Volume 4, Issue 5, Pages 931-937 (September 2013) The RNA Methyltransferase Dnmt2 Is Required for Efficient Dicer-2-Dependent siRNA Pathway Activity in Drosophila  Zeljko Durdevic, Mehrpouya Balaghy Mobin, Katharina Hanna, Frank Lyko, Matthias Schaefer  Cell Reports  Volume 4, Issue 5, Pages 931-937 (September 2013) DOI: 10.1016/j.celrep.2013.07.046 Copyright © 2013 The Authors Terms and Conditions

Figure 1 RNA Fragmentation Patterns in Dnmt2 Mutant Flies Indicate Specificity for tRNA (A) Northern blot (15 μg RNA) for 5′-fragments (<70 nt) of tRNAAspGTC before and after heat shock (37°C, 1 hr) of control (D2+/−) and Dnmt2 mutant (D2−/−) male flies, followed by 2 days of recovery (R). AC, cleavage in the anti-codon; smaller tRNA-derived fragments are marked by black arrowhead. (B) Presentation of tRNA-derived reads (normalized to the total read number of individual experiments; see Tables S1, S2, S3, and S4) after sRNA (25–70 nt) sequencing of a heat-shock experiment as in (A). (C) Overview of sRNA sequencing data from a heat-shock experiment as in (A). Percentages of mapped reads from individual experiments are displayed as pie charts and color-coded for rRNA, mRNA, tRNA, and RNAs from other sources (i.e., microbes). See also Figure S1 and Tables S1, S2, S3, and S4. Cell Reports 2013 4, 931-937DOI: (10.1016/j.celrep.2013.07.046) Copyright © 2013 The Authors Terms and Conditions

Figure 2 Dcr-2 Processes tRNAs into Halves and siRNA-Sized Fragments (A) Northern blot (using 5′ probes) for tRNAAspGTC and tRNAGlyGCC in Dcr-2-FLAG complexes after RNA immunoprecipitation. Arrowheads indicate unspecific tRNA binding (black), tRNA halves (gray), smaller tRNA-derived RNAs (white). IgG, immunoglobulin G control. (B) RNA in vitro cleavage assay using wild-type (+) and Dcr-2 catalytic mutant (−) ovary protein extracts (15 μg) on purified tRNA preparations followed by northern blotting for tRNAs using 5′ probes against tRNAAspGTC (upper) and tRNAGlyGCC (lower). Arrowheads indicate tRNA (black) and tRNA fragmentation products (gray). HS, heat shock; PE, protein extract; R-Inh, RNase inhibitor. (C) Northern blot on RNA (15 μg) from S2 cells that express endogenous (−) or ectopic Dcr-2-FLAG protein (+) using probes against tRNAAspGTC and tRNAGlyGCC. (D) RNA in vitro cleavage assay using protein extracts as in (B) on tRNAAspGTC oligonucleotides (nt 1–38; 1 μM). Upper part shows SYBR-stained Urea-PAGE-gel. Lower part shows northern blot using 5′ probes against tRNAAspGTC. Arrowheads indicate tRNA (black) and tRNA oligonucleotide (gray). Mg++, Mg ions. See also Figure S2. Cell Reports 2013 4, 931-937DOI: (10.1016/j.celrep.2013.07.046) Copyright © 2013 The Authors Terms and Conditions

Figure 3 Dnmt2 Mutants Show Reduced Dcr-2 Activity on Long dsRNA (A) Dcr-2 cleavage assay on 32P-labeled egfp-derived dsRNA (5 ng) using protein extracts (15 μg) from wild-type (WT) and Dcr-2 catalytic mutant (Dcr-2catΔ) ovaries. Arrowheads indicate long dsRNAs (black) and sRNA duplexes (sRNA, gray). (B) Dcr-2 cleavage assay on egfp-derived dsRNAs using protein extracts (15 μg) from WT ovaries and increasing amounts of tRNAAspGTC oligonucleotides (3, 6, 10 μM). Arrowheads indicate RNAs as in (A). (C) Dcr-2 cleavage assay on egfp-derived dsRNA using protein extracts from WT, Dnmt2 mutant (D2−/−), and Dcr-2 catalytic mutant (Dcr-2catΔ) ovaries before and after a heat shock (37°C, 1 hr). Increasing amounts of protein extract (7.5, 15, and 30 μg) from WT and Dnmt2 mutants were added to the reaction to recover the heat-shock-induced decrease of Dcr-2 activity on dsRNA (see Figure S3B). Arrowheads indicate RNAs as in (A). HS, heat shock; PE, protein extract. (D) Dcr-2 cleavage assay on egfp-derived dsRNA using protein extracts (15 μg) from WT and Dnmt2 mutant (D2−/−) ovaries after a heat shock (37°C, 1 hr) and increasing incubation times (1, 2, and 3 hr). Arrowheads indicate RNAs as in (A). (E) Western blot of protein extracts (25, 50, 80 μg), which were used in Dcr-2 cleavage assays as in (C) and probed for Dcr-2, Dnmt2, Hsp70, and Tubulin levels. (F) Northern blot of RNA (5, 10, and 15 μg) extracted from protein extracts used in Dcr-2 cleavage assays (C) with 5′ probes against tRNAAspGTC. Arrowheads indicate full-length tRNA (black) and tRNA halves (gray). See also Figure S3. Cell Reports 2013 4, 931-937DOI: (10.1016/j.celrep.2013.07.046) Copyright © 2013 The Authors Terms and Conditions

