Volume 25, Issue 4, Pages 575-586 (February 2007) Ribonuclease Dicer Cleaves Triplet Repeat Hairpins into Shorter Repeats that Silence Specific Targets  Jacek Krol, Agnieszka Fiszer, Agnieszka Mykowska, Krzysztof Sobczak, Mateusz de Mezer, Wlodzimierz J. Krzyzosiak  Molecular Cell  Volume 25, Issue 4, Pages 575-586 (February 2007) DOI: 10.1016/j.molcel.2007.01.031 Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 1 Transcripts Containing Long CNG Repeats Are Cleaved by Recombinant Dicer Reactions of the 5′ end-labeled pre-miR-24 (A), CUG17_cl (B), and CUG35_cl (C) transcripts with Dicer. Lanes Ci, incubation control without Dicer; lanes F, formamide ladder; and lanes T, guanine-specific ladder. The positions of selected G residues are shown. Note that Dicer cleavage products having 3′OH groups migrate slower than the T1 and formamide products of the same lengths, which have 2′3′-cyclic phosphate, and this difference is about 1 nt in the length range of 20–40 nt under the conditions used (data not shown). The primary cleavage sites (marked with black boxes and black arrowheads) and secondary cleavage sites (marked with white arrowheads) are shown in the autoradiograms and secondary structure models. (D) The halftime (in minutes) of substrate disappearance for the 32P-labeled CNG25_cl (white bars) and CNG35_cl (gray bars) transcripts, calculated after background subtraction. The presented data are the average of two independent experiments. Cleavage patterns obtained for the 5′ end-labeled CUG25_cl (E), CUG25 (F), and CAG25_cl, CCG25_cl, and CGG25_cl (G) transcripts after reaction with Dicer. The cleavage products were analyzed as described in (A)–(C). The X3 and X8 designate three and eight repetitions of central CNG repeats in the CNG25 and CNG35 transcripts, respectively. Molecular Cell 2007 25, 575-586DOI: (10.1016/j.molcel.2007.01.031) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 2 Dicer Knockdown Changes the Cellular Levels of Mutant Transcripts Containing CAG or CUG Repeats (A) Cellular levels of Dicer mRNA examined by means of real-time RT-PCR from cDNA template obtained from cells treated with control siRNA (black bars) and Dicer-specific siRNA (gray bars). The presented data are the average of three independent experiments. Right panel, analysis of Dicer and GAPDH protein levels by western blot after Dicer knockdown. (B) Northern blot analysis of mutant (1000 CUG) and normal (15 CUG) DMPK transcript levels after treatment of the DM1 fibroblast cells with Dicer-specific siRNA (lane −Dicer) or control siRNA (lane +Dicer). Intensity of signals corresponding to normal (black) and mutant (gray) transcripts was measured densitometrically (ImageQuant 5.1, Molecular Dynamics) and is proportional to bar heights. Intensity of signals normalized to GAPDH mRNA level was compared by paired t test (Prism v. 4, GraphPad Software, San Diego, CA). (C) Cellular levels of DMPK mRNAs were examined by means of real-time RT-PCR using the cDNA as templates obtained from cells treated with control siRNA (DM1) and Dicer-specific siRNA (DM1-Dicer) and the TaqMan probes for the DMPK and GAPDH cDNA (Applied Biosystems). Levels of DMPK transcripts normalized to GAPDH mRNA level were compared by paired t test. The data shown are the average of four independent experiments. (D) Left panel, analysis of the STR region to determine transcript levels by semiquantitative RT-PCR after treatment of the HD and SCA1 fibroblast cells with siRNA against Dicer mRNA (lane cDNA-Dicer). DNA template and cDNA isolated from the HD and SCA1 cells treated with control siRNA (lane cDNA) were used as controls. The lengths of HD and SCA1 STR regions are shown. Right panel, analysis of STR regions to determine levels of HD and SCA1 transcripts after Dicer knockdown (lane cDNA-Dicer). Intensity of signals from five independent experiments corresponding to normal (black bars) and mutant (gray bars) transcripts was measured densitometrically (ImageQuant 5.1). (E) Schematic representation of the STR-SNP haplotypes in HD and SCA1 mutant and normal transcripts. (F) Allele-specific real-time RT-PCR analysis of expression levels of mutant and normal HD and SCA1 transcripts. The experiments were repeated at least three times. P value was calculated by using paired t test. In all graphs, the error bars represent standard deviations. Molecular Cell 2007 25, 575-586DOI: (10.1016/j.molcel.2007.01.031) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 