A constitutively active ubiquitin-proteasome pathway degrades Drosha.

<p>Whole cell lysates of the indicated cell types were prepared with IP Lysis Buffer (Pierce); 20 µg protein aliquots were used for western blots. All experiments were repeated three times with similar results (p<0.05 by Student’s t-test). (<b>A</b>) Abundant polyubiquitination was observed in multiple cell types using lysine 48-linkage specific polyubiquitin antibody. (<b>B</b>) Drosha is ubiquitinated in various cell types including HEK293T, HeLa and AGS. Left panel: HEK293T lysates were immunoprecipitated with control IgG or Rabbit Polyclonal Antibody to Drosha respectively. Right panel: Cell lysates were immunoprecipitated respectively with Rabbit Polyclonal Antibody to Drosha. The immunoprecipitates were resolved by SDS-PAGE and blotted with lysine 48-linkage specific polyubiquitin antibody. The same membrane was reblotted with Drosha Rabbit mAb. (<b>C</b>) Inhibition of the ubiquitin-proteasome pathway with MG132 (10 µM) increases endogenous Drosha protein level in AGS cells. GAPDH was used as a loading control. (<b>D</b>) Proteasomal inhibition increases exogenous GFP-Drosha expression level. HEK293T cells were transfected with GFP-Drosha. Twenty-four hours post-transfection, the cells were treated with 1 µM Clasto-Lactacystin-β-lactone (Omuralide) overnight. GFP-Drosha protein level was increased 3-fold following treatment.</p

Acetylation of Drosha increases its protein level.

<p>All experiments were repeated three times with similar results (p<0.05 by Student’s t-test). (<b>A</b>), Inhibition of deacetylases increases Drosha protein level measured by western blot. HEK293T cells were treated with vehicle, trichostatin A (TSA, 2 µM), or nicotinamide (NIA, 1 mM) overnight prior to harvest. (<b>B</b>) TSA, an HDAC inhibitor, increases ectopically expressed Drosha levels in HEK293T upon transfection with a GFP-Drosha construct. (<b>C</b>) Inhibition of deacetylases have no effects on Drosha mRNA level measured by RT-PCR. (<b>D</b>)Inhibition of deacetylases increases Drosha acetylation. HEK293T cells were transfected with empty vector (EV) or GFP-Drosha. Twenty-four hours post-transfection, the cells were treated with TSA (2 µM) or NIA (1 mM) overnight. Whole cell lysates were prepared and immunoprecipitated with GFP antibody conjugated sepharose beads. The immunoprecipitates were resolved and blotted with mouse monoclonal acetylated lysine antibody to detect Drosha acetylation. The same membrane was then reblotted to check Drosha protein level. (<b>E</b>) Multiple acetyl transferases acetylate Drosha. A GFP-Drosha construct was cotransfected with an empty vector, p300, CBP, PCAF or GCN5 construct respectively into HEK293T cells. Forty-eight hours post-transfection, half of the cells was used to extract total RNA for checking the mRNA levels of GFP-Drosha. Another half of the cells was used to prepare whole cell lysates for detecting Drosha acetylation as in Figure1C.</p

N-terminus of Drosha is the main part for acetylation.

<p>(<b>A</b>) <i>In vitro</i> acetylation assays revealed that N’-terminal but not C’-terminal Drosha is the major acetylation domain. Immunoprecipitated N-terminal Drosha (GFP-Drosha1-390 ) or C-terminal Drosha (GFP-Drosha391-1374) was co-incubated with 10 U of p300 HAT Domain and 2 µM Acetyl CoA at 37°C for I hr. (<b>B</b>) Identification of an acetylated lysine site by mass spectrometry. (C) TSA treatment increases the acetylation and expression levels of GFP-DroshaK382R (D) miRNA sensor assays revealed that compared to vehicle control, treatment of cells with TSA increased miRNA function. (E) TSA treatment increases miR-143 level in HEK293T cells. (F) TSA treatment decreases expression of fibronectin type III domain containing 3B (FNDC3B), a target of miR-143. All experiments were performed in triplicate (p<0.05 by Student’s t-test).</p

The N-terminus of Drosha is responsible for regulating its degradation.

