nucleotides (50 mM ATP, 50 mM GTP, 2 mM CTP, and 10 mCi [a-32P] CTP (3000Ci/mmol), followed by incubation for 30 min- utes. Chase was performed by the addition of 1 mM UTP and 50 mM unlabeled CTP [28]. Reactions were stopped by the addition of 10 mM Tris-HCl, pH 7.2, 0.5 mM EDTA and 0.3 M NaCl, 0.2% SDS and 20 mg proteinase K. The reaction mixture was extracted once with phenol-chloroform-isoamyl alcohol. Transcripts were resolved on an 8% polyacrylamide–7 M urea gel and then visualized using a PhosphorImager. Cka1 Phosphorylation and Phosphatase in vitro Assays 500 ng of recombinant Npl3, Npl3-S411A, -S411D, -S411E and Rna15 were phosphorylated in kinase buffer (20 mM HEPES- KOH, pH 7.0, 7.5 mM MgOAc, 100 mM KOAc, 2 mM DTT, 20% glycerol) at 30uC for 60 min with [c-32P] –ATP (or cold ATP for MS analysis) and 500 ng recombinant Cka1-His6. For protein phosphatase assays, reactions were carried out in 16 Lambda Phosphatase (l-PPase) reaction buffer [13] with 500 ng of His- Npl3 and titrated (200–600 U) Lambda Protein Phosphatase [13]. These were then incubated at 30uC for 60 min and stopped by the addition of SDS sample buffer. Proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and detected using a PhosphorImager. Mass spectrometry analysis For identification of endogenous phosphorylated peptides in Npl3, the proteinwas immunoprecipitated from whole cell extracts. This in vivo phosphorylated Npl3 and the in vitro phosphorylated protein were excised from Coomassie-stained gels, digested with trypsin, and analyzed by LC-MS/MS in a linear ion trap (LTQ) mass spectrometer. MS/MS spectra were searched against the S. cerevisiae protein database. Protein analysis For the preparation of yeast whole-cell extracts, 10 ml of cells were grown overnight, inoculated to an optical density (600 nm) of 1.5, allowed to grow at the permissive (25uC) or non-permissive (37uC) temperature for 2 hours, and pellet. Glass beads were used to disrupt cells in lysis buffer (20 mM HEPES [pH 7.6], 200 mM potassium acetate [KOAc], 10% glycerol, 1 mM EDTA) supple- mented with protease inhibitors (1 mM phenylmethylsulfonyl fluoride and 1 mg/ml of aprotinin, leupeptin, antipain and pepstatin-A). Equal amounts of protein were then resolved using SDS-PAGE. Immunoblotting was performed using standard methods with polyclonal rabbit anti-phosphorylated Npl3 (kindly provided by C. Guthrie, UCSF) and anti-Npl3. Polyclonal rabbit anti-Rpb3 was from Neoclone, Horseradish peroxidase-conjugat- ed polyclonal goat anti-rabbit was from SouthernBiotech, and goat anti-mouse HRP antibodies were from Jackson ImmunoResearch Laboratories. UV cross-linking assays UV cross-linking experiments were performed as described previously [12]. RNA oligos containing the sequences: 59- UAAUAAUGACUAUAUAUG-39 (N4) or 59-UUGCCUG- GUUGCCUGGUU-39 (N2) were synthesized (Invitrogen), and [a-32P]ATP 59 end-labeled using T4 Polynucleotide Kinase (Invitrogen) as described previously [33]. RNA (,100,000 cpm) was mixed with recombinant Npl3, Rna15, and Cka1 in kinase buffer (200 mM HEPES-KOH, pH 7.0, 75 mM MgOAc, 1 M KOAc, 20 mM DTT, 20% glycerol) with 200 mM ATP. All reactions were incubated for 20 minutes at room temperature and then UV irradiated in an Ultra-lum/UVC-515 ultraviolet multi- Npl3 Stimulates Elongation linker set at 1800 mJ (6100). Loading buffer was added and reactions were resolved on a 10% SDS-polyacrylamide gel (30:0.8 acrylamide:bis), dried and exposed to a PhosphorImager screen. Expression analysis with tiled arrays RNA for NPL3, npl3-120, npl3-S411A and rna15-2 was extracted as described previously [59]. Sense RNA was synthesized using whole transcript sense target labeling assay (Affymetrix, HMS Biopolymers Facility), and hybridized to S. cerevisiae Tiling 1.0R Arrays, and intensities were analyzed using the Tiling Analysis Software (TAS) (https://www.affymetrix.com/support/learning/ tiling_analysis/tiling_analysis_sw_tutorial.affx). Representative graphs were generated using the R software [60]. The microarray datasets in Figure 5 have been deposited in the GEO database (http://www.ncbi.nlm.nih.gov/projects/geo/) under accession number GSE12677. Supporting Information Table S1 Yeast strains used in this study. Found at: doi:10.1371/journal.pone.0003273.s001 (0.04 MB DOC) Table S2 Genes with increased Log2 Ratio at their 39UTR. Found at: doi:10.1371/journal.pone.0003273.s002 (1.47 MB DOC) Figure S1 Distribution of the log2 ratios of the peptide libraries phosphorylated in vitro with Cka1 and measured by MS. Kinase assays utilizing peptide libraries representing acidic (L[LE]D- [KDN]D[DA][LE][ST]D[EL]E[LEN][EL]K) and proline-direct- ed ([KP]L[VKE]L[AP][NE][ST]P[KI][LKP]VV[KL]) motifs were performed. 20 mg of the non-phosphorylated libraries were used, and 0.2 mg of the heavy phosphorylated standard library was added upon reaction quenching with 0.15 % TFA. Reactions were desalted in a tC18 SepPak and enriched for phosphopeptides with PhosSelect IMAC resin (Sigma). Samples were desalted again prior to injection into a capillary (125 mm618 cm) C18 column and analyzed by LC-MS/MS in a LTQ-Orbitrap mass spectrom- eter using a 60 minute-gradient and data dependent TOP10 method. MS/MS spectra were searched against a database containing the sequences for all the peptides in the libraries and all the sequences from E. coli protein sequence database (used for distraction purposes) in the forward and reverse directions. Results were filtered to ,1% false positives and peptides were further quantified using the VistaQUANT algorithm. Found at: doi:10.1371/journal.pone.0003273.s003 (0.07 MB TIF) Figure S2 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.s004 (0.17 MB TIF) Figure S3 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 PLoS ONE | www.plosone.org 10 September 2008 | Volume 3 | Issue 9 | e3273