3 Effects Observed in EGFP/CNG HeLa Cells after Dicer Knockdown and Exogenous (CAG)7 and (CUG)7 siRNA Transfection (A) Analysis of Dicer and GAPDH mRNA and protein levels in cells treated with control siRNA and Dicer-specific siRNA (lane +siDicer). (B) RT-PCR (top) and real-time RT-PCR (bottom) analysis of EGFP transcripts containing 30, 70, or 200 CAG or CUG repeats in EGFP/CNG HeLa cells after Dicer knockdown (lane +siDicer). Levels of EGFP/CNG transcripts normalized to GAPDH mRNA level were compared by paired t test. Error bars represent standard deviations. The presented data are the average of three independent experiments. (C) EGFP protein expression observed by fluorescence microscopy in EGFP/CNG HeLa cells treated with either control, (CUG)7, or (CAG)7 siRNAs. Scale bar, 200 μm. Molecular Cell 2007 25, 575-586DOI: (10.1016/j.molcel.2007.01.031) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 4 Short Dicer Cleavage Products Composed of CNG Repeats Are Produced in TRED Cells and EGFP/CNG HeLa Cells Northern blot hybridization of fibroblast total RNA from (A) WT, HD, SCA1, and DM1 cells or (B) EGFP/CNG HeLa cells, having either normal or decreased Dicer level (lanes −Dicer), with oligonucleotide probes complementary to (CAG)7 (upper panels) and (CUG)7 (central panels). Expression levels of let-7, miR-21, and miR-16 were also determined (lower panels), and hybridization with U6 probe served as RNA loading control (bottom panels). Molecular Cell 2007 25, 575-586DOI: (10.1016/j.molcel.2007.01.031) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 5 Dicer Knockdown Shows Downstream Silencing Effects (A) Analysis of the SCA1 and HD transcripts levels by semiquantitative RT-PCR after treatment of the DM1 fibroblast cells with siRNA against Dicer (lane DM1-Dicer) or control siRNA (lane DM1). Control lanes show the results obtained from cDNA prepared from the control fibroblast cells (lanes WT-Dicer; WT). The genotypes of the SCA1 and HD STR regions in DM1 and control cells are shown. (B) Bar graph shows the cellular levels of all analyzed transcripts, as determined by RT-PCR of their STR regions (SCA1, HD, SCA3, AR, and MAB21L1) or northern blot (DMPK) measured densitometrically (ImageQuant 5.1). Intensity of signals normalized to GAPDH mRNA level was compared by paired t test. (C) Left panel, (top) analysis of Ago2 and GAPDH protein levels by western blot after treatment of the DM1 and WT fibroblast cells with siRNA against Ago2 (lines DM1-Ago2; WT-Ago2); (bottom) levels of DMPK transcript as determined by real-time RT-PCR after Ago2 knockdown. Right panel, levels of SCA1 and HD transcripts determined by semiquantitative RT-PCR after treatment of the DM1 fibroblast cells with siRNA against Ago2 (DM1-Ago2) or control siRNA (DM1). Graphs represent averages of three independent experiments. P value was calculated by paired t test. Error bars in (B) and (C) represent standard deviations. Molecular Cell 2007 25, 575-586DOI: (10.1016/j.molcel.2007.01.031) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 6 Cellular Effects Caused by Exogenous siRNAs Composed of Repeats (A) The 21 nt (CAG)7 or (CUG)7 siRNAs used in the experiments. The structure in which these reagents exist in cells is not known, but the alternatives to unstable duplexes are single-stranded structures and unstable hairpins. (B) Analysis of the SCA1 and HD transcript levels by RT-PCR after treatment of the DM1, HD, and SCA1 cells with (CAG)7 or (CUG)7 siRNAs. (C) Bar graph shows the cellular levels of all analyzed transcripts, as determined by the RT-PCR of their STR regions (SCA1, HD, SCA3, AR, and MAB21L1) or northern blot (DMPK) measured densitometrically (ImageQuant 5.1). All experiments were performed at least three times. Intensity of signals normalized to GAPDH mRNA level was compared by paired t test. Error bars represent standard deviations. (D) Northern blot analysis of mutant and normal DMPK transcript levels after treatment of the DM1 cells with (CAG)7 (lane DM1-Dicer) or control siRNA (lane DM1). Molecular Cell 2007 25, 575-586DOI: (10.1016/j.molcel.2007.01.031) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 7 A Model Depicting the Downregulation of Mutant Transcripts by Dicer in Cells and Downstream Effects Caused by the Endogenous and Exogenous Twin Strand siRNAs Molecular Cell 2007 25, 575-586DOI: (10.1016/j.molcel.2007.01.031) Copyright © 2007 Elsevier Inc. Terms and Conditions