Rapid proteasome degradation of SMAD4 in DBA iPSCs.

<p>DBA iPSCs, and the corrected DBA cells were cultured in the iPSC medium, and treated with proteasome inhibitors MG132 (4 μM) for 12h or PS341 (20 μM) for 4h. A-B) Increased level of SMAD4 in both DBA cells after treatment with proteasome inhibitor MG132 for 12h. C) Increased level of nuclear SMAD4 in DBA iPSCs with <i>RPS19</i> mutation after PS341 treatment. D and E) Increase of nuclear and cytoplasmic TRIM33, and increase of nuclear USP9X in both DBA iPSCs. The PS341 treatment did not affect the levels of TRIM33 and USP9X.</p

Activation of TGFβ signaling in the DBA iPSCs with <i>RPS19</i> or <i>RPL5</i> mutations.

<p>Activation of TGFβ signaling in the DBA iPSCs with <i>RPS19</i> or <i>RPL5</i> mutations.</p

Activation of non-canonical TGFβ pathway through p-JNK.

<p>DBA iPSCs, the corrected DBA cells and the GFP cells were cultured, and RNA, total protein and the protein from nuclear and cytoplasmic fraction were extracted from iPSCs for analysis. Fibrillarin were used as loading controls. A) Overexpression of <i>JNK1</i> mRNA level in both DBA mutant lines. B) Elevation of JNK protein, especially p-JNK, in the total protein of DBA iPSCs with <i>RPS19</i> mutation. C) Accumulation of nuclear p-JNK in the DBA iPSCs and the GFP lines (especially in <i>RPS19</i> mutant line), and restoration of p-JNK levels in the corrected DBA lines. D) Increase of cytoplasmic p-JNK in the DBA iPSCs, and normal levels of p-JNK in the rescued DBA lines.</p

Significant decrease of SMAD4 protein in DBA iPSCs.

<p>DBA iPSCs (labelled as “<i>RPS19/RPL5</i> mutant” or “<i>RPS19/RPL5</i> mutant-”), the corrected DBA cells (expressing a WT version of the mutated gene, <i>RPL5</i> or <i>RPS19</i>, labelled as “<i>RPS19/RPL5</i> mutant <i>RPS19/RPL5</i>” or “corrected <i>RPS19/RPL5</i> mutant”) and the control GFP cells (expressing GFP, labelled as “<i>RPS19/RPL5</i> mutant GFP”) were cultured and the nuclear and cytoplasmic protein was extracted from iPSCs for western blot analysis, and RNA for q-PCR analysis. A) Slight decrease of mRNA level of <i>SMAD4</i> in the DBA iPSCs with <i>RPS19</i> or <i>RPL5</i> mutations. B-C) Dramatic decrease of cytoplasmic and nuclear SMAD4 protein levels in both DBA iPSC lines and SMAD4 levels returned to normal in the corrected lines. D) Immunofluorescence staining showed the decreased SMAD4 levels in DBA iPSCs with <i>RPS19</i> mutation compared with those in WT cells, and the corrected line partially restored the SMAD4 protein. Microscope pictures of SMAD4 stained slices were taken using a Leica DM4000B equipped with a 40X objective and image was captured with a Spot RT slider camera. E) No significant decrease of SMAD4 protein in the lymphoblastoid cells generated from the DBA patients, and wild type lymphoblastoid cells was from a healthy control.</p

Change of SMAD4 after TGFβ inhibitor /activator treatment.

<p>The iPSCs were cultured in iPSC medium, and treated with TGFβ inhibitor SB431542 (10μM) for 3 days or treated with TGFβ activator, TGFβ1 (20ng/ml) for 24h. DMSO treatment was used as the control. Protein levels were measured by Western blot analysis. A) Decrease of total p-SMAD2 after SB431542 treatment in all iPSCs, and increase of p-SMAD2 in both DBA iPSCs after the TGFβ1 treatment. B) Decrease of SMAD4 in the cytoplasmic fraction of all iPSCs after SB431542 treatment. C) Slight accumulation of SMAD4 in the nucleus after SB431542 treatment in DBA iPSCs with <i>RPS19</i> or <i>RPL5</i> mutations. D-E) Mild increase of cytoplasmic and nuclear SMAD4 in DBA iPSCs after the TGFβ1 treatment.</p

Decrease of SMAD3 protein, but not SMAD2 in DBA iPSCs.

