Onstruct was generated in pRS316 and is identical in sequence to

Onstruct was generated in pRS316 and is identical in sequence to Eap1p-HA with the exception of the Y109A;L114A amino acid changes. Preparation of protein extracts by glass bead lysis and subsequent immunoprecipitation experiments were preformed as previously described in Rendl et al. (2008). Briefly anti-Flag M2 Peptide M custom synthesis affinity gel (Sigma) was used to immunoprecipitate Flag-tagged proteins for 3 h at 4uC. After extensive washing Vts1p-Flag and associated proteins were eluted with Flag-peptide (Sigma). VSV antibodies (Bethyl Laboratories), HA antibodies (Santa Cruz) and eIF4E antibodies (a generous gift from N. Sonenberg [25]) were used to detect eluted proteins by Western Blot.Materials and Methods Yeast Strains and MediaYeast strains used in this study were derivatives of the wild-type BY4741 [40]. All purchase Sudan I deletion strains are as described by Winzeler et al., (1999), with the exception of the eap1D strain which was generated for this study by directed PCR-mediated gene deletion (MATa eap1D::KANMX6 leu2D0 his3D1 ura3D0 met15D0). The TetO7-DCP2 strain is described in Mnaimneh et 15481974 al., (2004). The strains were transformed by standard techniques, and plasmids were maintained by growth in selective media.mRNA AnalysisTranscriptional pulse-chase experiments employed GFP-SRE+, GFP-SRE-, and GFP-YIR016W reporters described in Rendl et al., [18]. For these experiments cells were grown in selective medium containing 2 raffinose at 30uC to mid-log phase and then cooled to 20uC. Due to the short half-life of the GFP-SRE+ reporter in wild-type cells, an accurate measure of reporter mRNA stability at 30uC was not possible. 1317923 Cooling cells to 20uC slowed the decay of GFP-SRE+ mRNA to allow for measurement of reporter mRNA half-lives. GFP reporter transcription was initiated by the addition of galactose to a final concentration of 2 for 16 min and then repressed by the addition of glucose to a final concentration of 4 . Total RNA was isolated by glass bead lysis in LET buffer (100 mM LiCl, 20 mM EDTA, 25 mM Tris-HCl at pH 8.0) and LET-equilibrated phenol at the indicated time points and analyzed by Northern blot. SCR1 or ACT1 RNA was used for normalization of transcript levels as indicated. RNase H cleavage assays were performed as described by Decker and Parker (1993), using an oligonucleotide that hybridized ,330 nt upstream of the GFP reporter poly(A) site. To control for differences in RNA concentration a portion of each RNA sample was analyzed via a standard Northern blot and probed for SCR1 RNA. Northern blots were exposed to PhosphorImager screens and analyzed with ImageQuant Software. For RNA immunoprecipitations total RNA was isolated from cells expressing the GFP-SRE+ reporter during transcriptional pulse-chase experiments 16 min after transcriptional shut-off with glucose. Immunoprecipitations were performed as described in Muhlrad et al. (1994) using monoclonal anti-m3G-/m7G-cap (Synaptic Systems) with the following modifications. Anti-m3G-/ m7G-cap antibodies were prebound at 4uC to protein G-Agarose (Roche) in IPPH (500 mM NaCl, 10 mM Tris-Cl at pH 7.5, 0.1Supporting InformationmRNA is degraded through a Ccr4p/Xrn1p-dependent pathway. GFP-SRE- gene transcription was induced in wild-type, ccr4D and xrn1D cells with galactose and then shut off with glucose and reporter mRNA levels were assayed at the times indicated after transcriptional shutoff by Northern blot. The results of at least two independent experiments were quantitated and normalized usin.Onstruct was generated in pRS316 and is identical in sequence to Eap1p-HA with the exception of the Y109A;L114A amino acid changes. Preparation of protein extracts by glass bead lysis and subsequent immunoprecipitation experiments were preformed as previously described in Rendl et al. (2008). Briefly anti-Flag M2 affinity gel (Sigma) was used to immunoprecipitate Flag-tagged proteins for 3 h at 4uC. After extensive washing Vts1p-Flag and associated proteins were eluted with Flag-peptide (Sigma). VSV antibodies (Bethyl Laboratories), HA antibodies (Santa Cruz) and eIF4E antibodies (a generous gift from N. Sonenberg [25]) were used to detect eluted proteins by Western Blot.Materials and Methods Yeast Strains and MediaYeast strains used in this study were derivatives of the wild-type BY4741 [40]. All deletion strains are as described by Winzeler et al., (1999), with the exception of the eap1D strain which was generated for this study by directed PCR-mediated gene deletion (MATa eap1D::KANMX6 leu2D0 his3D1 ura3D0 met15D0). The TetO7-DCP2 strain is described in Mnaimneh et 15481974 al., (2004). The strains were transformed by standard techniques, and plasmids were maintained by growth in selective media.mRNA AnalysisTranscriptional pulse-chase experiments employed GFP-SRE+, GFP-SRE-, and GFP-YIR016W reporters described in Rendl et al., [18]. For these experiments cells were grown in selective medium containing 2 raffinose at 30uC to mid-log phase and then cooled to 20uC. Due to the short half-life of the GFP-SRE+ reporter in wild-type cells, an accurate measure of reporter mRNA stability at 30uC was not possible. 1317923 Cooling cells to 20uC slowed the decay of GFP-SRE+ mRNA to allow for measurement of reporter mRNA half-lives. GFP reporter transcription was initiated by the addition of galactose to a final concentration of 2 for 16 min and then repressed by the addition of glucose to a final concentration of 4 . Total RNA was isolated by glass bead lysis in LET buffer (100 mM LiCl, 20 mM EDTA, 25 mM Tris-HCl at pH 8.0) and LET-equilibrated phenol at the indicated time points and analyzed by Northern blot. SCR1 or ACT1 RNA was used for normalization of transcript levels as indicated. RNase H cleavage assays were performed as described by Decker and Parker (1993), using an oligonucleotide that hybridized ,330 nt upstream of the GFP reporter poly(A) site. To control for differences in RNA concentration a portion of each RNA sample was analyzed via a standard Northern blot and probed for SCR1 RNA. Northern blots were exposed to PhosphorImager screens and analyzed with ImageQuant Software. For RNA immunoprecipitations total RNA was isolated from cells expressing the GFP-SRE+ reporter during transcriptional pulse-chase experiments 16 min after transcriptional shut-off with glucose. Immunoprecipitations were performed as described in Muhlrad et al. (1994) using monoclonal anti-m3G-/m7G-cap (Synaptic Systems) with the following modifications. Anti-m3G-/ m7G-cap antibodies were prebound at 4uC to protein G-Agarose (Roche) in IPPH (500 mM NaCl, 10 mM Tris-Cl at pH 7.5, 0.1Supporting InformationmRNA is degraded through a Ccr4p/Xrn1p-dependent pathway. GFP-SRE- gene transcription was induced in wild-type, ccr4D and xrn1D cells with galactose and then shut off with glucose and reporter mRNA levels were assayed at the times indicated after transcriptional shutoff by Northern blot. The results of at least two independent experiments were quantitated and normalized usin.

Leave a Reply