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

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

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

<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

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

<i>In vivo</i> study protocol in pigtailed macaques.

<p>(<b>A</b>) Two studies were performed. The first included five macaques, in which each monkey received placebo films followed six weeks later with RC-101 films. The second study included the five original macaques plus an additional monkey, in which half received RC-101 films and half received placebo films. Six weeks later, each monkey receiving the opposite film type. (<b>B</b>) Baseline measurements for colposcopy, vaginal pH, and microflora were obtained at day 0. At days 1–4, RC-101- or placebo-containing films were instilled intravaginally. At days 5 and 8, cytobrushes, blood, and pinch biopsies were obtained, and at day 8 CVL fluids were obtained from each monkey.</p

RC-101 films do not alter vaginal pH or microbiological profile of the cervicovagina.

<p>(<b>A</b>) Vaginal pH was measured prior to film instillation at days 1-4 (D1-D4) and at followup (D5 and D8). Each measurement represents one experimental condition in a pigtailed macaque. Horizontal lines represent the mean value for all 11 replicates. No differences were observed between monkeys that were receiving RC-101 films or placebo films. (<b>B</b>) represents the difference in vaginal pH between the time zero condition and 30 minutes after film instillation for each day films were applied (D1–D4). Horizontal lines represent the mean value for all 11 replicates. A significant difference was observed at Day 4 (<i>P</i> = 0.003), with the placebo films inducing a greater change in vaginal pH than the RC-101 films. (<b>C</b>) Four (of twenty) microbes are presented for both RC-101 films and placebo films. For each microbe, there are 10 vertical bars, representing the number of replicates (from a total of 11) that were positive for the microbe indicated. The ten bars for each microbe signify the following from left to right: Day 1 (D1) time zero (t0), D1, time 30 min (t30), D2 t0, D2 t30, D3 t0, D3 t30, D4 t0, D4 t30, D5 and D8.</p

RC-101 was retained in the cervicovagina up to four days post-application.

<p>RC-101 was assessed by quantitative western blot from acetic acid-extracted samples of cervical biopsies, vaginal biopsies and cytobrushes at day 5, and cytobrushes and CVL fluid at day 8 (<b>A</b>). Note that CVL fluid was subdivided into both the cellular portion and the fluid portion. A representative western blot for RC-101 in cytobrushes at day 5 is shown in (<b>B</b>), with “M1”, “M2”, and “M3” signifying three individual monkeys. Note that the same monkey received both RC-101 films and placebo films six weeks apart, and that RC-101 was absent in cytobrushes obtained from monkeys that received placebo films. The <i>P</i>-values presented were adjusted for multiple comparisons using Tukey method.</p

Colposcopy of the cervicovagina reveals no adverse effects of RC-101 films.

<p>Images from colposcopic examination of cervicovaginal mucosa of a representative pigtailed macaque obtained prior to film insertion, at the time of insertion (Time 0), and 30 min and 24 hr after film insertion. Note the absence of mucosal aberrations at all time points with either RC-101 films or placebo films.</p