<p>HEK293T cells were transfected with an empty vector, GFP-Drosha wild type or the various mutants as indicated. Forty-eight hours post-transfection, whole cell lysates were prepared for western blot. GAPDH was used as a loading control. All experiments were repeated three times with similar results. (<b>A&C</b>) Schematic illustration of domain deletion constructs of Drosha-GFP at the N-terminus. (<b>B</b>) GFP-Drosha 391-1374 expressed more protein than GFP-Drosha or GFP-Drosha1-850 indicating N-terminal Drosha harbors a domain regulating expression levels. The mRNA levels of GFP Drosha wt and mutants were measured to monitor the transfection efficiency of various constructs. (<b>D</b>) Deletion of additional lysines increased Drosha protein expression, indicating that multiple lysines on the N- terminus regulate Drosha degradation. The mRNA levels of GFP Drosha wt and mutants were measured to monitor the transfection efficiency of various constructs.</p

<i>H. pylori</i> infection degrades Drosha through the ubiquitin-proteasome pathway.

<p>AGS gastric epithelial cells were infected with <i>H. pylori</i> at a ratio of 100∶1 (bacterium:cell) for different time periods. All experiments were repeated three times with similar results (p<0.05 by Student’s t-test). (<b>A</b>) <i>H. pylori</i> increased levels of K48-linked polyubiquitin. AGS cells were infected with <i>H. pylori</i> for 6 h. Beta-Actin was used as a loading control. (<b>B</b>) Western blot revealed that Drosha protein is decreased by <i>H. pylori</i> infection. GAPDH was used as a loading control. Other miRNA related proteins were not affected. (<b>C</b>) <i>H. pylori</i> infection does not alter Drosha mRNA levels. Total mRNA from AGS cells was reverse transcribed and amplified using Drosha specific primers. Gelstar was used to stain PCR products. (<b>D</b>) MG132, a specific proteasome inhibitor, rescued Drosha from <i>H. pylori</i> induced degradation. AGS cells were infected with <i>H. pylori</i> for 6 h and GAPDH was used as a loading control. (<b>E</b>) Omuralide, another specific proteasome inhibitor, also rescued Drosha from <i>H. pylori</i> induced degradation. AGS cells were infected with <i>H. pylori</i> for 6 h and GAPDH was used as a loading control. (<b>F</b>) H. pylori infection doesn’t affect Drosha acetylation.</p

Acetylation and Ubiquitination regulate Drosha protein level.

<p>All experiments were repeated three times with similar results (p<0.05 by Student’s t-test). (<b>A</b>) Acetylation of endogenous Drosha decreases its ubiquitination. HEK293T cells were treated with or without 2 µM TSA overnight. Whole cell lysates were prepared with IP lysis buffer and 300 µg protein was immunoprecipitated with rabbit polyclonal antibody to Drosha. The immunoprecipitates were resolved and levels of acetylated Drosha were measured. The same membrane was reblotted with lysine 48-linkage specific polyubiquitin antibody to check the ubiquitination status of Drosha. A parallel IP experiment was performed to check Drosha pull down. GAPDH was used as a loading control in another parallel western blot. (<b>B</b>) TSA blocks the ubiquitination of exogenously expressed GFP-Drosha. HEK293T cells were transfected with a GFP-Drosha construct. Twenty-four hours post-transfection, the cells were treated with or without 2 µM TSA overnight. GFP antibody conjugated sepharose beads were used to immunoprecipitate GFP fusion proteins.</p

<i>H. pylori</i> infection results in increased RKIP transcription and nuclear localization.

<p>(A) RKIP transcription reporter assay of AGS cells transiently transfected with RKIP luciferase construct and HA-RKIP for 24 h, then co-cultured with <i>H. pylori</i> for 12 h in the presence or absence of the PKC inhibitor. In comparison to empty vector controls, relative transcriptional activity was significantly increased for * <i>H. pylori</i>, p<0.002; and ** <i>H. pylori</i>+RKIP, P<0.0003. Comparing the loss of relative luciferase activity of <i>H. pylori</i> and RKIP when compared to <i>H. pylori</i>, RKIP and Bis, p<0.0004. Data represents the mean +/− standard deviation (sd) of the fold increase relative to empty vector controls in 2 independent experiments performed in duplicate. (B) Western blot analysis of nuclear and cytosolic fractions of AGS cells co-cultured with <i>H. pylori</i> for 4 h for the expression of pRKIP and total RKIP. Actin and laminA provide verification of successful cytoplasmic and nuclear fraction separation.</p

The pathogenicity island of <i>H. pylori</i> is responsible for RKIP phosphorylation.

