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1 Supplementary Materials for Meiosis-Specific Noncoding RNA Mediates Robust Pairing of Homologous Chromosomes in Meiosis Da-Qiao Ding, Kasumi Okamasa, Miho Yamane, Chihiro Tsutsumi, Tokuko Haraguchi, Masayuki Yamamoto, Yasushi Hiraoka *To whom correspondence should be addressed. This PDF file includes: Materials and Methods Figs. S1 to S5 Table S1 Full References Published 11 May 2012, Science 336, 732 (2012) DOI: /science

2 Materials and Methods Strain construction The fission yeast S. pombe strains used are listed in Table S1. Gene disruption and transposition of the sme2 gene were carried out using a two-step polymerase chain reaction (PCR)-based gene targeting method (24). Labeling of chromosomal loci at sme2, ade8, and lys1 by the use of a lac repressor (laci)/lac operator (laco) recognition system has been described in previous papers (2, 14, 25). To visualize sme2-rna, a 4x U1A tag was integrated at the 5 end of the sme2 gene (Fig. 4A), and a U1Ap-gfp fusion gene (nmt1 promoter) was integrated at the aur1 locus. Expression of U1Ap-GFP resulted in uniform GFP staining throughout the cytoplasm and the nucleus. Co-expression of U1A tag -sme2 with U1Ap-GFP resulted in distinct sme2- RNA dots in the nucleus of meiotic cells. Live cell observations of homologous chromosome pairing Fluorescence microscope images were obtained using a DeltaVision microscope system (Applied Precision, Inc., Seattle, WA), set up in a temperature-controlled room as described previously (26). Data analysis was carried out using SoftWoRx software on the DeltaVision system. Cells were grown on solid YES medium at 33 C. To induce meiosis, the cells were transferred to solid ME medium and incubated at 26 C for about 12 hours. They were then suspended in liquid EMM-N medium supplemented with appropriate amino acids for live cell observations. The cell suspension was placed in a 35 mm glass-bottom culture dish (MatTek Corp., Ashland, MA) coated with 0.2% (w/v) lectin. The behavior of GFP-labeled chromosomal loci in meiotic cells was examined at 26 C as described previously (2). A set of images from 10 focal planes with 0.3 m intervals was taken every 5 minutes. At least 20 individual zygotes were observed for each experiment. Data analysis to determine frequency of pairing was performed as previously described (2). Briefly, we defined the period from the end of karyogamy to the end of oscillatory nuclear movement as the horsetail stage and divided the horsetail stage in each zygote cell equally into 5 substages (each substage is about 25 minutes on average), and counted in each substage the number of time points at which two homologous loci were associated with each other. The measured frequency of pairing was then plotted as a time course. We defined homologous loci as being paired when the distance between the center of the GFP signals was equal to or less than 0.35 m (the diameter of the GFP signal), that is, when the signals overlapped or were linked with each other. Northern blot analysis Total RNAs were isolated from meiotic cells by the acid-phenol method. In h 90 strains, meiosis was induced by incubating the cells at 26 C for approximately 12 hours on a ME plate. Zygotes were concentrated to 25~40% by a brief filtration step. For Northern analysis, 2 g of total RNA were separated by electrophoresis on 1.2% agarose formaldehyde gels and transferred to positively charged nylon membrane (Amersham Hybond TM -N+; GE healthcare UK Ltd, Buckinghamshire). DynaMarker RNA High (BioDynamics Laboratory Inc. Tokyo) was used as an RNA size marker. A DIG- 2

3 labeled RNA probe was generated by a DIG Northern Starter Kit (Roche, Basel). A plasmid, in which the DNA fragment of the sme2 gene was cloned, was digested with EcoRV and used as the template DNA for in vitro transcription using an SP6 promoter. The blots were incubated with the RNA probe overnight at 68 C, washed and the signals detected according to the manufacturer s protocol. 3

(B) The pairing frequency of Mei2-dots in the horsetail stage in wild type and rec12 -

The sme2 locus was visualized with laco/laci-gfp (green in the merged image).

