Figure S1. sporulation frequency (%) 80. sme2-5. sme2-3. sme2

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1 Figure S1 N WT DSRless mei4 100 ssm4 MS2loop () (kb) sporulation frequency (%) rrna WT DSRless Figure S1. The DSR motifs in are crucial for its function. Expression of the meiotic transcripts was examined by rthern blot analysis in mutants. Wildtype (JS41), -5 (JS42), -3 (JS43) and -DSRless (JS44) cells were grown in MM media at 30 C (N+) and then transferred to MM media without nitrogen source for four hours at 30 C (N-). The rrnas stained with ethidium bromide are shown in the bottom panel as loading controls. Sporulation frequencies of mutants. Wild-type (JS38), (JT926), -5 (JS39), -3' (JS40) and - DSRless (JS5) cells was measured after incubation on an SPA medium at 30 C for 24 hours (n > 200).

2 Figure S2 N WT mei4 mei4 mmi1 pab2 82% (23/28) 18% (5/28) pla1-ts 100% (7/7) rrna clr4 95% (19/20) Figure S2. Localization of in -related mutants. Expression of was examined by rthern blot analysis in mmi1-depleted cells. Wild-type (JY362), mei4 (JZ807) and mmi1 mei4 (JS45) cells were grown in MM media at 30 C (N+) and then transferred to MM media without nitrogen source for four hours at 30 C (N-). The rrnas stained with ethidium bromide are shown in the bottom panel as loading controls. Localization of in mutants involved in -mediated meiotic mrna elimination and facultative heterochromatin formation. pab2 (JS46), pla1-ts (JS47) and clr4 (JS48) cells expressing MS2 loop-tagged from its endogeus promoter and MS2-GFP from the expression vector prep81 were examined under meiotic conditions. Frequencies of meiotic prophase cells containing dot are indicated. Scale bar, 5 µm.

3 Figure S3 1,10- phenanthroline Red1 locus merge colocalization frequency - 94% + 81% 2.0 % input red1 + (untagged) red1-13myc act1 mei Figure S3. Localization of and Red1 at the locus. Localization of and Red1 when transcription is inhibited. Wild-type cells (JS8) expressing Red1-YFP and CFP- from the respective endogeus promoters were grown in YE media and then added with 1,10-phenanthroline (250 µg/ml at final concentration) to inhibit transcription. The LacI-lacO system were used to visualize the locus. Images of the nuclear region after one-hour incubation with or without 1,10-phenanthroline are shown. In the merged image, green indicates Red1-YFP, red indicates the locus, and blue indicates CFP-. Scale bar, 2 µm. Frequencies of cells in which and Red1 localized to the locus are indicated (n > 100). Chromatin immuprecipitation (ChIP) analysis of Red1 accumulation on the act1 locus, the mei4 locus and the 5 or 3 region of the locus in untagged (JY450) and Myc-tagged (JS12) red1 + cells under mitotic conditions. Results represent the mean ± standard deviation from three reactions.

4 Figure S4 locus merge dot formation frequency 1% -5-3 (c) -5 1 dot 2 dots 3 dots 4 dots (%) Figure S4. does t form a dot in mitotic cells without overexpression. Localization of in mitotic cells in which overexpression was t induced. cells (JS16) were incubated in YE medium. The LacI-lacO system were used to visualize the locus. In the merged image, green indicates (MS2-GFP), and red indicates the locus. Scale bar, 5 µm. Frequency of cells containing a dot is indicated (n > 100). Localization of mitotic in cells in which overexpression of variants were t induced. (JS18), -5 (JS19) and -3 (JS20) cells expressing CFP- were incubated in YE media. Scale bar, 5 µm. (c) Percentages of cells containing 1, 2, 3, or 4 and more dots when overexpression of variants were t induced. More than 100 cells were counted in (JS18), -5 (JS19) and -3 (JS20) strains incubated in YE media.

5 Figure S5 + 1 dot 2 dots 3 dots 4 dots + (c) (%) (d) (e) 1 dot 2 dots 3 dots 4 dots (%) (f) -N Figure S5. Mei2 is t involved in the convergence of dots by the overexpression of. Localization of mitotic in cells overexpressing. + (JS21) and (JS22) cells expressing CFP- with the deletion of mei2 were examined under mitotic conditions. Scale bar, 5 µm. Percentages of cells containing 1, 2, 3, or 4 and more dots when variants were overexpressed in cells. More than 100 cells were counted in the + (JS21), (JS22), -5 (JS23), and -3 (JS24) strains with the deletion of mei2 under mitotic conditions. (c) Localization of mitotic in cells overexpressing -5 or (JS23) and -3 (JS24) cells expressing CFP- with the deletion of mei2 were examined under mitotic conditions. Scale bar, 5 µm. (d) Localization of mitotic in cells in which overexpression of variant was t induced. (JS22), -5 (JS23) and -3 (JS24) cells expressing CFP- with the deletion of mei2 were incubated in YE media. Scale bar, 5 µm. (e) Percentages of cells containing 1, 2, 3, or 4 and more dots when overexpression of variants were t induced. More than 100 cells were counted in (JS22), -5 (JS23) and -3 (JS24) strains incubated in YE media. (f) Localization of meiotic in cells overexpressing variants. + (JS21), (JS22), -5 (JS23), and -3 (JS24) cells expressing CFP- with the deletion of mei2 were examined under meiotic conditions. Scale bar, 5 µm.

