Cell Reports Supplemental Information Somatic Primary pirna Biogenesis Driven by cis-acting RNA Elements and Trans-Acting Yb Hirotsugu Ishizu, Yuka W. Iwasaki, Shigeki Hirakata, Haruka Ozaki, Wataru Iwasaki, Haruhiko Siomi, and Mikiko C. Siomi
Supplemental Data A (nt) 4, 2, 1, 5 2 EGFP-tj MT MT-1 MT-2 MT-3 MT-4 egfp probe gapdh probe B (nt) 4, 2, 1, 5 2 MT-2 MT-2-1 MT-2-2 MT-2-3 egfp probe gapdh probe Figure S1. Northern blotting, related to Figure 2. (A) Northern blotting using an egfp probe shows the expression levels of EGFP-tj MT and its mutants. The gapdh transcript was visualized as a loading control. (B) Northern blotting using an egfp probe shows the expression levels of MT-2 and its mutants. The gapdh transcript was visualized as a loading control.
A sgrna: 1+2 1+3 (bp) 8 7 6 5 4 3 664 bp B OSC-Δtj-cis 1 target site 1 traget site 2 5 -GATTCCAAGAATGTTTTCCAAATAGTCTCCACTCGAAAGAACATGTTTTCCAAAAAGAGTTTGTTAAAGCGTTTCCAAGGGGTCA-3 3 -CTAAGGTTCTTACAAAAGGTTTATCAGAGGTGAGCTTTCTTGTACAAAAGGTTTTTCTCAAACAATTTCGCAAAGGTTCCCCAGT-5 65 bp deletion TTGGTTTGATTCCAAG A C AAGGGGT C A G A T G T C A G OSC-Δtj-cis 2 target site 1 target site 3 5 -GATTCCAAGAATGTTTTCCAAATAGTCTCCACTCGAAAGAAC...ATTTGTGTTATACCACGCGTTATTGTAAGTTTGTCCCTA-3 3 -CTAAGGTTCTTACAAAAGGTTTATCAGAGGTGAGCTTTCTTG...TAAACACAATATGGTGCGCAATAACATTCAAACAGGGAT-5 134 bp deletion T T GGTTT G AT TCCAAG A G TTAT TG T AAG TTTG T CCCT A TTT TTG T G T Figure S2. Targeted deletion of the Tj-cis-element by CRISPR/Cas9 from the Drosophila genome, related to Figure 3. (A) PCR on the genomic DNA isolated from OSC cells transfected with the CRISPR/Cas9 vector detected shorter DNA fragments (red arrowheads) (599 nt and 53 nt). A single DNA fragment (664 nt) appeared when genomic DNA from parental OSC cells was used (black arrowhead). (B) Sequencing of the PCR bands in Fig.3A confirms the genomic deletion in the mutant cell lines. Target sites of three sgrnas are shown in blue. Proto-spacer adjacent motifs are shown in red.
(nt) 4, 2, 1, 5 2 EGFP-tj-cis MT-5 egfp probe gapdh probe Figure S3. Northern blotting, related to Figure 4. Northern blotting using an egfp probe shows the expression levels of EGFP-tj-cis and MT-5. The gapdh transcript was visualized as a loading control.
