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SUPPLEMENTARY DATA (Gallardo et al.) SUPPLEMENTARY METHODS Growth media, drug treatment and yeast strains. Yeast strains used in this study are listed in Table SI and plasmids in Table SII. The yeast cells were grown either in synthetic growth media lacking the nutrients indicated or in rich media. Transformations were performed according to the protocol described by (Schiestl & Gietz, 1989). Yeast gene disruption cassettes were created by PCR amplification of the loxP-KAN-loxP construct in plasmid pUG6 and pFA6a and primers specific for the gene of interest (Longtine et al, 1998). Strains were then selected on the appropriate selective media and specific disruption was confirmed by PCR analysis of genomic DNA. For the LMB treatment, cells were exposed to 20 to 100ng/mL of HPLC purified LMB (LC Labs) for 2h at 30°C. Plasmid construction To generate the deletion of the yKu binding stem in the TCL1 RNA (tlc1- stemyku), two PCR reactions (PCR1 and PCR2) were performed using the appropriate primers (see Table SIII) as follows: typically, each 25-µl reaction contained 10 mM TrisCl, pH 8.8, 25 mM KCl, 5 mM (NH4)2SO4, 2.5 mM MgCl2, 0.2 mM each dNTP, 1.52.5U Pwo polymerase (Roche), 20 pmol of each primer and 10 ng of template DNA pADCEN36 (Dandjinou et al, 2004). Amplification was carried out with 1 cycle at 94˚C for 2 min, 35 cycles at 94˚C for 15 s, 50˚C for 30 sec and 72˚C for 2 min, and one final 1 cycle at 72˚C for 5 min. PCR1: primers SPE-931TLC1FWD and TLC1 329:282 REV; PCR2 : primers +1502TLC1ECOREV and TLC1 269:323 FWD PCR1 and PCR2 products were gel-purified with QIAquick gel extraction kits (Qiagen) and joined during the PCR3 reaction as follows: typically, each 25-µl reaction contained 10 mM Tris-Cl, pH 8.8, 25 mM KCl, 5 mM (NH4)2SO4, 2.5 mM MgCl2, 0.2 mM each dNTP, 1.5-2.5 U Pwo polymerase (Roche), 20 pmol of each primer and 30 to 120 ng of equimolar amounts of purified PCR1 and PCR2 products. Joining was carried out with 1 cycle at 94˚C for 2 min, 10 cycles at 94˚C for 15 s, 50˚C for 30 sec and 72˚C for 2 min, followed by 25 amplification cycles at 94˚C for 15 s, 50˚C for 30 sec and 72˚C for 2 min and one final cycle at 72˚C for 5 min. 20 pmoles of each external primer (SPE931TLC1FWD and +1502TLC1ECOREV) were added just before the amplification cycles. The mutated PCR3 fragments were digested with SpeI and EcoRI, gel purified as previously and cloned into the SpeI-EcoRI site of pADCEN26 (Dandjinou et al, 2004). Expected deletion in each construct was confirmed by sequencing. Plasmid pADCEN26 (negative control) was transformed into the yeast diploid strain CSHY76. The diploid CSHY76 was sporulated, dissected and a tlc1 haploid spore was selected. This haploid strain was transformed with pADCEN36 (WT TLC1) and pADCEN63 (TLC1 deleted from the KU stem – tlc1-stemyku). All the mutants can complement the tlc1 rad52 deletion up to 100 generations. Preparation of the TLC1 fluorescent probes 2 A set of 5 oligonucleotide probes was used to detect the endogenous TLC1 RNA. Each probe is about 50nt long and contains five modified T (aminoallyl-T), distanced by about 10 nt each (Sequence of the probes are in the Table SIV). The probes were labeled with FluoroLink Cy3 monofunctionnal reactive dye (Amersham Biosciences, Piscataway,NJ) with at least two Cy3 incorporation per probes (usually 3 to 4) and used to detect the endogenous TLC1 RNA. Briefly, 10μg of probes were lyophylized in a speed-vac and resuspended in 35 μL of 0, 1M sodium carbonate pH 8,8. The Cy3 reactive pack was resuspended in 30μL of DEPC-treated water. 