Supplemental Figures Legends and Supplemental Figures. for. pirna-guided slicing of transposon transcripts enforces their transcriptional

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1 Supplemental Figures Legends and Supplemental Figures for pirn-guided slicing of transposon transcripts enforces their transcriptional silencing via specifying the nuclear pirn repertoire Kirsten-ndré Senti, Daniel Jurczak, Ravi Sachidanandam & Julius rennecke

2 Supplemental Figure S: Efficient /ub/go3 depletions by germline specific RNi () olor-inverted confocal images depict stage 7 egg chambers of indicated genotypes stained for, ub or go3 (black; scale bar = µm). () Shown are fold changes of piwi,, and mrn levels (relative to rpl3) in the indicated genotypes (n = 3; error bars indicate SD). () Images show loading patterns of Ponceau stained membranes used for western blots shown in Fig.. (D) Scheme showing the two-step IP normalization strategy (details see methods) exemplified by pirn profiles for the blood transposon. In step a mirn normalized total small RN-seq library was used to obtain normalization factors for go3-, ub and total--ip libraries (v, w, x). In step the normalized total- IP library was used to obtain normalization factors for germline -IP and soma -IP libraries (y, z). Supplemental Figure S: Impact of cytoplasmic ping-pong on germline -bound pirn populations () olor-inverted confocal images shows germline GFP- fluorescence (black) in stage 7 egg chambers of indicated genotype (scale bar = µm). () Shown is the average nuclear GFP- fluorescence intensity (in % of ) of germline GFP- in ovaries of the indicated genotypes.

3 () ar diagram shows the ' Uridine-bias (U; %) of germline -bound pirns isolated from ovaries of indicated genotypes. (D) Histograms shows length profiles of germline -bound pirns isolated from ovaries of indicated genotypes. (E) Scatterplot of the sum of normalized and anti germline -bound pirns versus the sum of normalized and anti ub- and go3-bound pirns mapping to individual TEs. (F) US browser shots shows normalized ub (orange) germline (green), and go3-bound (blue) pirns obtained from ovaries of indicated genotype and mapping to trn-glu-t (all mappers), the pasha hairpin, or the oskar mrn (unique mappers). Supplemental Figure S3: Determination of TEs exhibiting strong pirn pathway repression in the germline () Shown are profiles of RN Pol II occupancy (top) and ap-seq reads (bottom) mapping to blood from or from ovaries depleted for in the germline. The determined transcription start site (TSS) is indicated (red arrowheads). () Flow chart illustrating the parameters that were used to classify TEs as pirn pathway repressed. First, TE copy number was used to exclude ancient and probably inactive TEs (< genomic copies). Next, only TEs with a fold increase in Pol II occupancy over TE promoters larger than. in any genotype were considered. Finally, we restricted the analysis to TEs exhibiting steady state RN levels > RPKM= under de-repressed conditions and exhibiting a minimal fold change in RN levels in any > 3. 3

4 (, D) Venn diagrams showing the number of TEs with Pol II occupancy changes of at least. fold in the respective genotypes. Note that of 7 TEs shown in (D) are specifically deregulated in germline pirn pathway depletions, but not in soma pirn pathway depletions (our unpublished data). Exceptions are blood and rover, which are active and repressed in germline and somatic ovarian tissues alike. Supplemental Figure S. Silencing patterns of the I-element and urdock, two /ub/go3 TEs (, ) Shown are normalized profiles of pirns bound to germline (green), ub (orange) or go3 (blue) mapping to the I-element () or urdock () obtained from ovaries of indicated genotypes. ar diagrams display the sum of respective pirn populations ( and anti; in x ppm). (, D) olor-inverted confocal images of stage 7 egg chambers depicting I-element (), urdock (D) RN-FISH (black) and I-element ORF protein immuno-localization () in ovaries of indicated genotype (scale bar = µm). Red asterisks mark single nuclei shown enlarged (scale bar = µm) with RN-FISH (black) and GFP-Nup7 (green) outlines nuclei. (E) Plots showing the ping-pong signatures and ping-pong Z-values (calculated from total small RN-seq libraries) of all /ub/go3 group TEs in the indicated genotypes. Supplemental Figure S. Silencing patterns of 3S/el, a /ub-dominant TE () Plots showing the ping-pong signatures and ping-pong Z-values (calculated from total small RN-seq libraries) of all /ub dominant TEs in the indicated genotypes.