A (kd) Yb CLIP 1 Yb CLIP 2 B 3 26 16 11 8 Yb CLIP1 2 1 6 5 4 1 R=.98 2 3 3 Yb CLIP2 C Tj locus 5 bp RefSeq 2 pirna 1 Yb CLIP1 D (nt) MT-8 EGFP-tj-cis MT-6 MT-7 E 3 4, 2, 1, egfp probe Count 2 1 base A C G U 5 2 gapdh probe -1-5 5 1 Distance from crosslinked sites [nt] F Tj locus 5 bp RefSeq 1 Yb CLIP1 CIMS Tj-R2 Tj-R1 Tj-cis-element CIMS 344 Tj-cis-element Yb CLIP1 Tj-R1 CIMS 31 Yb CLIP1 Tj-R2 CIMS 712 Yb CLIP1
Figure S4. Yb binds pirna cluster transcripts, related to Figure 5. (A) Autoradiograph of Yb RNA complexes used for HITS-CLIP library generation. Library construction was performed twice. (B) The reproducibility of all Yb binding sites, when comparing two CLIP experiments was high (Pearson correlation co-efficient R =.98). The axis shows the amount of reads in each of the multisample clusters in log1 scale. (C) A browser view of Yb-CLIP tags and pirna sequences in wild-type OSCs (Figure S1A) mapped onto the Tj locus. The red dashed line indicates the Tj-cis-element. Signals are displayed as read counts. (D) Northern blotting using an egfp probe shows the expression levels of the constructs shown in Figure 5B. (E) Nucleotide composition around the CIMS identified in Yb HITS-CLIP data. The x-axis represents coordinates relative to the CIMS, where negative and positive values represent the upstream and downstream positions, respectively. The y-axis represents raw counts of each base. (F) CIMS defined with FDR <.1 mapped to Tj locus. Top panel: the Tj locus, with the number of Yb-CLIP tags and CIMS. Bottom panel: an enlarged view of the Tj-cis-element, Tj-R1 and Tj-R2.
A (nt) 4, 2, 1, MT-9 EGFP-tj-cis EGFP-flam-e1 EGFP-flam-e2 egfp probe B (nt) 4, 2, 1, MT-9 EGFP-tj-cis EGFP-flam-e1 EGFP-flam-e1-R2 EGFP-flam-e1-R1 egfp probe 5 2 gapdh probe 5 2 gapdh probe Figure S5. Northern blotting, related to Figure 6. (A and B) Northern blotting using an egfp probe shows the expression levels of the constructs shown in Figure 6B (A) and Figure 6D (B). The gapdh transcript was visualized as a loading control.
Figure S6. Artificial pirnas cause transcriptional silencing though Piwi-dependent pathway, related to Figure 7. (A) Scheme of the experiment to assess the exogenous pirna-induced gene silencing. (B) The efficiency of Krimper gene silencing through Krimper targeting pirnas as shown in Figure 7B was validated by the following experiments. Western blotting was performed to assess Krimp protein levels in blasticidin-selected exogenous pirna-expressing OSCs. Krimp protein levels were normalized to β-tubulin. Relative expression of OSCs transfected with the EGFP-tj-cis construct was presented as 1.. Bars represent means ± SD of three independent experiments. **P <.1. (C) The percentage of Krimp body containing EGFP-positive cells was calculated. Bars represent means ± SD of three independent experiments. (D) Western blot analysis of exogenous pirna-induced silencing in Piwi-depleted cells. Bars represent means ± SD of three independent experiments. *P <.5. (E) ChIP-qPCR analysis of RNA Pol II and H3K9me3 occupancy on the Krimp promoter following expression of Krimp targeting exogenous pirnas (Krimp-CDS-1 and Krimp-3 ). No targeting was used as a negative control. Bars represent means ± SD of three independent experiments. *P <.5. (F) The impact of strand orientation on the occupancy of RNA Pol II and H3K9me3 on the target region (left panel). Bars represent means ± SD of three independent experiments. *P <.5. Northern Blot analysis showed that sense or antisense pirnas were produced (right panel).
nucleus Yb primary pirna source Yb Yb primary binding of Yb pirna precursors target gene pirisc secondary binding of Yb Flam body Yb body primary pirnas Zuc MITO Figure S7. Model of somatic primary pirna biogenesis in Drosophila, related to all Figures. Our present study suggests that Yb determines primary pirna sources by two sequential modes of action: primary binding to cis-elements, representing the selection of pirna precursors among cellular RNAs, then secondary binding to downstream regions that represents the defining domains to be processed. The secondary binding may be accomplished either by 5 to 3 translocation or multimerization of Yb on the substrate. These implications are based on the fact that Yb is a member of the DEAD-box RNA helicases and our experimental observation that Yb self-associates in OSC cells (data not shown), respectively.