Figure 4 Dnmt2 Mutants Accumulate DsRNAs and Show Reduced sRNA Production (A) Dot-blot analysis of decreasing amounts of total RNA (1 μg to 125 ng) from WT and Dcr-2 catalytic mutant (Dcr-2catΔ) flies. Blots were stained with methylene blue for loading control, followed by probing with antibodies against dsRNA. (B) Dot-blot analysis of total RNA (2 μg) from a heat-shock experiment with WT and Dnmt2 mutant (D2−/−) flies. Blots were stained with methylene blue to control loading, followed by probing with antibodies against dsRNA. (C) Northern blot of RNA (15 μg) from adult WT (w1118, yw), Dcr-2, Ago-2, and Dnmt2 mutant flies using probes for esi-2.1 RNA, miR-bantam, and 2S rRNA. (D) Northern blot of RNA (15 μg) from WT and Dnmt2 mutant (D2−/−) flies during a heat-shock experiment probed for esi-2.1, miR-bantam and 2S rRNA. (E) qPCR analysis for mus308 mRNA in adult WT, Dcr-2 (Dcr-2catΔ) and Dnmt2 mutant (D2−/−) flies during a heat-shock experiment (hs = 37°C, R = recovery in days). Three independent experiments were performed for quantification (mean ± SD). (F) qPCR analysis for mus308 mRNA in WT and Dnmt2 mutant (D2−/−) male soma (left) and germline tissue (right) during a heat-shock experiment as in (E). Three independent experiments were performed for quantification (mean ± SD). See also Figure S4. Cell Reports 2013 4, 931-937DOI: (10.1016/j.celrep.2013.07.046) Copyright © 2013 The Authors Terms and Conditions

Figure S1 RNA Fragmentation Analysis during Heat Shock of Adult Somatic Tissues, Related to Figure 1 (A) Box plot displays tRNA read differences between genotypes and experimental conditions (see Tables S1–S3). P-values were calculated using Wilcoxon’s matched pair test. (B) Fold change of reads corresponding to Dnmt2-substrate tRNAs (GlyGCC, AspGTC) and tRNAGluCTC(TTC) between genotypes and experimental conditions as in Figure 1A (up). Fold change of reads corresponding to other abundant tRNAs (including non-Dnmt2 substrate tRNAs) between genotypes and experimental conditions as in Figure 2 (bottom). Reads were normalized to reads from control flies set to 1. (C) Breakpoints in individual 5′-derived fragments of tRNAAspGTC were mapped and visualized as percentage of all 5′-derived fragments (see Tables S2 and S3). Cartoon introduces color coding for individual fragments breaking at specific nucleotides. (D) Presentation of ribosomal RNA-derived reads (normalized to the total read number in individual experiments) after small RNA sequencing of RNA from a heat shock experiment as in Figure 1A. (E) Box plot displays rRNA read differences between genotypes and experimental conditions as in (D). P-values were calculated using Wilcoxon’s matched pair test. (F) List of mRNAs with top scores of mRNA-derived reads from a heat shock experiment as in Figure 1C (see GEO database accession GSE35981). (G) Presentation of mRNA-derived sequences from small RNA sequencing of a heat shock experiment as in Figure 1A and after mapping reads to annotated gene regions (UTRs, introns, exons). (H) Box plot displays mRNA read differences for the mRNAs as described in (F and G). P-values were calculated using Wilcoxon’s matched pair test. ctrl = control, hs = heat shock, R = recovery in days. Cell Reports 2013 4, 931-937DOI: (10.1016/j.celrep.2013.07.046) Copyright © 2013 The Authors Terms and Conditions

