Supplemental Data. Distinct Pathways for snorna and mrna Termination

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Molecular Cell, Volume 24 Supplemental Data Distinct Pathways for snorna and mrna Termination Minkyu Kim, Lidia Vasiljeva, Oliver J. Rando, Alexander Zhelkovsky, Claire Moore, and Stephen Buratowski

A B C 23 C Pcf pcf-2 pcf-9 pcf-3 2 3 4 5 6 7 8 9 02 2 3 4 5 6 7 8 9 02 2 3 4 5 6 7 8 9 02 2 3 4 5 6 7 8 9 02 2 0 8 6 4 2 Pcf (23 C) pcf-2 (23 C) pcf-9 (23 C) pcf-3 (23 C) 2 3 4 5 6 7 8 9 0 2 37 C, 30 min Pcf pcf-2 pcf-9 pcf-3 2 3 4 5 6 7 8 9 02 2 3 4 5 6 7 8 9 02 2 3 4 5 6 7 8 9 02 2 3 4 5 6 7 8 9 02 4 3 2 Pcf (37 C) pcf-2 (37 C) pcf-9 (37 C) pcf-3 (37 C) 0 2 2 3 4 5 6 7 8 9 2 3 4 5-47 68 376 584 807 00 250 208 2290 6 2866 3098 3287 3500 7 9 (ATG) 2757(STOP) poly(a) 405(ATG) 6354(STOP) 2823 3277 8 3448 3662 369 3905 0 2 479 4353 5350 5566 6445 667 PMA (95) LEU (7.9)

Figure S. Effect of PCF Mutant Alleles on Termination at PMA Chromatin used in Fig. 4 was used to assay Rpb3 on the PMA gene. ChIP gels are shown at left and quantitation at right. (A) Permissive temperature. (B) Non-permissive temperature. (C) Schematic of PMA and PCR primers used.

60 00 Brr5 (23 C) 30 Brr5 0 2 (ATG) 584 3 807 PMA (95) 00 250 4 2 3 4 5 6 7 8 9 0 2 0-304 -47 68 376 B A α-rna5 α-fip α-cft2 α-brr5 00 min at 37 C brr5-td (23 C) 60 2290 2757(STOP) 208 5 3098 2823 8 2 3 9 3448 3662 3905 4353 405(ATG) 479 0 4 5 6 7 8 9 0 2 Brr5 (37 C) 3500 369 7 3277 poly(a) 2866 6 3287 2 3 4 5 6 7 8 9 0 2 30 brr5-td 5350 5566 6354(STOP) 6445 2 667 2 3 4 5 6 7 8 9 0 2 LEU (7.9) brr5-td (37 C)

Figure S2. Brr5 Depletion Causes Readthrough of the PMA Gene Terminator (A) Wild-type and temperature sensitive degron-tagged Brr5 (Brr5-td) strains were shifted to 37 C and incubated for 0, 30, 60, and 00 min. Extracts were prepared and analyzed for depletion of Brr5 by western blot. Brr5-td was depleted after 00 min, but other 3 -processing factors (Cft2, Fip, and Rna5) were unaffected. (B) Wild-type and brr5-td cells were shifted to 37 C for 00 min and ChIP assays were carried out on PMA gene. the scheme below the gel. The positions of PCR products used in the ChIP assays are shown in Numbers in parentheses show the transcription rates (mrna/hr) as in Figure.

2 3 4 5 2 3 4 5 2 3 4 5 cbc rrp6 rnt ADH 2 3 4 5 rnt cbc 2 3 4 5 2 3 4 5 rrp6 snr3 2 3 4 5 rnt cbc 2 3 4 5 2 3 4 5 rrp6 snr33

Figure S3. Neither Rnt, the nuclear exosome subunit Rrp6, nor the cap binding complex subunit Cbc are required for transcription termination at mrnas or snornas. rnt, rrp6, or cbc cells were analyzed by ChIP assays on ADH, snr3, and snr33 genes.

-304-47 Sen (37 C, 30 min) sen- (37 C, 30 min) 2 3 4 5 6 7 8 9 0 2 2 3 4 5 6 7 8 9 0 2 2 3 4 5 68 376 584 807 00 250 208 2290 3287 3500 7 9 (ATG) 2757(STOP) poly(a) 405(ATG) 2823 3277 6 2866 3098 8 3448 3662 369 3905 0 479 4353 5350 5566 PMA (95) LEU (7.9) 2 6445 667 6354(STOP)