5 () Shown are normalized profiles of pirns bound to germline (green), ub (orange), or go3 (blue) mapping to 3S/el. ar diagrams indicate the sum of respective pirn populations ( and anti; in x ppm). () olor-inverted confocal images of stage 7 egg chambers depicting 3S/el RN- FISH signals (black) in ovaries of indicated genotype (scale bar = µm). Enlarged captures (scale bar = µm) show RN-FISH signal (black) and GFP-Nup7 marked nuclear envelope (green) for individual nurse cell nuclei (respective nuclei marked in left panel by red asterisks). Supplemental Figure S6. Silencing patterns of mdg3 and Max, two -dominant TEs (, ) Shown are normalized profiles of pirns bound to germline (green), ub (orange) or go3 (blue) mapping to mdg3 () or Max () obtained from ovaries of indicated genotypes. ar diagrams indicate the sum of respective pirn populations ( and anti; in x ppm). (, D) olor-inverted confocal images showing stage /6 egg chambers depicting mdg3 () and showing stage 7 egg chambers with Max (D) RN-FISH signals (black) as well as immuno-localization of the mdg3 ORF protein (black) () in ovaries of indicated genotype (scale bar = µm). Enlarged captures (scale bar = µm) show RN-FISH signal (black) and GFP-Nup7 marked nuclear envelope (green) for individual nurse cell nuclei (respective nuclei marked in left panel by red asterisks). (E) Plots showing the ping-pong signatures and ping-pong Z-values (calculated from total small RN-seq libraries) of all dominant group TEs in the indicated genotypes.

6 Supplemental Figure S7. Higher TE sequence divergence in the dominant group of TEs. ( and ) Scatter plots of normalized total small RNs from mapped with and 3 mismatches (x and y axis respectively) to those 7 TEs that make up more than 9% of all germline- pirns. dominant TEs are shown in blue, /ub dominant TEs in red, /ub/go3 TEs in green and TEs not deregulated by the germline-pirn pathway in grey. () Jitter plots with median bars show the ratio of total small RNs mapping with 3 versus mismatches. Labels as in (, ). (D and E) Scatter plots of normalized germline- pirns from mapped with and 3 mismatches (x and y axis respectively) to the same 7 TEs as in (- ). (F) Jitter plots with median bars of the ratio of 3 versus mismatches as in (, D). Supplemental Figure S. haracterization of novel piwi,, and null alleles () Shown are protein cartoons of, ub, and go3 with PZ and PIWI domains, RISPR/as9 induced frame shifts (FS; black arrows), and short hairpin mediated RN cleavage sites (blue arrows) at the indicated amino acid positions. Sequence changes between white and the indicated homozygous mutant alleles of piwi, and are indicated. () µg total protein from ovary lysates of the indicated genotypes were resolved on % SDS-PGE gels, transferred and probed for, ub, or GO3 and then re-probed for rmitage as a loading ( indicates an unspecific band detected by the rabbit anti- antibody). 6

7 () Shown is the morphology of ovaries of the indicated genotype (scale bar: µm). (D) Shown are confocal images of stage 7 egg chambers of indicated genotype stained for, ub, or go3 (black; scale bar: µm). (E) Shown are confocal images of stage 7 egg chambers of indicated genotype detecting germline GFP- fluorescence (black; scale bar: µm). (F) Plot showing the average nuclear GFP- fluorescence intensities (in % of ) from a germline driven GFP- construct in the indicated genotypes. Error bars indicate the SD of at least imaged egg chambers per genotype. (G) Graphs showing fold changes of expression (relative to rpl3) of the indicated TEs by RT-qPR (n=3; error bars indicate SD). 7