15μL of resuspended Cy3 was mixed to the 35μL of probes, vortexed, and the reaction was incubated at room temperature for 16-24 hours in the dark. To remove the unincorporated Cy3, the reaction mix was purified using a Sephadex G25 column. The absorbance in the pellet was estimated by measuring the OD at 260nm for the probe and the Cy3 concentration at 552nm. Production of oxalyticase The oxalyticase was prepared using DH5α cells containing the pUV5 plasmid expressing this lyticase under the control of the T7 promoter. A single colony was first inoculated in 2ml of LB medium in presence of 100μg/ml of ampicilin (Roche) and incubated at 37°C overnight. To produce the enzyme, 1ml of the overnight culture was inoculated in 500ml of LB medium in presence of ampicillin and brought to a OD of 0,5 at 600nm before protein induction by Isopropyl--thiogalactoside (IPTG) (Sigma). The cells were induced with a final concentration of 0,4mM IPTG during 5 hours at 37°C. After which, the cells were harvested by centrifugation for 30 min at 3400rpm and washed once with 25mM Tris pH 7,4. The cells were resuspended in 10ml of 25mM Tris pH 7,4 containing 2mM 3 of EDTA. An equal volume of 40% sucrose buffer (40% sucrose in 25mM Tris pH 7,4) was added and the cells were mixed gently for 20 min. The sucrose buffer was removed following centrifugation at 7500rpm for 10 min in a GSA rotor (Sorvall). All previous steps were performed at room temperature to ensure better shocking for the cells. The cells were submitted to a cold shock in 10ml of ice cold 0,5mM MgSO4 and mixed gently on ice for 20 minutes. Finally, the cells were spun at 10 000rpm for 10 minutes in a SS34 rotor (Sorvall) and the supernatant was separated in 200μl aliquots. The oxalyticase containing aliquots were lyophylized to a volume of 20μl and stored at -20°C until use. 4 SUPPLEMENTARY TABLES TABLE SI: Yeast strains used in this study ________________________________________________________________________ Strain genotype source W303 Mat a, ura3-1, leu2-3, his3-11, trp1-1, ade2-1 R. Jansen BY4742 Mat α, his3Δ1, leu2Δ0, lys2Δ0, ura3Δ0 Open Biosystems BY4743 Mat a/α his3/his3 leu2/leu2 lys2/LYS2 Open Biosystems MET15/met15Δ0, ura3Δ0/ura3Δ0 CSHY76 tlc1 MATa/ ade2/ade2 ura3/ura3 leu2/leu2 his3/his3 trp1/trp1 tlc1::LEU2/TLC1 rad52::TRP1/RAD52. C. Greider Mata, ura3-1, leu2-3, his3-11, trp1-1, ade2-1, R. Wellinger TLC1::LEU2, RAD52::TRP1 tgs1 W303 ΔTGS1::KAN this study RAP1 Myc13 W303, RAP1-13xMyc KAN this study est1 BY4743, EST1/EST1::KAN Open Biosystems est2 BY4743, EST2/EST2::KAN R. Wellinger est3 BY4743, EST3/EST3::KAN Open Biosystems yku70 W303 YKU70::KAN R. Wellinger Y464 his, leu, trp, CRM1::KAN (pDC-crm1T539C-LEU2) M. Rosbash Y464yku70 Y464 YKU70::HIS3 this study xpo1-1 W303 XPO1::LEU2 (pKW456-xpo1-1-HIS) C. Guthrie xpo1-1 yku70 xpo1-1 YKU70::TRP1 this study Nsp1ts10A Mat? Ade2-1, can1-100, leu2-3, lys1-1, ura3-52, 5 P-E Gleizes NSP1:URA3, nsp1Ts10A-URA3 Nsp1ts10A yku70 Nsp1ts 10A YKU70::KAN this study mex67-5 Mat a, ade2, his3, leu2, trp1, ura3, MEX67::HIS C. Cole (pun100-leu2-mex67-5) mex67-5 yku70 mex67-5 YKU70::TRP1 this study MS739 Mat α, leu2-3, ura3-52, ade2-101, kar1-1 kap123 BY4742 KAP123::KAN Open Biosystems kap120 BY4742 KAP120::KAN Open Biosystems msn5 BY4742 MSN5::KAN Open Biosystems sxm1 BY4742 SXM1::KAN Open Biosystems kap122 BY4742 KAP122::KAN Open Biosystems kap114 BY4742 KAP114::KAN Open Biosystems tel1 BY4742 TEL1::KAN Open Biosystems mre11 BY4742 MRE11::KAN Open Biosystems xrs2 BY4742 XRS2::KAN Open Biosystems PSY1201 ura3-52 trp1-63 leu2-1 pse1-1 P. Silver PSY1199 nmd5::HIS3 in PSY1183 P. Silver Y1171 Mat ade2-1 his3-11,15 ura3-52 leu2-3,112 mtr10::HIS3 E. Hurt L5850 leu2-3,112 ura3-1 ade2-1 his3-11,15 trp1 srp1-31 G. Fink P. Silver ________________________________________________________________________ 6 TABLE SII: Plasmids used in this study Plasmids Description Source pADCEN36 TLC1, CEN, URA3 pADCEN63 tlc1ΔstemIIc(Stem Ku) in pADCEN36 pNop1-GFP NOP1-GFP in pRS315 URA3 S. Abou Elela. pRL134 LOC1-6xMyc cloned in pESC-URA3 R. Long pVL399 Empty 2µ vector, pADHTerm-ADH, LEU2. V. Lundbald. pVL784 EST1 in pVL399 V. Lundbald. pVL999 EST2 in pVL399 V. Lundbald. 