expression intensities for each strain, with the black and gray dotted lines showing two independent experiments. The bottom panel shows the ratio of each mutant vs. wild-type; a solid line for npl3-120 vs. NPL3, or dashed line for npl3-S411A vs. NPL3, and the corresponding position and orientation (Watson (W+), shown for reference only, or Crick (C-) strand) of the genes (black solid bars). Found at: doi:10.1371/journal.pone.0003273.s005 (0.18 MB TIF) Figure S4 Opposing effects in termination demonstrated using RNA-binding or phosphorylation defective npl3 alleles. RNA was extracted from NPL3, npl3-120 and npl3-S411A and whole transcript sense strand cDNA was synthesized and used for hybridization to tiled arrays. Shown are the hybridization signals for twelve genes. The three top panels represent the individual expression intensities for each strain, with the black and gray dotted lines showing two independent experiments. The bottom panel shows the ratio of each mutant vs. wild-type; a solid line for npl3-120 vs. NPL3, or dashed line for npl3-S411A vs. NPL3, and the corresponding position and orientation (Watson (W+), shown for reference only, or Crick (C-) strand) of the genes (black solid bars). Found at: doi:10.1371/journal.pone.0003273.s006 (0.17 MB TIF) References 1. Proudfoot NJ (1989) How RNA polymerase II terminates transcription in higher eukaryotes. Trends Biochem Sci 14: 105–110. 2. Hirose Y, Manley JL (2000) RNA polymerase II and the integration of nuclear events. Genes Dev 14: 1415–1429. 3. McCracken S, Fong N, Yankulov K, Ballantyne S, Pan G, et al. (1997) The C- terminal domain of RNA polymerase II couples mRNA processing to transcription. Nature 385: 357–361. 4. Zhao J, Hyman L, Moore C (1999) Formation of mRNA 39 ends in eukaryotes: mechanism, regulation, and interrelationships with other steps in mRNA synthesis. Microbiol Mol Biol Rev 63: 405–445. 5. Sadowski M, Dichtl B, Hubner W, Keller W (2003) Independent functions of yeast Pcf11p in pre-mRNA 39 end processing and in transcription termination. Embo J 22: 2167–2177. 6. Rosonina E, Kaneko S, Manley JL (2006) Terminating the transcript: breaking up is hard to do. Genes Dev 20: 1050–1056. 7. Gross S, Moore CL (2001) Rna15 interaction with the A-rich yeast polyadenylation signal is an essential step in mRNA 39-end formation. Mol Cell Biol 21: 8045–8055. 8. Gilbert W, Siebel CW, Guthrie C (2001) Phosphorylation by Sky1p promotes Npl3p shuttling and mRNA dissociation. Rna 7: 302–313. 9. Lei EP, Krebber H, Silver PA (2001) Messenger RNAs are recruited for nuclear export during transcription. Genes Dev 15: 1771–1782. 10. Gilbert W, Guthrie C (2004) The Glc7p nuclear phosphatase promotes mRNA export by facilitating association of Mex67p with mRNA. Mol Cell 13: 201–212. 11. Bucheli ME, Buratowski S (2005) Npl3 is an antagonist of mRNA 39 end formation by RNA polymerase II. Embo J 24: 2150–2160. 12. Bucheli ME, He X, Kaplan CD, Moore CL, Buratowski S (2007) Polyadenylation site choice in yeast is affected by competition between Npl3 and polyadenylation factor CFI. Rna. 13. Wong CM, Qiu H, Hu C, Dong J, Hinnebusch AG (2007) Yeast cap binding complex (CBC) impedes recruitment of cleavage factor IA to weak termination sites. Mol Cell Biol. 14. Komarnitsky P, Cho EJ, Buratowski S (2000) Different phosphorylated forms of RNA polymerase II and associated mRNA processing factors during transcription. Genes Dev 14: 2452–2460. 15. Phatnani HP, Greenleaf AL (2006) Phosphorylation and functions of the RNA polymerase II CTD. Genes Dev 20: 2922–2936. 16. Bentley DL (2005) Rules of engagement: co-transcriptional recruitment of pre- mRNA processing factors. Curr Opin Cell Biol 17: 251–256. 17. Buratowski S (2003) The CTD code. Nat Struct Biol 10: 679–680. 18. Krogan NJ, Kim M, Ahn SH, Zhong G, Kobor MS, et al. (2002) RNA polymerase II elongation factors of Saccharomyces cerevisiae: a targeted proteomics approach. Mol Cell Biol 22: 6979–6992. 19. Krogan NJ, Peng WT, Cagney G, Robinson MD, Haw R, et al. (2004) High- definition macromolecular composition of yeast RNA-processing complexes. Mol Cell 13: 225–239. 20. Meggio F, Pinna LA (2003) One-thousand-and-one substrates of protein kinase CK2? Faseb J 17: 349–368. 21. Lewis BA, Sims RJ, 3rd, Lane WS, Reinberg D (2005) Functional characterization of core promoter elements: DPE-specific transcription requires the protein kinase CK2 and the PC4 coactivator. Mol Cell 18: 471–481. 