8 67, Supplemental Figure, Senti et al. / fold change to rpl3 piwi RN. piwi ex piwi -6 ub fold change to rpl3 RN /. GO3 fold change to rpl3 RT RN. RT. D μg M / μg /. against Ponceau stain go3 membrane piwi against Ponceau stain ub membrane piwi / piwi M against Ponceau stain membrane. M μg blood pirn profiles in s norm. go3 pirns mirn norm. total small RN-seq Σ = 3 Σ anti = 3 v Σ = 7 norm. ub pirns w Σ = 6 norm. total pirns Σ = 369 y Σ anti = 73 norm. germline- pirns Σ = 63 Σ anti = Σ anti = 337 Σ anti = 76 x norm. soma- pirns Σ = 9 norm. total pirns Σ = 369 IP normalization z Σ anti = 76 Σ anti = IP normalization

germline GFP- n = w piwi / U bias in germline pirns [%] 6 / D

length [nt] against against E F 3 U G G G U U U G U U U U G U G U

pirn pasha hairpin pirn U G U U U G U G G U U G U U U U G G U U U

9 67, Supplemental Figure, Senti et al. nos-gfp- / normalized nuclear GFP- levels (in % of ) 6 expressing germline GFP- n = w piwi / U bias in germline pirns [%] 6 / D germline- pirn reads (in % of total) 3 / pirn length [nt] against against E F 3 U G G G U U U G U U U U G U G U U G G G G U G G G U U U U G U G G G G G G U U pirn trn Glu 6b pirn pasha hairpin pirn U G U U U G U G G U U G U U U U G G U U U G U U G U G G U U G U 3 G U G G pirn Σ norm. and anti ub and go3. normalized (ppm) R =.93. Σ norm. and anti GL pirns ub pirns go3 pirns trn Glu 6b 3 3 pasha oskar (short) 3 K K K K zuc K K pirn nts pirn 7 nts pirn 6 nts pirn nts kb

10 67, Supplemental Figure 3, Senti et al. TSS LTR blood ORF ORF Pol II hip-seq LTR ap-seq 3 6 7nt ll TEs () determine approximate # of TE copies per haploid genome (genomic DN-seq) < copies/genome (3) > copies/genome (9) determine whether repressed at transcriptional level by the pirn pathway (Pol II hip-seq) fold change Pol II <. in any () fold change Pol II >. in any () dominant (3) ub go3 (7) go3 () go3 () apply threshold for levels in steady state RN levels (poly plus RN-seq) RPKM < or fold change < 3 in any () RPKM > and fold change > 3 in any (7) D dom. (3) ub go3 (7) ub dominant () go3 () go3 () ub dominant ()

11 67, Supplemental Figure, Senti et al. go3 pirns [ppm] anti PP = ppmkc ORF protein RN in situs / GFP-Nup7 Σ in k ppm ub pirns [ppm] I-element (LINE element) GL pirns [ppm] I-element (LINE element) PP = ppmkc D burdock (LTR element) anti PP = 6 ppmkc PP = ppmkc PP = ppmkc PP = ppmkc Ping Pong signatures ub go3 TEs 3 E Z-values ub go3 TEs 6 burdock (LTR element) RN in situs / GFP-Nup7 Σ in k ppm go3 pirns [ppm] ub pirns [ppm] ping-pong signature in % ping-pong Z-values PP = ppmkc / PP = ppmkc GL pirns [ppm] / / /

12 67, Supplemental Figure, Senti et al. Ping Pong signature ub dominant TEs Z-values ub dominant TEs 3 ping-pong signature in % ping-pong Z values 6 3S / el (LTR element) GL pirns [ppm] ub pirns [ppm] go3 pirns [ppm] Σ 3S / el (LTR element) RN in situs / GFP-Nup7 in k ppm anti PP = 6 ppmkc PP= ppmkc 3 67 PP = 3 ppmkc 3 / PP = ppmkc /