6 Figure S6 N WT mmi1-ts6 30 C 25 C 28 C 30 C - + mei4 ssm4 rrna rrna Figure S6. The overexpression of reduces the activity of. Expressions of in meiotic wild-type cells and mitotic -overexpressing cells were examined by rthern blot analysis. Wild-type (JY362) cells were grown in MM media at 30 C (N+) and then transferred to MM media without nitrogen source for four hours at 30 C (N-). (JS34) cells were grown in MM media. The rrnas stained with ethidium bromide are shown in the bottom panel as loading controls. Note that overexpressed carries the MS2 loop sequence and is about 130 nt longer than wild-type. Expression of mei4 and ssm4 mrnas was examined by rthern blot analysis in (JS34) and mmi1-ts (JS35) cells with or without overexpression. These cells were grown in YE media (-) and MM media (+) at indicated temperatures. The rrnas stained with ethidium bromide are shown in the bottom panel as loading controls.

7 Table S1. S. pombe strains used in this study Name JS1 JS2 JS3 JS4 JS5 JS6 JS8 JS9 JS10 JS12 JS13 JS14 JS16 JS17 JS18 JS19 JS20 JS21 JS22 JS23 JS24 JS31 JS32 JS33 Getype h 90 lacox32-kan R -ura4 -MS2loop-2 arg1::padh41-4mcherry-laci-nls-hph R CO2::Padh41-MS2-YFP-nat R LEU2-CFP-mmi1 ade6-m216 leu1 ura4-d18 h 90 bsd R --MS2loop-2 CO2::Padh41-MS2-YFP-nat R LEU2-CFP-mmi1 mei2-mcherry-hph R ade6-m216 leu1 ura4-d18 h MS2loop-2-Tnmt CO2::Padh41-MS2-YFP-nat R LEU2-CFP-mmi1 mei2-mcherry-hph R ade6-m216 leu1 ura4-d18 h 90 MS2loop-2--3 CO2::Padh41-MS2-YFP-nat R LEU2-CFP-mmi1 mei2-mcherry-hph R ade6-m216 leu1 ura4-d18 h 90 bsd R --DSRless-MS2loop-2 ade6-m216 leu1 ura4-d18 h 90 bsd R --DSRless-MS2loop-2 CO2::Padh41-MS2-YFP-nat R LEU2-CFP-mmi1 mei2-mcherryhph R ade6-m216 leu1 ura4-d18 h 90 lacox32-kan R -ura4 -MS2loop-2 arg1::padh41-4mcherry-laci-nls-hph R LEU2-CFP-mmi1 red1-yfp-nat R ade6-m216 leu1 ura4-d18 h 90 lacox32-kan R -ura4 LEU2--m nat R -CFP-mmi1 red1-mcherry-hph R his7 + <<GFP-lacI ade6-m210 leu1 ura4-d18 h 90 lacox32-kan R -ura4 -DSRless-MS2loop-2 arg1::padh41-4mcherry-laci-nls-hph R LEU2-CFP-mmi1 red1-yfp-nat R ade6-m216 leu1 ura4-d18 h 90 red1-13myc-nat R ade6-m216 leu1 h 90 lacox32-kan R -ura4 LEU2--m red1-13myc-nat R ade6-m216 leu1 ura4-d18 h 90 bsd R --DSRless-MS2loop-2 red1-13myc-nat R ade6-m216 leu1 h 90 lacox32-kan R -ura4 bsd R --MS2loop-2 arg1::padh41-4mcherry-laci-nls-hph R CO2::Padh81-MS2-GFP-nat R ade6-m216 leu1 ura4-d18 h 90 -MS2loop-2-bsd R nat R -CFP-mmi1 ade6-m216 leu1 ura4-d18 h 90 kan R --MS2loop-2 nat R -CFP-mmi1 ade6-m216 leu1 ura4-d18 h 90 kan R --5 -MS2loop-2-Tnmt nat R -CFP-mmi1 ade6-m216 leu1 ura4-d18 h 90 kan R -MS2loop-2--3 nat R -CFP-mmi1 ade6-m216 leu1 ura4-d18 h 90 mei2::ura4 + nat R -CFP-mmi1 ade6-m216 leu1 ura4-d18 h 90 mei2::ura4 + kan R --MS2loop-2 nat R -CFP-mmi1 ade6-m216 leu1 ura4-d18 h 90 mei2::ura4 + kan R --5 -MS2loop-2-Tnmt nat R -CFP-mmi1 ade6-m216 leu1 ura4-d18 h 90 mei2::ura4 + kan R -MS2loop-2--3 nat R -CFP-mmi1 ade6-m216 leu1 ura4-d18 h 90 CO2::MS2loop-2-ura4-nat R ade6-m216 leu1 ura4-d18 h 90 CO2::MS2loop-2-ssm4-nat R ade6-m216 leu1 ura4-d18 h 90 -MS2loop-2 ade6-m216 leu1 ura4-d18