22. Calvo O, Manley JL (2005) The transcriptional coactivator PC4/Sub1 has multiple functions in RNA polymerase II transcription. Embo J 24: 1009–1020. Npl3 Stimulates Elongation Acknowledgments We are grateful to D. Brow J. Manley, and B. Schwartz for comments. We thank M. Lund/C. Guthrie lab for yeast strain; L. Vasilieva, Bret Redwine, the Conaway lab, and S. Annis (HMS Biopolymers Facility) for technical advice; and K. Struhl and E. Harlow for their support. Author Contributions Conceived and designed the experiments: MEB. Performed the experi- ments: JLD JV BO MEB. Analyzed the data: JLD JD JV MEB. Contributed reagents/materials/analysis tools: JD JV SPG PJP ASP CM SB MEB. Wrote the paper: JLD MEB. Figures 2 & 4: JLD. Oligo(dC)-tail template elongation assay: JLD. Figure 5, Expression analysis: JD PJP. Figure S2, S3 and S4, Expression analysis: JD PJP. Figure S1: JV SPG. Mass spectrometry analysis of Cka1 phosphorylation specificity using peptide libraries: JV. Analysis of Npl3 phosphorylation sites: JV. Editing of paper: JV. Figure 3: BO. Cka1 phosphorylation in vitro analysis: BO. Cka1 phosphorylation in vivo analysis: BO. Mass spectrometry analysis of Cka1 phosphorylation specificity using peptide libraries: SPG. Analysis of Npl3 phosphorylation sites: SPG. Figure 2: ASP. RNAP II purification: ASP. Editing of paper and guidance: CM SB. 23. Thomas MC, Chiang CM (2006) The general transcription machinery and general cofactors. Crit Rev Biochem Mol Biol 41: 105–178. 24. He X, Moore C (2005) Regulation of yeast mRNA 39 end processing by phosphorylation. Mol Cell 19: 619–629. 25. Ryan K (2007) Pre-mRNA 39 cleavage is reversibly inhibited in vitro by cleavage factor dephosphorylation. RNA Biol 4: 26–33. 26. Kadesch TR, Chamberlin MJ (1982) Studies of in vitro transcription by calf thymus RNA polymerase II using a novel duplex DNA template. J Biol Chem 257: 5286–5295. 27. Ziegler LM, Khaperskyy DA, Ammerman ML, Ponticelli AS (2003) Yeast RNA polymerase II lacking the Rpb9 subunit is impaired for interaction with transcription factor IIF. J Biol Chem 278: 48950–48956. 28. Kong SE, Banks CA, Shilatifard A, Conaway JW, Conaway RC (2005) ELL- associated factors 1 and 2 are positive regulators of RNA polymerase II elongation factor ELL. Proc Natl Acad Sci U S A 102: 10094–10098. 29. Misteli T, Caceres JF, Clement JQ, Krainer AR, Wilkinson MF, et al. (1998) Serine phosphorylation of SR proteins is required for their recruitment to sites of transcription in vivo. J Cell Biol 143: 297–307. 30. Xiao SH, Manley JL (1997) Phosphorylation of the ASF/SF2 RS domain affects both protein-protein and protein-RNA interactions and is necessary for splicing. Genes Dev 11: 334–344. 31. Shen H, Green MR (2004) A pathway of sequential arginine-serine-rich domain- splicing signal interactions during mammalian spliceosome assembly. Mol Cell 16: 363–373. 32. Lund MK, Kress TL, Guthrie C (2008) Autoregulation of Npl3, a yeast SR protein, requires a novel downstream region and serine-phosphorylation. Mol Cell Biol. 33. Deka P, Bucheli ME, Moore C, Buratowski S, Varani G (2008) Structure of the yeast SR protein Npl3 and Interaction with mRNA 39-end processing signals. J Mol Biol 375: 136–150. 34. Li X, Gerber SA, Rudner AD, Beausoleil SA, Haas W, et al. (2007) Large-scale phosphorylation analysis of alpha-factor-arrested Saccharomyces cerevisiae. J Proteome Res 6: 1190–1197. 35. Ficarro SB, McCleland ML, Stukenberg PT, Burke DJ, Ross MM, et al. (2002) Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae. Nat Biotechnol 20: 301–305. 36. Zielinski R, Hellman U, Kubinski K, Szyszka R (2006) Fip1–an essential component of the Saccharomyces cerevisiae polyadenylation machinery is phosophorylated by protein kinase CK2. Mol Cell Biochem 286: 191–197. 37. Sarno S, Vaglio P, Marin O, Issinger OG, Ruffato K, et al. (1997) Mutational analysis of residues implicated in the interaction between protein kinase CK2 and peptide substrates. Biochemistry 36: 11717–11724. 38. McBride AE, Cook JT, Stemmler EA, Rutledge KL, McGrath KA, et al. (2005) Arginine methylation of yeast mRNA-binding protein Npl3 directly affects its function, nuclear export, and intranuclear protein interactions. J Biol Chem 280: 30888–30898. 39. Pike AC, Rellos P, Niesen FH, Turnbull A, Oliver AW, et al. (2008) Activation segment dimerization: a mechanism for kinase autophosphorylation of non- consensus sites. Embo J 27: 704–714. 40. Sawa C, Nedea E, Krogan N, Wada T, Handa H, et al. (2004) Bromodomain factor 1 (Bdf1) is phosphorylated by protein kinase CK2. Mol Cell Biol 24: 4734–4742. 41. Flach J, Bossie M, Vogel J, Corbett A, Jinks T, et al. (1994) A yeast RNA-binding protein shuttles between the nucleus and the cytoplasm. Mol Cell Biol 14: 8399–8407. PLoS ONE | www.plosone.org 11 September 2008 | Volume 3 | Issue 9 | e3273