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The kinase ABL phosphorylates the microprocessor subunit DGCR8 to stimulate primary microRNA processing in response to DNA damage

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Sci. Signal.  30 Jun 2015:
Vol. 8, Issue 383, pp. ra64
DOI: 10.1126/scisignal.aaa4468
  • Fig. 1 ABL tyrosine kinase stimulates the expression of miR-34c.

    (A) Unsupervised hierarchical clustering of miRNAseq results from 293T cells treated for 24 hours with vehicle (V), imatinib (IM; 10 μM), doxorubicin (D; 0.5 μg/ml), or a combination of imatinib and doxorubicin (I + D). Ninety-three miRs were clustered into the “D, but not IM + D” category. (B) Top five miRNAs from (A) increased by doxorubicin but not by the imatinib + doxorubicin combination. The P values were calculated as described in Materials and Methods. (C and D) Production of miR-34a and miR-34c in MCF7 (C) or HCT116 (D) cells at 24 hours after the indicated treatments with imatinib and/or doxorubicin relative to vehicle treatment. (E) Immunoblotting of total lysates from the indicated HCT116 cells at 24 hours of the indicated treatments. kd, knockdown; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (F) Production of miR-34c in cells treated as in (E) relative to vehicle treatment. (G) Schematic of the human miR-34b/c gene locus. E1, upstream exon; EST, expressed sequence tag. (H) Expression of miR34b/c-E1 in cells treated as in (E) relative to vehicle treatment. Relative abundance values are means ± SD of at least three independent experiments. *P < 0.05, **P < 0.01, two-tailed t tests within cell line (C, D, F, and H); ***P < 0.001, two-way analysis of variance (ANOVA) test of interaction, with Bonferroni correction for the two main effect tests on the two cell lines (F).

  • Fig. 2 ABL kinase stimulates Drosha-dependent processing of pri-miR-34c from a minigene.

    (A) Diagrams of vector (Vec) and the minigenes (34a and 34c). The primer sets for the mature miRNAs and the uncut pri-miRNAs are shown. Numbers are genomic sequences (nt) flanking the pre-miRNA stem-loop. (B) Schematic of AblPPn depicting the activating mutations (42, 43). (C) Immunoblotting for ABL in HCT116 cells transfected with the indicated constructs. (D) Production of miR-34a and miR-34c in HCT116 cells transfected as indicated relative to vector-only–transfected cells. (E) Northern blots of total RNA from transfected HCT116 cells probed for miR-34a, miR-34c, and U6. (F) Expression of indicated mRNA in HCT116 relative to vector-only–transfected cells. (G and H) Production of miR-34c in 34c minigene–transfected 293T cells with the indicated cotransfections of AblPPn and Dicer–small interfering RNA (siRNA) (G) or with TN-Drosha or wild-type (WT) Drosha (H) relative to minigene-only–transfected cells. (I and J) Expression of pri-miR-34c in 34c minigene–transfected 293T cells with the indicated cotransfections of AblPPn, Drosha, or TN-Drosha (I) or treated with imatinib (J) relative to minigene-only–transfected cells. pTyr, phosphotyrosine. Data are means ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed t tests.

  • Fig. 3 Sequences flanking pre-miR-34c stem-loop determine the miRNA-selective effect of ABL.

    (A) Schematics of pri-miR-34a and pri-miR-34c transcribed from the minigenes. Arrowheads denote the predicted Drosha cleavage sites. The pri-miR-34a contains upstream (light blue) and downstream (purple) extensions not in pri-miR-34c. Flanking sequences are indicated in orange (34a) or dark blue (34c). (B) Schematics of the hybrid minigenes with color-coding of sequence blocks shown in (A). Primers for measuring the uncut pri-miRNAs are shown as orange (34a-flanking) and blue (34c-flanking) arrowheads. (C) Expression of the indicated pri-miRNA in HCT116 cells cotransfected with the indicated minigenes and AblPPn relative to vector cotransfected cells. (D) Immunoblotting of lysates from HCT116 cells cotransfected with AblPPn and TN-Drosha as indicated. (E) Expression of the indicated pri-miRNA in HCT116 cells cotransfected with minigenes and the indicated plasmids relative to vector cotransfected cells. Data are means ± SD from three independent experiments. **P < 0.01, ***P < 0.001, two-tailed t tests.

  • Fig. 4 Flanking sequences promote and tyrosine phosphorylation reduces DGCR8 interaction with pri-miR-34c.