<p>Western blot analysis of (A) AGS cells co-cultured with <i>H. pylori</i> strains for 6 h and examined for the indicated proteins. C = control (uninfected), WT = AGS cells infected with wild type <i>H. pylori</i> for 6 h, <i>PAI</i>- and <i>oipA</i>- represent isogenic mutants lacking these genes. (B) AGS cells transiently transfected for 24 h with RKIP S153 cDNA or 24 h and co-cultured with <i>H. pylori</i> for 6 h. (C) RKIP luciferase reporter assay of AGS cells transiently transfected with S153V RKIP in the presence or absence of <i>H. pylori</i> infection. In comparison to empty vector controls, the relative activity of RKIP transcription was increased by: *<i>H. pylori</i>, p<0.002; **RKIP, p<0.002; ***S153V, p<0.03, ****<i>H. pylori</i> and RKIP, p<0.0005; *****<i>H. pylori</i> and S153V, p<0.003. **, RKIP transcriptional activity was significantly decreased by the S153V compared with the wild type RKIP construct in response to <i>H. pylori, #</i>, p<0.0003. The data represents the mean +/− sd of 2 independent experiments performed in duplicate.</p

IL-6 promotes RKIP and STAT 3 transcription and phosphorylation; <i>H. pylori</i> infection induces STAT3 transcription and phosphorylation.

<p>(A) Western blot analysis of AGS cells treated with the indicated concentrations of IL-6 and for 6 hours. Densitometry was performed and for pRKIP expression, our results indicate a 1.8 fold incerase of pRKIP (average intensity 0.59 vs 1.051) in cells treated with 25 ng/ml IL-6 and a 1.35 fold increase (average intensity 0.59 vs 0.772) in cells treated with 50 ng/ml IL-6. For RKIP expression, our results indicate a 0.8 fold incerase of pRKIP (average intensity 0.69 vs 0.563) in cells treated with 25 ng/ml IL-6 and a 1.05 fold increase (average intensity 0.69 vs 0.745) in cells treated with 50 ng/ml IL-6 when normalized to actin at each time point. (B) Western blot analysis of AGS co-cultured with <i>H. pylori</i> and examined for pY705 STAT3 for the indicated times; (C) STAT3 luciferase reporter transcriptional assay of AGS cells co-cultured with <i>H. pylori</i> at the indicated MOI; (D) STAT3 luciferase reporter assay of AGS cells transiently transfected with STAT3 for 24 h and co-cultured with <i>H. pylori</i> and/or treated with IL-6 for 6 h. A paired t-test was performed to analyze the increase or decrease in STAT3 transcription of experimental samples when compared to empty vector (EV): *IL-6, p<0.0003; **STAT3, p<0.009; *** <i>H. pylori</i> p<0.0005; **** STAT3 and <i>H. pylori</i> p<0.0000023.</p

<i>H. pylori</i> infection results in RKIP phosphorylation.

<p>(A) Western blot analysis of AGS cells co-cultured with <i>H. pylori</i> (HP) at MOI of 100∶1 for 2 and 6 h and examined for pRKIP, RKIP, and actin expression. Densitometry was performed on three independent experiments and band intensities normalized in comparison to Actin for each time point. Our results indicated a 3.126 fold increase (average intensity 0.44 vs 1.376) of pRKIP after 2 h and 1.384 fold increase (average intensity 0.6774 vs 0.938) of RKIP after 2 h of <i>H. pylori</i> infection. (B) AGS cells co-cultured for 6 h in the presence or absence of the PKC inhibitor bisindolylmaleimide (Bis), were examined for the expression of pRKIP, RKIP and actin.All treatments were performed in 1% DMSO as a vehicle control (Bis).</p