4 Fig. S1. Dynamics of Mei2 dots in meiotic prophase (A) Selected frames from time-lapse observations of Mei2-dots in a living cell. A Mei2 dot appears in karyogamy and remains as a bright dot during the horsetail stage. (B) The pairing frequency of Mei2-dots in the horsetail stage in wild type and rec12 - mutant cells. (C) Double staining of a Mei2-dot and the sme2 locus. The sme2 locus was visualized with laco/laci-gfp (green in the merged image). The Mei2-dot was visualized with a Mei2-mCherry fusion protein (magenta in the merged image). The inset images are enlarged three times. A single Mei2 dot is localized between as yet unpaired sme2 loci (0 min inset), or is associated with a paired sme2 locus (20 min inset). Bar, 5 m. 4

5 Fig. S2 Pairing frequencies of the sme2 locus in various mutant strains (A) Pairing of chromosome loci at 100 kb and 200 kb (as shown in the upper schematic diagram) from the sme2 locus in wild type (WT) and the sme2-deletion ( A) strain. (B) Pairing frequencies at the sme2 locus in defective mutants of RNAi machinery dcr1 and ago1. 5

In contrast with the single bright dot of sme2 RNA observed in the wild type nucleus, multiple tiny dots were frequently observed in the mutant. Bar, 5 m.

6 Fig. S3 sme2 RNA dots released from the sme2 locus in 3 end deletion mutants. (A) Double staining of sme2 RNA stained with U1Ap-GFP (top row; green in bottom row) and chromosome DNA stained with Hoechst (magenta in bottom row) in wild type and E mutant cells. In contrast with the single bright dot of sme2 RNA observed in the wild type nucleus, multiple tiny dots were frequently observed in the mutant. Bar, 5 m. (B) Frequencies of the number of RNA dots. In wild type cells, only one dot was observed for 80% of cells in the horsetail stage. In E and F mutant cells more than 64% of the nuclei had two dots or more; in 5~12% of the mutant cells no apparent dots were detected. More than 100 horsetail nuclei were observed for each strain. 6

Fig. S4 A model for homologous chromosome recognition. (A) Complexes containing RNA transcripts act as chromosome recognition sites along each of the chromosomes aligned by telomeres.

7 Fig. S4 A model for homologous chromosome recognition. (A) Complexes containing RNA transcripts act as chromosome recognition sites along each of the chromosomes aligned by telomeres. (B) Recognition complexes (yellow, green and orange bulbs) on homologous chromosome arms, as shown in A, are aligned by telomere-mediated chromosome movements to find a homologous chromosome partner. Telomeres and centromeres are indicated by blue and red filled small spheres. 7

The number of cells and the number of timepoints examined are shown under each bar graph; p-values based on Student s t test between wild type and mutant

8 Fig. S5 Statistical analyses of pairing frequency in wild type and mutant cells. Mean and standard deviation of pairing frequency in the first three horsetail substages are shown in bar graphs. The number of cells and the number of timepoints examined are shown under each bar graph; p-values based on Student s t test between wild type and mutant strains are shown under the bar graph of the respective mutant. Respective data set in Fig. 1C is shown in (A), Fig. 2A in (B), Fig. 3B-D in (C), Fig. 4D in (D), Fig. S2A in (E), and Fig. S2B in (F). In (B), p-value* indicates that calculated between rec12 + and rec12 - cells. 8

