Fig 2: Up-regulation of full-length Dicer and Dicer fragments, induced by zymosan or LPS during differentiation of MM6 cells. (A) Scheme of Dicer domains. Epitope regions of antibodies used in this study are indicated. Most data were obtained with antibody (Ab) 600 to 650 and Ab 1701 to 1912. (B) Analysis of Dicer expression with Dicer antibodies 1701 to 1912 and 600 to 650. Lane 1, molecular weight markers; lane 2, undifferentiated MM6 cells; lane 3, MM6 cells differentiated with TGF-β (5 ng/mL) and 1,25diOHvitD3 (50 nM) (VD3) for 96 h; lane 4, zymosan (25 µg/mL) present during 96 h differentiation; and lane 5, LPS (1 µg/mL) present during 96 h differentiation. Whole-cell lysates (∼50 µg protein) were analyzed by Western blot (WB) using Dicer Ab 1701 to 1912. The membrane was reblotted with Dicer Ab 600 to 650. (C) Relative expression levels of full-length Dicer and Dicer fragments (∼50 and ∼170 kDa). In undifferentiated MM6 cells (Undiff), MM6 cells differentiated with TGF-β + 1,25diOHvitD3 (Diff) for 96 h, with zymosan present during differentiation, and with LPS present during differentiation. Band intensities were normalized to β-actin, before comparisons between samples. Data are from B and six additional experiments. Mean ± SE, n = 7.

Fig 3: DICER and major satellite repeat transcripts associate with chromatin. (A) In situ hybridization using a LNA probe for the forward strand-derived MSR transcripts (MSR-tr Fw, red) on WT testis cryosections. Strong signal was detected in the cytoplasm of late pachytene spermatocytes at stage IX of the seminiferous epithelial cycle. MSR-tr signal was also detected in the nucleus (arrows). Nuclei were stained by DAPI (grey). (B) The forward MSR transcript signal disappeared when the sections were treated with RNase prior to hybridization, demonstrating that the signal originated from RNA transcripts. No signal was detected using a scramble probe. Scale bars for A and B: 10 µm. (C) Relatively stronger expression of the forward MSR transcripts (red) was detected by in situ hybridization in Dicer1 cKO spermatocytes compared to the control. To facilitate the recognition of pachytene spermatocytes, the sections were immunostained using an antibody against phosphorylated gamma-H2AX (?H2AX, green), which detects double-strand breaks and labels the sex body (asterisks) in pachytene spermatocytes. Scale bar: 10 µm. The graph shows quantification of average signal intensity of cytoplasmic MSR Fw transcripts signal in CTRL and Dicer1 cKO pachytene spermatocytes. The signal was quantified from three independent technical replicates (15–20 cells each); SEM, ***P< 0.001. See also Supplementary Figure S1G. (D) Chromatin fractionation was performed from nuclear extracts of 18 dpp mouse testicular cells using increasing concentrations of NaCl, and RNA was extracted from the cytoplasmic fraction as well as from 300 mM and 600 mM NaCl chromatin fractions. MSR transcripts were detected separately from long (>200 nt) or short (<200 nt) RNA fraction with the LNA probe against the forward MSR transcript. Long MSR transcripts were found both in the cytoplasmic and chromatin fractions, while MSR transcript-derived small RNAs were mainly detected in the chromatin fractions. The numbers below the anti-DIG signal indicate relative intensities of MSR-tr Fw signal quantified by ImageJ. The signal intensities are shown relative to the cytoplasmic long RNA sample that was set as ‘1’. (E) Separation of total RNA into long and small RNA fractions was validated by agarose gel electrophoresis followed by ethidium bromide staining. (F) Immunoblotting of cytoplasmic and chromatin fractions with anti-DICER antibody demonstrated the presence of DICER in all fractions. Cellular fractions were validated by immunoblotting with antibodies against cytoplasmic proteins (ß-TUBULIN, GAPDH) and chromatin-associated proteins (YY1, H3). All panels show a representative figure of an experiment that was independently repeated at least two times.

Fig 4: Increased processivity of AGO2-associated DICER1 in the presence of target mRNA.(a) In vitro pre-miRNA processing assay with rAGO2 and rDICER1 reconfirmed target mRNA-driven increase in AGO2-associated DICER1 activity. In vitro pre-miRNA processing assay with rDICER1 and rAGO2 (native or heat-denatured) to quantify miR-122 biogenesis in the presence of target mRNA. Heat denaturation of rAGO2 was carried out at 95 °C for 5 min followed by rapid chilling. (b) In vitro pre-miRNA processing assay of pre-let-7a with rDICER1 and increasing concentrations of rAGO2 (10, 25 and 50 ng) in the presence of RL-con or RL-3 × bulge-let-7a (25 ng ml−1). Mature let-7a levels were measured and plotted. (c) Schematic representation of RL-3 × bulge-let-7a_5BoxB mRNA used in the in vitro assays. In vitro pre-miRNA processing assay of pre-let-7a with rDICER1 and 50 ng rAGO2 in the presence of RL-3 × bulge-let-7a or RL-3 × bulge-let-7a_5BoxB mRNA (both at 25 ng μl−1). Mature let-7a levels after the reaction were measured and plotted. (d) In vitro assay to measure the association of AGO2 and DICER1 along the 3′-UTR of target mRNAs. FH-AGO2 immunoprecipitated from HEK293 cells transiently expressing NHA-DICER1 was subjected to in vitro pre-miRNA processing assay with pre-miR-122 and RL-3 × bulge-miR-122 as described earlier, followed by immunoprecipitation of AGO2 and DICER1 with antibodies specific to endogenous proteins. Quantitative reverse transcriptase PCR (qRT-PCR) was done with indicated primers. (e) In vitro assay to measure processivity of DICER1. Immunopurified AGO2 (let-7a miRISC) incubated with 25 ng ml−1 RL-con or RL-3 × bulge-let-7a in the presence of pre-miR-122 (10 nM) at 37 °C for 30 min followed by RNA isolation and quantification of mature miR-122 formed by qRT-PCR. Paired two-tailed Student's t-tests were used for all comparisons. *P<0.05, **P<0.01 and ***P<0.001. In a–e, values are means from at least three biological replicates. Error bars represent s.d.

Fig 5: Dicer acts as a transcriptional repressor of p53.(A) Western blot analysis of the effect of different Dicer CRISPR-Cas9 knockout clones on p53 activity in HCT116 cells. sgRNA, single-guide RNA. (B) Western blot analysis of the effect of Dicer knockout in p53+/+ or p53−/− HCT116 cells. (C) Western blot analysis of the DNA damage response effect of Dicer knockout in p53 wild-type HCT116 cells. CRISPR-Cas9 negative control cells treated with doxorubicin (0.2 μg/ml) are positive control in DNA damage response. DMSO, dimethyl sulfoxide. sgNC, sgRNA Negative Control. (D) Western blot analysis of H1299 cells transfected with p53 or cotransfected with p53 and increasing amount of Dicer. (E) Quantitative polymerase chain reaction (qPCR) assay of the p21, PUMA, and Mdm2 genes regulated by the p53 and/or Dicer transfection in H1299 cells. (F and G) Western blot analysis and qPCR assay for detecting p21 level in H1299 cells transfected with p53 or cotransfected with p53 and Dicer or Dicer-∆. (H and I) Western blot analysis and qPCR assay for detecting p21 level in H1299 cells transfected with p53 or cotransfected with p53 and Dicer CT or CT-D1320A. Error bars in qPCR assay indicate means ± SD. n = 3 technical replicates. All data are shown as representative of three experiments.