Figure S4. Sen Is Not Required for Transcription Termination of the PMA Gene Isogenic wild-type and sen- cells were shifted to 37 C for 30 min, and ChIP assays were carried out on PMA gene. The positions of PCR products used in the ChIP assays are shown in the scheme below the gel. Table S. Strains Used in This Study Genotype Source BY474 FY23 DAt-2 YSB42 YSB43 SC629 SC0976 SC0878 SC0294 W303-B YEN3 YEN25 YEN24 Sen-Myc YSB2084 YEN2 YEN2 YEN0 YF566 LRB535 YZS84 YDP70 YDP74 YJC60 YJC88 YJC07 FWY97 FWYv556 BMA64a rnt YSB2002 YSB962 YSB970 MATa, ura3 0, leu2 0, his3, met5 0 MATa, ura3-52, leu2, trp 63 MATa, ura3-52, leu2, trp 63, rat- MATa, ura3 0, leu2 0, trp ::LEU2/KanR, his3, met5 0, rtt03 ::KanMX MATa, ura3 0, leu2 0, trp ::LEU2/KanR, his3, met5 0, rai ::KanMX MATa, ura3-52, leu2-3,2, trp-289, arg4-, ade2-, RNA4-TAP::URA3 MATa, ura3-52, leu2-3,2, trp-289, arg4-, ade2-, RNA5- TAP::URA3 MATa, ura3-52, leu2-3,2, trp-289, arg4-, ade2-, PCF- TAP::URA3 MATa, ura3-52, leu2-3,2, trp-289, arg4-, ade2-, CFT2- TAP::URA3 MATα, ura3-, leu2-3,2, trp-, his3-,5, can-00, ade2- MATa, ura3-, leu2-3,2, trp-0, his3-,5, can-00, ade2-, HRP-TAP::TRP(K. lactis) MATa, ura3-, leu2-3,2, trp-0, his3-,5, can-00, ade2-, NRD-TAP::TRP(K. lactis) MATa, ura3-, leu2-3,2, trp-0, his3-,5, can-00, ade2-, NAB3-TAP::TRP(K. lactis) MATα, ura3-, leu2-, trp-,his3-, lys2-, ade2-, cup, Sen-Myc3::KanMX6 MATa, ura3-52, leu2, trp 63, Sen-Myc3::KanMX6 MATa, ura3-, leu2-3,2, trp-0, his3-,5, can-00, ade2-, CBC-TAP::TRP(K. lactis) MATa, ura3-, leu2-3,2, trp-0, his3-,5, can-00, ade2-, PTI-TAP::TRP(K. lactis) MATa, ura3-, leu2-3,2, trp-0, his3-,5, can-00, ade2-, REF2-TAP::TRP(K. lactis) MATa, ura3 0, leu2 0, his3, met5 0, RAT-TAP::HIS3 MATa, ura3-52, leu2-3,2, his3 200 MATa, ura3-52, leu2-3,2, his3 200, ssu72-2 MATα, ura3-52, leu2-3,2, his3, trp, ssu72 ::LEU2 [SSU72, TRP, CEN/ARS] MATα, ura3-52, leu2-3,2, his3, trp, ssu72 ::LEU2 [ssu72-3, TRP, CEN/ARS] MATα, ura3 0, leu2 0, his3, met5 0, nrd ::KAN [NRD-HA, LEU2, CEN/ARS] MATα, ura3 0, leu2 0, his3, met5 0, nrd ::KAN [nrd-5, LEU2, CEN/ARS] MATα, ura3 0, leu2 0, his3, met5 0, nrd ::KAN [nrd-02-ha, LEU2, CEN/ARS] MATa, ura3-52, leu2-3,2, trp-, pep4-3, SEN MATa, ura3-52, leu2-3,2, trp-, pep4-3, sen- MATa, ura3-, leu2-3,2, trp, his3-,5, ade2-, RNT MATa, ura3-, leu2-3,2, trp, his3-,5, ade2-, rnt ::HIS3 MATa, ura3-, leu2-3,2, trp, his3-,5, ade2-, rnt ::HIS3, rrp6 ::KanMX MATa, ura3 0, leu2 0, his3, met5 0, rrp6 ::KanMX MATα, ura3-, trp-, rna4-, rrp6 ::KanMX F. Winston [] [2] [2] CellZome CellZome CellZome CellZome [3] [3] [3] [4] This study [3] [3] [3] [2] [5] [5] [5] [5] [6] [6] [6] [7] [7] [8] [8] This study This study [9]