9 Table S1 Strain list Strain Genotype Figure 1 YY548-13C h 90 ade6-216 leu1-32 ura4-d18 sme2proxy[::ura4 + -kan r -lacop] his7 + :: gfp-laci YY417-A h 90 ade6-210 leu1-32 lys1-131 rec12::kan r sme2proxy[::ura4 + -kan r - YY656-7B h 90 ade6-216 leu1-32 lys1 + ::A A::hyg r sme2proxy[::ura4 + -kan r -lacop] his7 + :: gfp-laci YY h 90 ade6-210 leu1-32 lys1 + ::A B::LEU2 sme2proxy[::ura4 + -kan r -lacop] his7 + :: gfp-laci YY665-16C h 90 ade6-216 leu1-32 rec12-152::leu2 lys1 + ::A A::hyg r sme2proxy[::ura4 + -kan r - YY551-3C h 90 ade6-210 leu1-32 ura4-d18 dhc1-d3::leu2 sme2proxy[::ura4 + -kan r - YY555-3C h 90 ade6-210 leu1-32 ura4-d18 bqt1::leu2 sme2proxy[::ura4 + -kan r - Figure 2 CT050-2B h 90 leu1-32 lys1-131 ura4-d18 ade8[::ura4 + -kan r -lacop] his7 + ::gfp-laci YY650-31A h 90 leu1-32 lys1-131 ura4-d18 A::NAT ade8proxy::a-hyg r ade8[::ura4 + - kan r - YY647-2C h 90 leu1-32 lys1-131 ura4-d18 rec12-152::leu2 A::NAT ade8proxy::ahyg r ade8[::ura4 + -kan r -lacop] his7 + : gfp-laci YY516 h 90 ura4-d18 lys1 + ::lacop his7 + :: gfp-laci YY612-18B h 90 ade6-210 leu1-32 lys1-131 ura4-d18 A::NAT lys1::a- ura4 + -lacop his7 + :: gfp-laci YY610-11B h 90 ade6-210 leu1-32 lys1-131 ura4-d18 rec12-152::leu2 A::NAT lys1::a - ura4 + -lacop his7 + :: gfp-laci YY369-17C h - ade6-210 leu1-32 sme2proxy[::ura4 + -kan r - AY193-10C h + ura4-d18 lys1 + ::lacop his7 + ::laci-gfp YY479-6B h + ade6-216 leu1-32 ura4-d18 sme2::ura4 + lys1::a-ura4 + -lacop his7 + ::gfp-laci YY532-1 h + leu1-32 kes1 + ::hyg r HR908 h - leu1-32 lys1-131 ura4-d18 GFP-atb2 + ::kan r YY534-1 h - leu1-32 sme2::ura4 + lys1 + ::A kes1 + ::hyg r YY530-1 h + leu1-32 sme2::ura4 + lys1 + ::A GFP-atb2 + ::kan r YY525-6 h + lys1 + ::lacop ctr5 + -GFP::kan r YY545-1 h - leu1-32 lys1 + ::lacop sgo2 + :: hyg r YY582-3A h + leu1-32 A::NAT lys1::a-ura4 + -lacop ctr5 + -GFP::kan r YY581-5A h - A::NAT lys1::a-ura4 + -lacop sgo2 + :: hyg r 9

10 YY350-16C h + ade8 trp1 972 h - YY668-16B h + ade8 trp1 A::NAT lys1 + ::A YY664-6 h - A::NAT lys1 + ::A Figure 3 YY632-1 h 90 ade6-210 leu1-32 lys1 + ::A C::hyg r sme2proxy[::ura4 + -kan r -lacop] his7 + :: gfp-laci YY608-2 h 90 ade6-216 leu1-32 ura4-d18 D::hyg r sme2proxy[::ura4 + -kan r -lacop] his7 + :: gfp-laci YY832-3 h 90 ade6-210 leu1-32 lys1 + ::A sme2-m::leu2 sme2proxy[::ura4 + -kan r - YY733-1D h + ade6-216 leu1-32 C::hyg r sme2proxy[::ura4 + -kan r -lacop] his7 + :: gfplaci YY834-2B h + ade6-210 leu1-32 lys1-131 sme2-m::leu2 sme2proxy[::ura4 + -kan r - YY369-17A h - ade6-210 leu1-32 sme2proxy[::ura4 + -kan r -lacop] his7 + ::gfp-laci YY633-6 h 90 ade6-216 leu1-32 ura4-d18 E::hyg r sme2proxy[::ura4 + -kan r -lacop] his7 + ::gfp-laci YY652-3 h 90 ade6-216 leu1-32 ura4-d18 F::hyg r sme2proxy[::ura4 + -kan r -lacop] his7 + ::gfp-laci JZ464 h 90 ade6-216 leu1-32 ura4-d18 sme2::ura4 + YY655-1 h 90 ade6-216 leu1-32 ura4-d18 A::hyg r sme2proxy[::ura4 + -kan r -lacop] his7 + :: gfp-laci YY597-1 h 90 ade6-210 leu1-32 D::hyg r sme2proxy[::ura4 + -kan r -lacop] his7 + :: gfplaci YY494-2 h 90 ade6-210 leu1-32 lys1-131sme2-m::leu2 sme2proxy[::ura4 + -kan r - Figure 4 YY h 90 ade6-216 leu1-32 lys1-131 mei2 + -CFP::kan r 4XU1A tag -sme2::hyg r aur1::pnmt41-u1ap-gfp YY h 90 ade6-216 leu1-32 lys1-131 mei2 + -mcherry::hyg r 4XU1A tag - sme2::leu2 aur1::pnmt1-u1ap-gfp YY852-1D h 90 ade6 leu1-32 lys1-131 mei2 + -mcherry::hyg r 4XU1A tag ::LEU2 aur1::pnmt1-u1ap-gfp YY908 h 90 ade6-216 leu1-32 lys1-13 ura4-d18 LEU2-gfp-mmi1 + 4XU1A tag - sme2::hyg r aur1::pnmt1-u1ap-mcherry YY905 h 90 ade6-210 leu1-32 LEU2-gfp-mmi1 + 4XU1A tag ::hyg r aur1::pnmt1-u1ap-mcherry YY814-2 h 90 ade6-216 leu1-32 ura4-d ::hyg r sme2proxy[::ura4 + -kan r - YY897-25C h 90 ade6-216 leu1-32 sme2proxy[::ura4 + -kan r - mmi1-48::kan r 10