Figure S2 Identification of Dcr-2-Associated Activities that Degrade tRNAs, Related to Figure 2 (A) RNA cleavage assay on tRNAAspGTC oligonucleotides (1-38 nt, 1 μM). SYBR-stained Urea-PAGE-gel reveals a heat shock-dependent activity that degrades tRNA-derived sequences. Arrowheads indicate tRNA (black) and tRNA oligonucleotides (gray). Bracket indicates degradation products. (B) SYBR-stained Urea-PAGE-gel after in vitro RNA cleavage of tRNAAspGTC oligonucleotides (1 μM) using protein extracts depleted of Dcr-1 or Dcr-2 by dsRNA interference. Arrowheads indicate tRNA (black) and tRNAAspGTC oligo (gray). Bracket indicates size marker oligo (19 nt). Asterisks indicate loss of cleavage products. (C) Northern blot of the in vitro RNA cleavage assay (B) using 5′ probes against tRNAAspGTC. Arrowhead indicates tRNA oligonucleotide (gray) and arrow shows size marker tRNAAspGTC (1-19 nt). Asteriks denote missing cleavage products in dcr-2 dsRNA treated cell extracts. (D) Western blot of hypotonic S2 cell protein extracts (100 μg) used in (A and B), which were treated with dsRNA against egfp, dcr-1 or dcr-2. (E) DsRNAi-mediated knock-down of various ribonuclease gene products in S2 cells. Cells were heat shocked or left untreated (+/−). Hypotonic protein extracts (15 μg) were incubated with tRNAAspGTC oligonucleotides (1 μM), the cleavage products were analyzed on Urea-PAGE gels and stained with SYBR. Asterisks indicate loss of cleavage products. (F) Western blot on protein extracts (25-100 μg) from wild-type (+) and Dcr-2 catalytic mutant (-) ovaries that were used for RNA cleavage assays in Figure 2D. PE = protein extract, R-Inh = RNase Inhibitor; HS = heat shock, nt = nucleotides, dsRNA = double-stranded RNA. Cell Reports 2013 4, 931-937DOI: (10.1016/j.celrep.2013.07.046) Copyright © 2013 The Authors Terms and Conditions

Figure S3 Dcr2 Activities Are Transiently Reduced After Heat Shock, Related to Figure 3 (A) Western blotting for Dcr-2, Ago-2, Hsp70 and Tubulin protein levels in S2 cells (80 μg protein) during a heat shock experiment (hs = 37°C, 1 hr, R = recovery in hours). (B) Dcr-2 cleavage assay on egfp-derived dsRNA (5 ng) using protein extracts (15 μg) from S2 cells during a heat shock experiment as in (A). (C) Northern blot on RNA (5 μg) extracted from S2 cells during a heat shock experiment as in (A) and probed for 5′-fragments (<70 nt) of tRNAAspGTC. (D) Q-PCR analysis for siRNA components in adult wild-type (wt) and Dnmt2 mutant (D2−/−) flies during a heat shock experiment. Three independent experiments were performed for quantification (mean ± SD). (E) Western blotting for Dcr-1, Dcr-2, Ago-2, Hsp70 and Tubulin protein levels in adult wild-type (wt) and Dnmt2 mutant (D2−/−) flies (80 μg) during a heat shock experiment as in (D). AC = products from cleavage in the anti-codon; small tRNA-derived fragments are marked by gray arrowhead. Cell Reports 2013 4, 931-937DOI: (10.1016/j.celrep.2013.07.046) Copyright © 2013 The Authors Terms and Conditions

Figure S4 Ectopic tRNA Fragments Induce siRNA Pathway Mutant Conditions, Related to Figure 4 (A) Western blotting for Dcr-2, Ago-2 and Tubulin expression in S2 cells that were treated with dsRNAs against egfp, dcr-2 and ago-2 before (ctrl) and 24 hr after a heat shock (hs = 37°C). (B) Q-PCR analysis for various genes that are regulated by Dcr-2 or Ago-2 function in S2 cells that were pre-treated with dsRNAs as in (A) before (ctrl) and 6 or 24 hr after a heat shock (hs = 37°C). Error bars represent normalized Cp errors of three technical replicates from three independent dsRNA treatments that were pooled before cDNA synthesis. (C) SYBR-stained urea-PAGE with total RNA from adult wild-type (wt) and Dnmt2 mutant (D2−/−) flies during a heat shock experiment as in (A) indicating the regions between 10-65 nucleotides that were extracted to enrich for small RNAs (top). Northern blot on extracted small RNAs (100 ng per lane) using a probe against 2S rRNA (bottom). (D) Q-PCR analysis for Dcr-2 or Ago-2-regulated genes after treatment of S2 cells for 24 hr with small RNAs that were extracted as in (C). Three independent experiments were performed for quantification (mean ± SD). (E) Q-PCR analysis for Dcr-2 or Ago-2-regulated genes after treatment of S2 cells for 24 hr with two concentrations (10 nM, 100 nM) of small tRNAAspGTC oligonucleotides (1-38 nt). Three independent experiments were performed for quantification (mean ± SD). Cell Reports 2013 4, 931-937DOI: (10.1016/j.celrep.2013.07.046) Copyright © 2013 The Authors Terms and Conditions

Cell Reports 2013 4, 931-937DOI: (10.1016/j.celrep.2013.07.046) Copyright © 2013 The Authors Terms and Conditions