rna4- W rna5-2 W NA53 NA65 NA67 YMS800 YAZ26 YAZ22 MATα, ura3-, trp-, rna4- MATα, ura3-, rna5-2 MATa, ura3-, leu2-3,2, trp, his3-,5, ade2-, pcf ::TRP [pfl38-pcf] MATa, ura3-, leu2-3,2, trp, his3-,5, ade2-, pcf-2 MATa, ura3-, leu2-3,2, trp, his3-,5, ade2-, pcf-9 MATa, ura3-, leu2-3,2, trp, his3-,5, ade2-, pcf ::TRP [pnopl-pcf-3] MATa, ura3-52, leu2, trp 63, brr5::dhfr ts -BRR5 [pse360-brr5] MATa, ura3-52, leu2, trp 63, brr5::dhfr ts -BRR5 [9] [9] [0] [0] [0] [0] [] []. Amberg, D.C., A.L. Goldstein, and C.N. Cole, Isolation and characterization of RAT: an essential gene of Saccharomyces cerevisiae required for the efficient nucleocytoplasmic trafficking of mrna. Genes Dev, 992. 6(7): p. 73-89. 2. Kim, M., et al., The yeast Rat exonuclease promotes transcription termination by RNA polymerase II. Nature, 2004. 432(706): p. 57-22. 3. Nedea, E., et al., Organization and function of APT, a subcomplex of the yeast cleavage and polyadenylation factor involved in the formation of mrna and small nucleolar RNA 3'-ends. J Biol Chem, 2003. 278(35): p. 33000-0. 4. Steinmetz, E.J., et al., Genome-wide distribution of yeast RNA polymerase II and its control by Sen helicase. Mol Cell, 2006. in press. 5. Sun, Z.W. and M. Hampsey, Synthetic enhancement of a TFIIB defect by a mutation in SSU72, an essential yeast gene encoding a novel protein that affects transcription start site selection in vivo. Mol. Cell. Biol., 996. 6(4): p. 557-566. 6. Conrad, N.K., et al., A yeast heterogeneous nuclear ribonucleoprotein complex associated with RNA polymerase II. Genetics, 2000. 54(2): p. 557-7. 7. Ursic, D., et al., Multiple protein/protein and protein/rna interactions suggest roles for yeast DNA/RNA helicase Senp in transcription, transcription-coupled DNA repair and RNA processing. Nucleic Acids Res, 2004. 32(8): p. 244-52. 8. Lee, C.Y., A. Lee, and G. Chanfreau, The roles of endonucleolytic cleavage and exonucleolytic digestion in the 5'-end processing of S. cerevisiae box C/D snornas. RNA, 2003. 9(): p. 362-70. 9. Minvielle-Sebastia, L., et al., Mutations in the yeast RNA4 and RNA5 genes result in an abnormal mrna decay rate; sequence analysis reveals an RNAbinding domain in the RNA5 protein. Mol Cell Biol, 99. (6): p. 3075-87. 0. Sadowski, M., et al., Independent functions of yeast Pcfp in pre-mrna 3' end processing and in transcription termination. EMBO J, 2003. 22(9): p. 267-77.

. Zhelkovsky, A., et al., The role of the Brr5/Ysh C-terminal domain and its homolog Syc in mrna 3'-end processing in Saccharomyces cerevisiae. RNA, 2006. 2(3): p. 435-45.

Table S2. Oligos Used in This Study Gene Sequences ADH snr3 snr33 Ch V # up (5 -TTC CTT CCT TCA TTC ACG CAC ACT-3 ) # low (5 -GTT GAT TGT ATG CTT GGT ATA GCT TG-3 ) #2 up (5 -TTC AAC CAA GTC GTC AAG TCC ATC TCT A-3 ) #2 low (5 -ATT TGA CCC TTT TCC ATC TTT TCG TAA-3 ) #3 up (5 -ACC GGC ATG CCG AGC AAA TGC CTG-3 ) #3 low (5 -CCC AAC TGA AGG CTA GGC TGT GG-3 ) #4 up (5 -CAA GTA ACT ACC AGC ACC ATA ACA CCG-3 ) #4 low (5 -CTC CGG CTT TTT ATT GGC TTG CTC-3 ) #5 up (5 -CAT TTA GGT CTA CTT CTG GTC GTG C-3 ) #5 low (5 -CAG ATG ATC TTA GTT TGT TAT GAG TG-3') # up (5 -TTA TAA ATG GCA TCT CAA ATC GTC-3 ) # low (5 -GGT CAG ATA AAA GTA AAA AAA GGT AGC-3 ) #2 up (5 -CTG ACC TTT TAA CTT CCC CGT AG-3 ) #2 low (5 -CTG TCG CTT CCG TGT CTC TTG TCC TG-3 ) #3 up (5 -CAC GGA AGC GAC AGA AAG ACA GGG AG-3 ) #3 low (5 -CTA GAG GGA ATG TAT GTT GTT GAA G-3 ) #4 up (5 -GAG CAT CTG CTT TCC TTT CAC-3 ) #4 low (5 -ATC ACG GCG CCT CAT CTT TG-3 ) #5 up (5 -AAA ACC AAG AAA AGG ATA AAG AG-3 ) #5 low (5 -TCG GTG TCT ACA AAA TGA TAC GC-3 ) # up (5 -CGG AAC GGT ACA TAA GAA TAG AAG AG-3 ) # low (5 -TTC AAT CTC TGC TCC TCC AAA C-3 ) #2 up (5 -GCC TAG CTT TTA CAC CGG TTT GAG TC-3 ) #2 low (5 -TAA AGA AAA CGA TAA GAA CTA ACC-3 ) #3 up (5 -CAA TTG CAG TAA CCA GAA GCG AAC AG-3 ) #3 low (5 -CGG CAG TGA TCT CGT TCC ATT G-3 ) #4 up (5 -GCT AAT AAA CCC GCA AGA AAA TC-3 ) #4 low (5 -AGA GTC CCA GGA TTT CAC AAG GTA G-3 ) #5 up (5 -GTA TTT CAC TTG CAC TAT TCT TCT ATC-3 ) #5 low (5 -TGG AAG ATG TGT TAG GAT TAG G-3 ) no ORF up (5 -GGC TGT CAG AAT ATG GGG CCG TAG TA-3 ) no ORF low (5 -CAC CCC GAA GCT GCT TTC ACA ATA C-3 )

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