Affymetrix GeneChip Yeast Genome S98 [YG_S98]

Identifying eIF4E-binding protein translationally-controlled transcripts reveals links to mRNAs bound by specific PUF proteins.

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One common form of translational control is mediated by proteins that bind to the mRNA 5' cap-binding protein eIF4E. These proteins are collectively called 4E binding proteins (4EBPs). Saccharomyces cerevisiae possesses two 4EBPs that are encoded by non-essential genes called CAF20 and EAP1. To determine the impact of gene deletion on gene expression, we monitored the transcript level and also the translation status for each RNA using cycloheximide to freeze elongating ribosomes in wild-type, caf20 and eap1 cells. Polyribosome fractionation of cell extracts was used to separate highly translated and poorly translated mRNAs that were then separately analyzed.

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Transcription and translation profiling by array of yeast eap1 mutants

"One common form of translational control is mediated by proteins that bind to the mRNA 5' cap-binding protein eIF4E. These proteins are collectively called 4E binding proteins (4EBPs). Saccharomyces cerevisiae possesses two 4EBPs that are encoded by non-essential genes called CAF20 and EAP1. To determine the impact of gene deletion on gene expression, we monitored the transcript level and also the translation status for each RNA using cycloheximide to freeze elongating ribosomes in wild-type, caf20 and eap1 cells. Polyribosome fractionation of cell extracts was used to separate highly translated and poorly translated mRNAs that were then separately analyzed."

One common form of translational control is mediated by proteins that bind to the mRNA 5' cap-binding protein eIF4E. These proteins are collectively called 4E binding proteins (4EBPs). Saccharomyces cerevisiae possesses two 4EBPs that are encoded by non-essential genes called CAF20 and EAP1. To determine the impact of gene deletion on gene expression, we monitored the transcript level and also the translation status for each RNA using cycloheximide to freeze elongating ribosomes in wild-type, caf20 and eap1 cells. Polyribosome fractionation of cell extracts was used to separate highly translated and poorly translated mRNAs that were then separately analyzed.


Affymetrix:Protocol:Hybridization-Unknown

P-AFFY-6

Title: Affymetrix CEL analysis. Description:

P-MEXP-8413b

 Pooled fractions were collected directly into 2 volumes of Trizol (Invitrogen), mixed and  1 volume of chloroform was added with glycogen (50-150mg/ml) and sodium acetate (0.1 M pH 4.5).  After centrifugation, the aqueous phase was collected and 0.7 volumes of isopropanol added.  The RNA precipitate was pelleted, washed in 80% ethanol and resuspended in water. Equal amounts were analysed by northern blot analysis.  For microarrays the RNA samples were diluted, precipitated again by the addition of an equal volume of LiCl buffer (4 M LiCl, 20 mM Tris-HCl pH 7.5, 10 mM EDTA) and stored overnight at -20°C.  The RNA pellet was washed twice with 80% ethanol and resuspended in water. 

P-MEXP-8415

Cell lysates were prepared essentially as described previously (Ashe et al. 2000).  Briefly, after incubation with cycloheximide 100mg /ml at 4C, cells were pelleted and washed twice in 50ml of ice-cold lysis buffer (Ashe et al. 2000). Cell pellets were resuspended in 500ml of lysis buffer and rapidly frozen in liquid nitrogen.  Lysates were prepared by grinding under liquid nitrogen.  The ground yeast were thawed on ice and spun at 16000g at 4C for 5 min.  The extract was cleared at 16000g for 30 min at 4C.  60 A260 units were layered onto 35 ml 15-50% sucrose gradients. The gradients were sedimented via centrifugation at 16900 rpm for 13 hours.  Gradients were collected as described previously (Ashe et al. 2000) and fractionated into 15 2.2 ml aliquots. For comparison of polysomal RNA levels, fractions 4-8 and 11-15 from the gradient were pooled together for monosomal and polysomal RNA samples respectively.  As a control all 15 fractions from a second gradient were pooled. 

P-MEXP-8418

In vitro transcription by using double-stranded cDNA as a template in the presence of biotinylated UTP and CTP, using an ENZO BioArray HighYield RNA Transcript Labeling Kit (Affymetrix, Santa Clara, CA, and ENZO Diagnostics, Farmingdale, NY) according to the manufacturer's instructions.

P-MEXP-8427

Total RNA samples were prepared according standard protocols.

P-MEXP-8507

P-MEXP-89963

The isogenic yeast strains BY4742 (MATa his3d1 leu2d0 lys2d0 ura3d0) and Y17036 (MATa his3d1 leu2d0 lys2d0 ura3d0 eap1::kanMX4) (obtained from Euroscarf (Frankfurt, Germany)) were grown at 30C in a synthetic minimal media containing auxotrophic suppliments. Mid-log phase (optical density at 600nm) of 0.7 was used. The BY4742 stain was used as the control strain and the Y17334 as the mutant.

Transcription and translation profiling by array of yeast eap1 mutants

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