Phosphorylation of RNA polymerase CTD dictates transcription

Transcription

1 Supplementary Information Phosphorylation of RNA polymerase CTD dictates transcription termination choice Rajani Kanth Gudipati 1,2,3, Tommaso Villa 1,2,3, Jocelyne Boulay 1,2 and Domenico Libri 1,2. 1 LEA Laboratory of Nuclear RNA metabolism, Centre de Génétique Moléculaire, C.N.R.S.- UPR2167, 1, av de la Terrasse, 91190, Gif sur Yvette, France 2 Centre for mrnp Biogenesis and Metabolism, Department of Molecular Biology, Aarhus University, C.F. Møllers Alle, Bldg. 130, DK-8000 Aarhus C., Denmark. 3 These authors contributed equally to this work.

2 1 W303, no NEL W303 2 Supplementary Figure 1 (Libri) rrp6 rrp6 rrp6 Gal Glu

3 Supplementary Figure 1. Northern blot analysis of transcripts derived from the chromosomally integrated NEL-5 -HSP104 construct. As in Figure 1b, except that an oligonucleotide probe directed against the 5 end of the HSP104 RNA was used to reveal the short primary termination products of the HSP NEL chimeric gene. These unstable transcripts are only visible in rrp6 strains. Note that in P GAL -NRD1/rrp6 in the presence of glucose these short RNAs are not visible due to Nrd1p depletion and transcription termination failure.

4 a. NRD1-TAP FCP1 25 C fcp C 25 C 30 C 25 C 30 C Ser2-P-CTD (H5) Rpb1p Rpb3p Nrd1-TAP b. 1.2 Ser2-P-CTD /total Pol II Fold enrichment fcp1-1 Supplementary Figure 2 (Libri)

5 Supplementary Figure 2 (a) Western blot analysis of the CTD phosphorylation status in an fcp1-1 mutant background at the permissive (25 C) and semi-permissive temperature (30 C, two hours). Nrd1-TAP and Rpb3p were used as a loading control. (b) Chromatin immunoprecipitation analysis of CTD Ser2-phosphorylation at the NEL025c locus. Immunoprecipitations were performed with the H5 antibody to evaluate the levels of Ser2 phosphorylated CTD and the signals were normalized to total Pol II occupancy. Note that, although the H5 antibody recognizes both Ser2-phosphorylated and Ser2- + Ser5- phosphorylated CTD, no changes in Ser5-phosphorylated CTD were observed in the fcp1-1 strain (data not shown). Average of three independent experiments. Error bars represent standard deviation.

6 1.2 NRD1/ACT1 mrna (relative to levels in galactose) C -fcp Time (min) in glucose Supplementary Figure 3 (Libri)

7 Supplementary Figure 3 RT-real time PCR analysis of NRD1 RNAs during transcriptional repression by glucose addition. Steady state levels of the NRD1 mrna depend on a regulatory feedback loop that leads to Nrd1p-dependent premature transcription termination and nuclear degradation of the resulting truncated RNA. We expected that the effects of alterations of Nrd1p-dependent termination (e.g. due to mutation of Fcp1p) on the NRD1 mrna steady state levels would be masked by the feedback regulatory loop. In fact, decreased premature termination would ultimately lead to increased levels of Nrd1p, which, in turn, is expected to enhance termination. To optimize detection of a termination defect induced by mutation of Fcp1p we analyzed the production of full-length NRD1 mrna during transcriptional repression in the P GAL NRD1 and P GAL NRD1/fcp1-1 strains. In these conditions, we expected that the levels of full length NRD1 mrna would be more strongly dependent on the efficiency of premature termination. Primers are located in the 3 end of the NRD1 gene and detect transcripts that have not been terminated by the Nrd1- complex pathway, i.e. full-length NRD1 mrnas. Signals are normalized to ACT1 RNAs and expressed relative to levels in galactose. The experiment was performed at the permissive temperature for fcp1-1 cells. Average of two experiments. Error bars represent standard deviations