7 R.J Wellinger this study TABLE SIII: Sequences of the primers used for cloning Primer Sequence SPE-931TLC1FWD 5’-GGGTACACTAGTAGCCTTTCTAGAGGTTCC-3’ TLC1 329:282 REV TLC1 269:323 FWD 5’-CGGTTTGATAAAAAACCACAAATTGCGCACACACAAGC-3’ 5’-GGTTTTTTATCAAACCGTAAATTCTTAAACACTGCTATTGC-3’ +1502TLC1ECOREV 5’-AACAGAATTCGGGAAGGTAAATACCACC-3’ ________________________________________________________________________ Underlined nucleotides indicate either Spe1 and EcoR1 restriction sites. 8 TABLE SIV: Sequences of the oligonucleotide probes against TLC1 Probe Sequence TLC1-1 5’-t*gcgcacacacaagcat*ctacactgacaccagcat*actcgaaattctt*tg-3’ TLC1-2 5’-ct*aataaacaatt*agctgtaacatt*tgtgtgtggggt*gtggtgatggt*aggc-3’ TLC1-3 5’-tt*ccagagttaacgat*aagatagacat*aaagtgacagcgct*tagcaccgt*c-3’ TLC1-4 5’-ttacgt*tcttgatctt*gtgtcattgtt*cagttactgat*cgcccgcaaacct*-3’ TLC1-5 5’-tgcat*cgaaggcat*taggagaagt*agctgtgaat*acaacaccaagat*tca-3’ ________________________________________________________________________ * aminoallyl modified-T 9 SUPPLEMENTARY FIGURES LEGENDS Figure S1: The TLC1 RNA is exported from the nucleus independently from the ribosomal RNA and mRNA export pathways. The nuclear export mutants nsp1ts10a, specific for rRNA export (Gleizes et al, 2001) and mex67-5, specific for mRNA export (Segref et al, 1997), were used to define the nuclear export pathway of TLC1 RNA. A) FISH against the TLC1 RNA in a nsp1ts10a yku70 strain was performed at T= 0h or 2hrs of shift at restrictive temperature (37ºC). TLC1 RNA keeps its cytoplasmic accumulation even after 2 hours at 37°C. Scale bar = 1µm. B) FISH against the TLC1 RNA in a mex67-5 yku70 strain was performed at T= 0h or 2hrs of shift at restrictive temperature (37ºC). TLC1 RNA keeps its cytoplasmic accumulation even after 3hrs at restrictive temperature (data not shown). Scale bar = 1µm. Figure S2: Control for nucleus leakage during the heterokaryon shuttling assay. Heterokaryons were created by mating a Mata strain deleted of the TLC1 gene (tlc1) with a Mat strain carrying the kar1-1 allele, a wild-type TLC1 gene and a galactose-inducible Loc1p-myc (kar1-1+pRL134). After transient expression of Loc1p-myc, mating and immunofluorescence, the distribution of Loc1p-myc in the nuclei of the heterokaryons was determined. In all heterokaryons observed, Loc1p-myc was detected in only one nucleus. Scale bar = 1µm. 10 SUPPLEMENTARY REFERENCES Dandjinou AT, Levesque N, Larose S, Lucier J-F, Elela SA, Wellinger RJ (2004) A Phylogenetically Based Secondary Structure for the Yeast Telomerase RNA. Current Biology 14(13): 1148-1158 Gleizes P-E, Noaillac-Depeyre J, Leger-Silvestre I, Teulieres F, Dauxois J-Y, Pommet D, Azum-Gelade M-C, Gas N (2001) Ultrastructural localization of rRNA shows defective nuclear export of preribosomes in mutants of the Nup82p complex. J Cell Science 155(6): 923-936 Longtine MS, McKenzie A, 3rd, Demarini DJ, Shah NG, Wach A, Brachat A, Philippsen P, Pringle JR (1998) Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast (Chichester, England) 14(10): 953-961 Schiestl RH, Gietz RD (1989) High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Curr Genet 16(5-6): 339-346 Segref A, Sharma K, Doye V, Hellwig A, Huber J, Luhrmann R, Hurt E (1997) Mex67p, a novel factor for nuclear mRNA export, binds to both poly(A)+ RNA and nuclear pores. EMBO J 16(11): 3256-3271 11