13 67, Supplemental Figure 6, Senti et al. D Max-element (LTR element) 3 PP = ppkm ub pirns [ppm] PP = 33 ppkm 3 in k ppm 3 PP = ppkm / PP = ppkm 3 3 / PIng Pong signature dominant TEs Z-values dominant TEs E RN in situs / GFP-Nup7 Σ go3 pirns [ppm] GL pirns [ppm] Max-element (LTR element) PP = ppkm ping-pong singnature in % ping-pong Z-values PP = 7 ppkm PP = ppkm ORF protein in k ppm / PP = 7 ppkm RN in situs / GFP-Nup7 Σ / anti 6 go3 pirns [ppm] mdg3 (LTR element) ub pirns [ppm] GL pirns [ppm] mdg3 (LTR element) 6

14 67, Supplemental Figure 7, Senti et al. Mio. normalized anti total small RNs Mio. normalized total small RNs total srns from mapped with 3 mismatches Max GTE K HeT- THRE blood Inv K I-element 3S flea transpac K K K K Mio. Max K GTE HeT- not-deregulated THRE dominant Inv I-element /ub dominant K 3S /ub/go3 blood transpac K flea K K K Mio. total srns from mapped with mismatches ratio of total small RNs mapping with 3MM versus MM anti total small RNs 3/ mismatch ratios anti anti anti not-deregulated dominant /ub dominant /ub/go3 D normalized anti germline- pirns E normalized germline- pirns germline- pirns from mapped with 3 mismatches K K K HeT- THRE Inv I-element Max GTE blood flea 3S transpac K K K THRE I-element Inv GTE HeT- flea transpac Max blood 3S F ratio of germline- pirns mapping witb 3MM versus MM anti K K K germline- pirns from mapped with mismatches germline- pirns 3/ mismatch ratios anti anti anti K K K not-deregulated dominant /ub dominant /ub/go3

15 w piwi g w piwi g sh grns/ N- N- FS@63/6aa FS@/7aa grns/ grns3/ sh go3 PZ domain 67aa - w g w g white domain : TTGGTGGG : G (-) : GGGGTGTGG : G GG (-) 6aa w g3 w g : TGGTTTGGTG : TG GTG (-) : GTGGTGG : GT-GGTGG (-) g/g anti-go3 anti-rmi anti-rmi anti-rmi g/g g3/ 7 anti-ub anti- g/ white g/g; g/g morphology piwi g/g g/ - : GTGGGGT : G GGT (-) : GGGTTGGGGG : GGG GG (-) w g w g sh grns/ aa : TTGTTGGG : TTG TGGG (-) : TTGTTGGGT : TTGGTTGGT (+7/-6) ub PZ FS@3/aa domain piwi g/ PZ FS@/7aa N- white white 67, Supplemental Figure, Senti et al. D piwi g/g g/g g/g g/g g/g g3/g g/g; g/g go3 ub white 6 g/g fold change ub dominant group of TEs fold change dominant group of TEs burdock q-pr target blood diver 3S qpr target - days fold change relative to RpL3 fold change ub go3 group of TEs white piwi g/g white 3- days G HeT- GTE qpr target g/g g/g g3/g g/g ite ite wh wh expressing germline-gfp- nos - GFP- n g/g ; expressing germline GFP- g/g F ago 3 ; ag o3 g/g g/g white normalized nuclear GFP- levels (in % of ) white E g/g; g/g

Nature Structural & Molecular Biology: doi: /nsmb Supplementary Figure 1

Nature Structural & Molecular Biology: doi: /nsmb Supplementary Figure 1 Supplementary Figure 1 U1 inhibition causes a shift of RNA-seq reads from exons to introns. (a) Evidence for the high purity of 4-shU-labeled RNAs used for RNA-seq. HeLa cells transfected with control