8 Supplementary Table 1. Yeast strains used in this study Name Genotype Source W303 ura3-1, ade2-1, his3-11,5, trp1-1, leu2-3,112, can1- (Thomas and Rothstein, 1989) 100 DLY883 HIS::Pgal::NRD1 (Thiebaut et al., 2006) DLY885 HIS::Pgal::NRD1, rrp6::kan (Thiebaut et al., 2006) DLY878 fcp1::leu2, prs314/fcp1-1/trp1 T.H. Jensen DLY1086 HIS::Pgal::NRD1, fcp1::leu2, prs314/fcp1-1/trp1 DLY1092 HIS::Pgal::NRD1, fcp1::leu2, prs314/fcp1-1/trp1, rrp6::kan DLY125 rrp6::kan (Libri et al., 2002) DLY142 rna14-1 F.Lacroute DLY987 HSP NEL DLY1054 NEL-5 -HSP104 DLY998 HSP NEL, HIS::Pgal::NRD1 DLY1056 NEL-5 -HSP104, HIS::Pgal::NRD1, rrp6::kan DLY1057 NEL-5 -HSP104, HIS::Pgal::NRD1 DLY1103 HSP NEL, HIS::Pgal::NRD1, rrp6::kan DLY1146 NRD1::KAN, prs415/nrd1-102/leu2 DLY1147 NRD1::KAN, prs415 /nrd1-51/leu2 DLY1174 NRD1:: KAN, prs415/nrd1-102/leu2, fcp1::leu2, prs314/fcp1-1/trp1 DLY1175 NRD1:: KAN, prs415/ nrd1-51/ LEU2, fcp1::leu2, prs314/fcp1-1/trp1 DLY1126 NRD1::hU1A-RBD::TRP1-Kl DLY1161 NRD1::hU1ARBD::TRP1-Kl, rrp6::kan DLY1162 NRD1::hU1ARBD::TRP1-Kl, trf4::kan DLY267 ctk1::trp V. Goguel DLY1123 ctk1::trp, rrp6::kan DLY1197 kin28::ura, psf19/kin28/trp, HIS:: DLY1198 kin28::ura, psf19/kin28-ts16/trp, HIS:: BSY1476 upf1::tap::trp1kl B. Seraphin

9 Supplementary Table 2. Oligonucleotides used in this study Region Name Sequence (5' to 3') ACT1 Forward ATGTTCCCAGGTATTGCCGA Reverse ACACTTGTGGTGAACGATAG SNR13 RT Forward AAGTGACGAAGTTCATGCTA Reverse TCCGTGTCTCTTGTCCTGCA NRD1 Forward GCAGCAACCGTATGGTTATG Reverse GTTGGTTAAGCATATTCATC HSP104 DL163 ACATTTCATCACGAGATTTACCC HSP104 RT D2 ATAATGGACCAATCCGCGTGTG HSP104 5, forward ATATGAACGACCAAACGC HSP104 5, reverse AGATCATAGTCGTAACGGC HSP104 3, forward TTGAACTTGACACCCGAGCAA HSP104 3, reverse TAAACTTTAGTTATCAACGCCA PGK1-pG DL1112 ATTCCCCCCCCCCCCCCCCCCA PGK1 5 -tag DL1103 CAATTCGTCGTCGTCGAATAAAG PP3, Forward CGCAGAGTTCTTACCAAACG NEL025c PP3, Reverse TAAATGGCCAACCGCTGTTG PP6, Forward TCAGCACAGAACGTAACGAC PP6, Reverse CAATTTTTGAGCCCACATGC

10 References Libri, D., Dower, K., Boulay, J., Thomsen, R., Rosbash, M., and Jensen, T. H. (2002). Interactions between mrna export commitment, 3'-end quality control, and nuclear degradation. Mol Cell Biol 22, Thiebaut, M., Kisseleva-Romanova, E., Roug le, M., Boulay, J., and Libri, D. (2006). Transcription termination and nuclear degradation of cryptic unstable transcripts: a role for the nrd1-nab3 pathway in genome surveillance. Mol Cell 23, Thomas, B. J., and Rothstein, R. (1989). Elevated recombination rates in transcriptionally active DNA. Cell 56,