    (A) Percentage of input pri-miRNA cross-linked to endogenous DGCR8 in HCT116 cells transfected with the indicated minigenes and expression plasmids. Primers for quantitative reverse transcription polymerase chain reaction (qRT-PCR) of the pri-miRNAs are shown. (B) Domains of DGCR8. Rhed, RNA binding heme domain; WW, WW domain; dsRBD1 and dsRBD2, double stranded RNA binding domain 1 and 2. Phosphorylation site Tyr267 (Y267) is located between the NLS and the Rhed domain. (C) Immunoblotting of total lysates or anti-FLAG pull-down fractions from HCT116 cells transfected with the indicated plasmids. IP, immunoprecipitation. (D) Abundance of pri-miR-34c (left) and miR-34c (right) in cells cotransfected as indicated with the 34c minigene relative to minigene-only–transfected cells. (E) Percentage of input pri-miR-34c cross-linked to FLAG-epitope in HCT116 cells cotransfected with the 34c minigene and the indicated plasmids. (F) Percentage of input pri-miR-34c cross-linked to Drosha in HCT116 cells cotransfected with the indicated plasmids. (G) Immunoblotting (IB) (left) and pri-miR-34c expression (right) of HCT116 cells cotransfected with the 34c minigene and the indicated plasmids relative to minigene-only–transfected cells. Data are means ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed t tests.

  • Fig. 5 Nuclear import of ABL is required for miR-34c production in mouse tissues.

    (A) Knock-in substitution mutations inactivate the three NLS in the mouse Abl-μNLS (μ) allele (27). (B) Expected and observed frequencies of Abl genotypes among 201 pups from breeding Abl+/μ mice with χ2 test and P value. (C to F) Abundance of miR-34a and miR-34c in mouse kidney (C), liver (D), thymus (E), and spleen (F) at 48 hours after intraperitoneal injection with vehicle (white bars) or cisplatin (gray bars) relative to vehicle-treated Abl+/+ tissues. Data are means ± SD of three mice. ***P < 0.001, two-way ANOVA test of interaction with Bonferroni corrections on the two main effect tests on the two mouse Abl genotypes in (C); *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed t tests of the cisplatin effects on miRNA expression (D to F).

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/8/383/ra64/DC1

    Fig. S1. Effects of ABL knockdown and imatinib on doxorubicin-induced miRNA expression.

    Fig. S2. Expression of mRNAs predicted to be targets of the miR-34 family of miRNAs.

    Fig. S3. Sequences flanking the pre-miRNA stem-loops did not affect DGCR8 binding in vitro.

    Fig. S4. Reduced expression of pri-miR-34a and pri-miR-34c in Ablμ/μ mouse embryo fibroblasts.

    Fig. S5. Limited overlaps between Mim23-DGCR8 stimulated progrowth miRNAs in HeLa cells and the miRNAs identified in this study.

    Table S1. Ranking of 22 miRNAs in the “induced by doxorubicin but not by imatinib + doxorubicin” cluster by the significance of doxorubicin effect (from the miRNAseq data).

    Table S2. Primers used in this study.

  • Supplementary Materials for:

    The kinase ABL phosphorylates the microprocessor subunit DGCR8 to stimulate primary microRNA processing in response to DNA damage

    Chi-Chiang Tu, Yan Zhong, Louis Nguyen, Aaron Tsai, Priya Sridevi, Woan-Yuh Tarn, Jean Y. J. Wang*

    *Corresponding author. E-mail: jywang{at}ucsd.edu

    This PDF file includes:

    • Fig. S1. Effects of ABL knockdown and imatinib on doxorubicin-induced miRNA expression.
    • Fig. S2. Expression of mRNAs predicted to be targets of the miR-34 family of miRNAs.
    • Fig. S3. Sequences flanking the pre-miRNA stem-loops did not affect DGCR8 binding in vitro.
    • Fig. S4. Reduced expression of pri-miR-34a and pri-miR-34c in Ablμ/μ mouse embryo fibroblasts.
    • Fig. S5. Limited overlaps between Mim23-DGCR8 stimulated progrowth miRNAs in HeLa cells and the miRNAs identified in this study.
    • Table S1. Ranking of 22 miRNAs in the “induced by doxorubicin but not by imatinib + doxorubicin” cluster by the significance of doxorubicin effect (from the miRNAseq data).
    • Table S2. Primers used in this study.

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    Citation: C.-C. Tu, Y. Zhong, L. Nguyen, A. Tsai, P. Sridevi, W.-Y. Tarn, J. Y. J. Wang, The kinase ABL phosphorylates the microprocessor subunit DGCR8 to stimulate primary microRNA processing in response to DNA damage. Sci. Signal. 8, ra64 (2015).

    © 2015 American Association for the Advancement of Science

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