8 JS34 JS35 JS36 JS37 JS38 JS39 JS40 JS41 JS42 JS43 JS44 JS45 JS46 JS47 JS48 JS50 JS51 JS52 JT926 JV579 JV582 JY362 JY450 JZ807 h 90 kan R --MS2loop-2 ade6-m216 leu1 ura4-d18 h 90 mmi1-ts6-kan R bsd R --MS2loop-2 ade6-m216 leu1 ura4-d18 h 90 mei4::ura4 + bsd R --MS2loop-2 ade6-m216 leu1 ura4-d18 h 90 mei4::ura4 + mmi1::kan R bsd R --MS2loop-2 ade6-m216 leu1 ura4-d18 h 90 bsd R --MS2loop-2 ade6-m216 leu1 ura4-d18 h 90 bsd R --5 -MS2loop-2-Tnmt ade6-m216 leu1 ura4-d18 h 90 bsd R --3 -MS2loop-2 ade6-m216 leu1 ura4-d18 h 90 /h 90 bsd R --MS2loop-2/bsd R --MS2loop-2 ade6-m216/ade6-m210 leu1/leu1 ura4-d18/ura4-d18 h 90 /h 90 bsd R --5 -MS2loop-2-Tnmt/bsd R --5 -MS2loop-2-Tnmt ade6-m216/ade6-m210 leu1/leu1 ura4-d18/ura4-d18 h 90 /h 90 bsd R --3 -MS2loop-2/bsd R --3 -MS2loop-2 ade6-m216/ade6-m210 leu1/leu1 ura4-d18/ura4-d18 h 90 /h 90 bsd R --DSRless-MS2loop-2/bsd R --DSRless-MS2loop-2 ade6-m216/ade6-m210 leu1/leu1 ura4-d18/ura4-d18 h + /h - mei4::ura4 + /mei4::ura4 + mmi1::kan R /mmi1::kan R ade6-m216/ade6-m210 leu1/leu1 ura4-d18/ura4-d18 h 90 pab2::hph R bsd R --MS2loop-2 ade6-m216 leu1 ura4-d18 h 90 pla1-41-kan R bsd R --MS2loop-2 ade6-m216 leu1 ura4-d18 h 90 clr4::kan R bsd R --MS2loop-2 ade6-m216 leu1 ura4-d18 h 90 CO2::MS2loop-2-ura4-nat R kan R -CFP-mmi1 ade6-m216 leu1 ura4-d18 h 90 CO2::MS2loop-2-ssm4-nat R kan R -CFP-mmi1 ade6-m216 leu1 ura4-d18 h 90 mei4-ura4 kan R -lacox32 arg1::padh41-4mcherry-laci-nls-hph R LEU2-CFP-mmi1 ade6-m210 leu1 ura4 h 90 ::ura4 + ade6-m216 leu1 ura4-d18 h 90 mmi1-ts3-kan R ade6-m216 leu1 h 90 mmi1-ts6-kan R ade6-m216 leu1 h - /h + ade6-m216/ade6-m210 leu1/leu1 h 90 ade6-m216 leu1 h - /h + mei4::ura4 + /mei4::ura4 + ade6-m216/ade6-m210 leu1/leu1 ura4-d18/ura4-d18

9 Table S2. Oligonucleotides used in this study For laco integration Name Sequence -laco F1 -laco F2 -laco R2 -laco R1 mei4-laco F1 mei4-laco F2 mei4-laco R2 mei4-laco R1 GTTGCTCCTATAGAAGAACA TTAATTAACCCGGGGATCCGTTCGCGCAGATAAGCGCAAG GTTTAAACGAGCTCGAATTCGATTAAATAGAATATTCGTACATG CACCAAGTTTCTTAAAAGCG ATGAGTCAAGTGGCTACTCAAGTGC TTAATTAACCCGGGGATCCGTTGTTTCGTCAACATATATTATTAC GTTTAAACGAGCTCGAATTCTTCTAAATAAAGTCATAGGAATGTA GCGAGATAGTTAGAAGCGGAAGTGG For quantitative PCR Name Sequence act1-f act1-r mei4-f mei4-r -5 -F -5 -R -3 -F -3 -R TGAGGAGCACCCTTGCTTGT TCTTCTCACGGTTGGATTTGG AATGCGAAACTGAAGCATTG TAGGATCGCCAAACCGATTA AAGACGGAATATGCATGCAAGA AACAAACCACAACACAAAGAAAGAGA GAAAATAAACAATAACCACAGCAAGCT ACAGCACAACCGAAGACCAAT

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