Table S1. DNA oligonucleotides and sirnas, related to experimental procedures. (Table S1.xlsx)
Supplemental Experimental Procedure Plasmid construction The EGFP-tj MT construct was generated using a KOD plus mutagenesis kit (Toyobo) with the primers EGFP-tj MT F/R. To generate the MT-1 construct, the Tj 3 UTR 51 1467 nt region of EGFP-tj MT was removed by inverse PCR using the primers MT-1 F/R. MT-2 and MT-3 constructs were also generated by inverse PCR using the MT-1 construct as a template with the primers MT-2 F/R and MT-3 F/MT-2 R. pace vector was generated by subcloning of the EGFP ORF of pegfp-c1 into NheI and HindIII sites of pacm. For MT-4 construct, the full-length actin42a 3 UTR was PCR-amplified with the primers act42a-3 UTR F/R from OSCs cdna samples using KOD plus DNA polymerase and then cloned between HindIII and NotI sites of pace. Mutations were introduced into the 171-196 nt region of the actin42a 3 UTR using a KOD plus mutagenesis kit with the primers MT-4 F/R. MT-2-1, MT-2-2 and MT-2-3 constructs were generated using the KOD plus mutagenesis kit with the primers MT-2-1 F/R, MT-2-2 F/R and MT-2-3 F/R. For sgrna expression constructs, target-specific sequences were synthesized as 5'-phosphorylated oligonucleotides, annealed and ligated into the BbsI sites of pu6-bbsi-chirna (Addgene ID 45956). To generate the EGFP-tj-cis construct, the Tj 3 UTR 22 31 nt region was first introduced downstream of the EGFP ORF of pace using the KOD plus mutagenesis kit with the primers EGFP-tj-cis F/R, and then a 25-nt tandem repeat was inserted into the construct by the same strategy using the primers R1 F/R. For the MT-5 construct, the Tj
3 UTR 22 31 nt region of EGFP-tj-cis was first removed by inverse PCR using the primers delta-tj-cis F/R and then inserted upstream of the polya signal using an In-Fusion HD Cloning Kit (Clontech) according to the manufacturer's instructions. The target fragment containing the Tj 3 UTR 22 31 nt region was PCR-amplified using the In-Fusion primers MT-5-insert F/R and then cloned into the linearized vector generated by PCR with the primers MT-5-vector F/R. For the MT-6 and MT-7 constructs, the target fragments of Tj 3 UTR were PCR-amplified with the In-Fusion primers MT-6-insert F/R and MT-7-insert F/R respectively, using a cdna library as a template and then cloned into the linearized vector generated by PCR with the primers delta-tj F/R. For the MT-8 construct, the multi-cloning site of pacm was introduced downstream of the EGFP ORF of EGFP-tj-cis construct using the KOD plus mutagenesis kit with the primers MT-8 F/R. For the constructs used in Figures 6, the target fragment of flam exon and CG9257 was PCR-amplified with the In-Fusion primers using a cdna library and genome DNA as a template and then cloned into the linearized vector generated by PCR with the primers delta-tj F/R. For Krimper and Tj targeting pirna expression constructs (Krimp-5, Krimp-CDS-1, Krimp-CDS-2, Krimp-3, Tj-5' and Tj-CDS), three repeat sequences of the pirna targeting regions were inserted into the BamHI site of EGFP-tj-cis in antisense orientation. To generate tandem repeats, the double-stranded monomer fragments were first generated by annealing two complementary single-stranded DNA fragments Krimp-5 S/AS, Krimp-CDS-1 S/AS, Krimp-CDS-2 S/AS, Krimp-3 S/AS, Tj-5 S/AS and Tj-CDS S/AS. Then, monomer fragments were ligated to form oligomers, followed by
digestion with BamHI and BclI to remove non-unidirectional repeats.