11 Figure S1 JW231 YY648-2D YY624-8A h 90 ade6-216 leu1-32 mei2 + -gfp:: kan r h 90 ade6-210 leu1-32 ura4-d18 lys1-131 mei2 + -mcherry:: hyg r rec12::kan r h 90 ade6-216 leu1-32 ura4-d18 lys1-131 mei2 + -mcherry:: hyg r sme2proxy[::ura4 + -kan r -lacop] his7 + ::gfp-laci Figure S2 YY902 h 90 ade6-216 leu1-32 lys kb[::ura4 + -kan r - YY915 h 90 ade6-216 leu1-32 lys1 + ::A A::hyg r 100kb[::ura4 + -kan r -lacop] his7 + :: gfp-laci YY909-8C h 90 ade6-210 leu1-32 lys kb[::ura4 + -kan r - YY924 h 90 ade6-216 leu1-32 lys1 + ::A A::hyg r 200kb[::ura4 + -kan r -lacop] his7 + :: gfp-laci YY433 h - ade6-216 leu1-32 ura4-d18 lys1-131 dcr1:: kan r sme2proxy[::ura4 + - kan r - YY439-2D h + ade6-216 leu1-32 ura4-d18 lys1-131 dcr1:: kan r sme2proxy[::ura4 + - kan r - YY662-8D h 90 ade6-216 leu1-32 ura4-d18 lys1-131 ago1:: kan r sme2proxy[::ura4 + - kan r - Figure S4 YY YY h 90 ade6-210 leu1-32 lys1-131mei2 + -CFP::kan r 4XU1A tag -sme2 E::NAT aur1::pnmt41-u1agfp h 90 ade6-210 leu1-32 lys1-131mei2 + -CFP::kan r 4XU1A tag -sme2 F::NAT aur1::pnmt41-u1agfp A, B represent fragments of sme2 shown in Fig.1A; C-F represent fragments of sme2 shown in Fig.3A and represent the sme2 5 -deletion mutant shown in Fig.4A 11