<p>DBA iPSCs, the corrected DBA cells, and the control GFP cells were cultured, and the RNA, total protein, protein from nuclear fraction were extracted from iPSCs. The ratio of protein level to WT is labelled under the image. A) No change of mRNA level of <i>SMAD2</i> or <i>SMAD3</i> in the DBA iPSCs with <i>RPS19</i> or <i>RPL5</i> mutations. B) Western blot analysis of total protein showing some decrease in SMAD3 protein but the change of SMAD2 was not obvious. C) Western blot analysis of nuclear protein showing decrease in SMAD3 protein in both DBA lines, and no change in SMAD2 protein. D) Western blot analysis showing slight change of p-SMAD2 level in the nuclear fractions from <i>RPS19</i> mutant compared to WT and the corrected lines. The nuclear p-SMAD2 decreased in <i>RPL5</i> mutant and corrected lines. E) Western blot showing the decrease of p-SMAD3 in the nuclear fraction in both DBA iPS lines, and the nuclear p-SMAD3 protein partially restored in the corrected DBA iPSCs in the <i>RPS19</i> corrected line, but not in <i>RPL5</i> corrected line.</p

Molecular model of dysregulation of TGFβ signaling in DBA iPSCs.

<p>Ribosomal haploinsufficiency may lead to an increasing expression of TGFβ downstream targets through non-canonical JNK signaling. We also identified an increased proteolytic turnover of SMAD4 in the DBA iPSCs. The activation of TGFβ signaling contributes to the reduced differentiation of erythroid cells, and the decrease of SMAD4 could be the major factor permitting DBA iPSCs to proliferate.</p

Fold changes of WNT related genes in <i>DKC1</i> mutant iPS cells.

<p>Fold changes of WNT related genes in <i>DKC1</i> mutant iPS cells.</p

<i>DKC1</i> mutant iPS cells show no significant defects in ribosome biogenesis.

<p>A: measurement of pseudouridine in 28S rRNA from WT and mutant iPS cells. iPS cells were labeled with ³²P-labeled orthophosphate and 28S rRNA was gel-purified. After digestion with RNase T₂, each sample was separated by two-dimensional TLC. The positions of the labeled ribonucleotides are indicated. Ap: Adenine, Cp: Cytosine, Gp: Guanine, Up: Uridine, Ψp: Pseudouridine: B: Pulse–chase labeling experiments of rRNA isolated from <i>WT</i>, <i>A353V</i> and <i>ΔL37</i> iPS cells. Cells were labeled with L-[³H-methyl] methionine for 30 min and then chased in nonradioactive medium for the times shown. The RNA was separated on a 1.25% agarose gel, transferred to a nylon filter, and exposed to x-ray film. C: Real-time RT/PCR results of some H/ACA snoRNAs of <i>WT</i> and <i>DKC1</i> mutant iPS cells. Results were expressed relative to <i>GAPDH</i> RNA. The combined results of 3 independent experiments are shown, the error bars show standard deviation.</p

Expression of WT dyskerin can rescue the expression of <i>LGR5</i>, <i>WLS</i> and <i>FRZB</i>.

<p>A: Real-Time RT/PCR experiments showed that, in <i>A353V</i> iPS cells, the mRNA expression of <i>LGR5</i>, <i>FRZB</i> and <i>WLS</i> was significantly increased after expressing WT dyskerin protein. The combined results of 3 independent experiments are shown, the error bars show standard deviation. * p<0.01. B: Knock down of dyskerin affects canonical WNT signaling. The relative luciferase activity in inducible <i>DKC1</i>-shRNA HEK293T cells transfected with plasmids containing a luciferase gene under the control of a WT (OT-flash) or mutant (OF-flash) β-catenin responsive promotor and pRL-SV40 plasmid. Luciferase activity was measured by using dual-luciferase system according to protocol from manufacturer. OT: WT TCF/LEF reporter, OF: Mutant TCF/LEF reporter, Dox: Doxycycline, CHIR: CHIR-99021.Four independent experiments were carried out and the mean of results are shown; the error bars showing standard deviation. * p<0.01.</p