analyzed by RT-PCR.</p

CNOT4 regulates the ubiquitination status of the PAF1 protein.

<p>(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>(</b>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. <b>(</b>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.</p

The mammalian Arg/N-end rule pathway and missense mutations in human <i>UBR1</i> that underlie specific cases of the Johanson-Blizzard syndrome (JBS).

<p>(A) The mammalian N-end rule pathway. N-terminal residues are indicated by single-letter abbreviations for amino acids. Yellow ovals denote the rest of a protein substrate. ‘Primary’, ‘secondary’ and ‘tertiary’ denote mechanistically distinct subsets of destabilizing N-terminal residues (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#s1" target="_blank">Introduction</a>). C* denotes oxidized Cys, either Cys-sulfinate or Cys-sulfonate. MetAPs, Met-aminopeptidases. (B) Single-residue mutations in the UBR1 proteins of JBS patients #1 and #2. The positions of mutant residues are indicated both for the original mutations in human UBR1 and for their mimics in <i>S. cerevisiae</i>. (C) Same as in B but the mutation in UBR1 of patient #3 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#s2" target="_blank">Results</a>).</p

Functional activity of yeast Ubr1 mimics of missense JBS-UBR1 mutants.

<p>(A) Relative enzymatic activity of βgal in extracts from <i>S. cerevisiae</i> JD55 (<i>ubr1Δ</i>) that expressed His-βgal or Tyr-βgal, and also carried an empty vector, or an otherwise identical plasmid expressing wild-type <i>S. cerevisiae</i> Ubr1, or (separately) its three missense mutants Ubr1^V146L, Ubr1^H160R, or Ubr1^Q1224E. The activity of βgal was measured in triplicates, with standard deviations shown. (B) Relative levels of induction of the peptide transporter Ptr2 were assayed by measuring the activity of a plasmid-borne <i>lacZ</i> (βgal-encoding) reporter that was expressed from the P<i>_PTR2</i> promoter in <i>ubr1Δ S. cerevisiae</i> that carried either an empty vector or otherwise identical plasmids that expressed either wild-type Ubr1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone.0024925-Hwang1" target="_blank">[27]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone.0024925-Xia1" target="_blank">[28]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone.0024925-Turner1" target="_blank">[52]</a> or its indicated mutants. Cells were grown to A₆₀₀ of ∼0.8 in SC(-Ura, -Leu) medium at 30°C, followed by measurements, in triplicate, of βgal activity in cell extracts, with standard deviations shown. (C) The lysine-requiring JD55 (<i>ubr1Δ</i>) <i>S. cerevisiae</i> strain was grown on plates containing 110 µM lysine (Lys) or 66 µM Lys-Ala dipeptide as the sole source of Lys in the medium <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone.0024925-Hwang1" target="_blank">[27]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone.0024925-Hwang3" target="_blank">[33]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone.0024925-Turner1" target="_blank">[52]</a>. JD52 (<i>ubr1Δ</i>) cells carried a vector plasmid or otherwise identical plasmids expressing wild-type Ubr1 or its missense mutants Ubr1^H160R, Ubr1^V146L and Ubr1^Q1224E. Cells were grown to A₆₀₀ of ∼1 in SC(-Leu) medium at 30°C, washed in sterile water, serially diluted 5-fold, spotted on SC(-Leu, -Lys) plates containing 110 µM Lys or 66 µM Lys-Ala, and incubated at 30°C for 3 days. (D) Cell extracts (equal total protein levels) from experiments described in panels A and B were subjected to SDS-PAGE, followed by immunoblotting with affinity-purified anti-Ubr1 antibody (upper panel) and anti-tubulin antibody (a loading control; lower panel). Asterisk indicates a protein that crossreacts with anti-Ubr1 antibody. (E) Extracts from human lymphocytes (equal amounts of total protein) were subjected to SDS-PAGE, followed by immunoblotting with antibody to human UBR1 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#s4" target="_blank">Materials and Methods</a>). Lane 1, wild-type lymphocytes. Lane 2, same as lane 1 but from lymphocytes of patient #2 (see the main text and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone-0024925-g001" target="_blank">Figs. 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone-0024925-g002" target="_blank">2</a>). Lane 3, same as lane 1 but with lymphocytes from patient #3. Lane 4, same as lane 1, but with lymphocytes from a JBS patient with a homozygous nonsense mutation in <i>UBR1</i>, previously shown to have no detectable UBR1 (null UBR1 control) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone.0024925-Varshavsky3" target="_blank">[17]</a>. Lane 5, same as a lane 1.</p

<i>S. cerevisiae</i> Ubr1 N-recognin.

<p>(A) A diagram of the 225 kDa <i>S. cerevisiae</i> Ubr1. The indicated evolutionarily conserved regions of Ubr1 are the UBR box, the BRR (basic residues-rich) domain, the Cys/His-rich RING-H2 domain, and the AI (<u>a</u>uto<u>in</u>hibitory) domain <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone.0024925-Tasaki1" target="_blank">[18]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone.0024925-Xie1" target="_blank">[30]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone.0024925-Hwang3" target="_blank">[33]</a>. Three missense mutations in patients #1-3 of the present work are indicated as well (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone-0024925-g001" target="_blank">Fig. 1B, C</a>). (B) Ribbon diagram of the <i>S. cerevisiae</i> UBR domain <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone.0024925-Choi1" target="_blank">[48]</a> in a complex with RLGES, the N-terminal region of the separase-produced fragment of Scc1, a subunit of cohesin <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone.0024925-Rao1" target="_blank">[75]</a>. The bound RLGES peptide is shown as a stick model, with carbon atoms colored yellow. Several residues are marked with a black sphere and numbered to facilitate the tracing of the polypeptide chain. The names of residues of the RLGES peptide are in red, with the letter ‘s’ (substrate) appended to their position numbers. Side-chains of residues in the UBR domain that are present near JBS mutations (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone-0024925-g001" target="_blank">Fig. 1B, C</a>) are shown in a stick form, with carbon atoms colored green. Three coordinated zinc ions of the UBR domain <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone.0024925-Choi1" target="_blank">[48]</a> are shown as red spheres. (C) Close-up view of the UBR region near the V146L mutation (patient #1; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone-0024925-g001" target="_blank">Fig. 1B</a>). In panel B, this region of UBR is boxed and labeled as ‘C’. The residues of UBR that accommodate the position-2 Leu residue (‘Leu2s’) of the RLGES peptide substrate are shown and labeled. The van der Waals sphere of the mutant Leu residue, in the UBR1^V146L mutant, is shown as purple dots. (D) Close-up view of the UBR region near the H160R mutation (patient #2, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone-0024925-g001" target="_blank">Fig. 1B</a>). In panel B, this region of UBR is boxed and labeled as ‘D’. The residues of UBR coordinating Zn3 atom are shown and labeled. The van der Waals sphere of the mutant Arg residue, in the UBR1^H160R mutant, is shown as purple dots. The views in (C) and (D) are oriented to maximize visibility of mutation-proximal residues.</p

Clinical features in JBS patients.

a<p>Ref. 1;</p>b<p>Ref. 6 and unpublished data.</p