12 References 1. Y. Chikashige et al., Telomere-led premeiotic chromosome movement in fission yeast. Science 264, 270 (1994). 2. D. Q. Ding, A. Yamamoto, T. Haraguchi, Y. Hiraoka, Dynamics of homologous chromosome pairing during meiotic prophase in fission yeast. Dev. Cell 6, 329 (2004). 3. Y. Chikashige, T. Haraguchi, Y. Hiraoka, Another way to move chromosomes. Chromosoma 116, 497 (2007). 4. Y. Hiraoka, A. F. Dernburg, The SUN rises on meiotic chromosome dynamics. Dev. Cell 17, 598 (2009). 5. R. Koszul, N. Kleckner, Dynamic chromosome movements during meiosis: A way to eliminate unwanted connections? Trends Cell Biol. 19, 716 (2009). 6. D. Q. Ding, T. Haraguchi, Y. Hiraoka, From meiosis to postmeiotic events: Alignment and recognition of homologous chromosomes in meiosis. FEBS J. 277, 565 (2010). 7. B. D. McKee, Homologous pairing and chromosome dynamics in meiosis and mitosis. Biochim. Biophys. Acta 1677, 165 (2004). 8. K. S. McKim, When size does not matter: Pairing sites during meiosis. Cell 123, 989 (2005). 9. J. L. Gerton, R. S. Hawley, Homologous chromosome interactions in meiosis: Diversity amidst conservation. Nat. Rev. Genet. 6, 477 (2005). 10. D. Zickler, From early homologue recognition to synaptonemal complex formation. Chromosoma 115, 158 (2006). 11. A. Barzel, M. Kupiec, Finding a match: How do homologous sequences get together for recombination? Nat. Rev. Genet. 9, 27 (2008). 12. Y. Watanabe, M. Yamamoto, S. pombe mei2+ encodes an RNA-binding protein essential for premeiotic DNA synthesis and meiosis I, which cooperates with a novel RNA species meirna. Cell 78, 487 (1994). 13. Y. Watanabe, S. Shinozaki-Yabana, Y. Chikashige, Y. Hiraoka, M. Yamamoto, Phosphorylation of RNA-binding protein controls cell cycle switch from mitotic to meiotic in fission yeast. Nature 386, 187 (1997). 14. T. Shimada, A. Yamashita, M. Yamamoto, The fission yeast meiotic regulator Mei2p forms a dot structure in the horse-tail nucleus in association with the sme2 locus on chromosome II. Mol. Biol. Cell 14, 2461 (2003). 15. A. Yamashita, Y. Watanabe, N. Nukina, M. Yamamoto, RNA-assisted nuclear transport of the meiotic regulator Mei2p in fission yeast. Cell 95, 115 (1998). 16. A. Yamashita et al., Hexanucleotide motifs mediate recruitment of the RNA elimination machinery to silent meiotic genes. Open Biol 2, (2012). 12

13 17. Y. Harigaya et al., Selective elimination of messenger RNA prevents an incidence of untimely meiosis. Nature 442, 45 (2006). 18. Y. Chikashige et al., Meiotic proteins Bqt1 and Bqt2 tether telomeres to form the bouquet arrangement of chromosomes. Cell 125, 59 (2006). 19. A. Yamamoto, R. R. West, J. R. McIntosh, Y. Hiraoka, A cytoplasmic dynein heavy chain is required for oscillatory nuclear movement of meiotic prophase and efficient meiotic recombination in fission yeast. J. Cell Biol. 145, 1233 (1999). 20. P. Provost et al., Dicer is required for chromosome segregation and gene silencing in fission yeast cells. Proc. Natl. Acad. Sci. U.S.A. 99, (2002). 21. T. Volpe et al., RNA interference is required for normal centromere function in fission yeast. Chromosome Res. 11, 137 (2003). 22. T. Andoh, Y. Oshiro, S. Hayashi, H. Takeo, T. Tani, Visual screening for localized RNAs in yeast revealed novel RNAs at the bud-tip. Biochem. Biophys. Res. Commun. 351, 999 (2006). 23. C. Ellermeier, G. R. Smith, Cohesins are required for meiotic DNA breakage and recombination in Schizosaccharomyces pombe. Proc. Natl. Acad. Sci. U.S.A. 102, (2005). 24. J. Bähler et al., Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe. Yeast 14, 943 (1998). 25. A. Yamamoto, Y. Hiraoka, Monopolar spindle attachment of sister chromatids is ensured by two distinct mechanisms at the first meiotic division in fission yeast. EMBO J. 22, 2284 (2003). 26. T. Haraguchi et al., Multiple-color fluorescence imaging of chromosomes and microtubules in living cells. Cell Struct. Funct. 24, 291 (1999). 13

7.06 Problem Set #4, Spring 2005

7.06 Problem Set #4, Spring 2005 1. You re doing a mutant hunt in S. cerevisiae (budding yeast), looking for temperaturesensitive mutants that are defective in the cell cycle. You discover a mutant strain