Quantified_Poly(A)_Interactome_Capture

This data is related to the publication 'Metabolic tuning enables immediate adaptation to energy stress in yeast', and is discussed there in detail.

Summarised hypothesis and experiment:
Recruitment of the 40S and 60S ribosomal subunits at the start codon of a transcript is mediated by ‘scanning’ initiation factors, which unwind secondary structures in the mRNA to grant access to this site. These are eIF4B and two DEAD-box RNA helicases, eIF4A and Ded1 that act to promote 43S-complex loading . In response to glucose withdrawal, binding of these factors to transcripts is lost, which directly blocks formation of new 80S ribosomes.

To confirm the timescale of initiation factor displacement from mRNA transcripts, we developed a poly(A) interactome capture approach. RNAs were UV crosslinked to bound proteins in cells grown in glucose or at timepoints immediately following transfer to Glc/EthOH medium. Poly(A)+ RNAs were purified from cell lysates using oligo[dT]25 beads.

Western blot analysis was used to compare amounts of Flag-tagged initiation factors (eIF4A/eIF4B) in inputs and eluates of poly(A) interactome capture. Poly(A)-binding protein (Pab1) acted as a control for input and pull-down efficiency.

Membranes were visualized by chemiluminescent imaging using the LiCor Odyssey CLx imaging system (LICORbio). Quantitative analysis of captured images was performed with the Image Studio software (LICORbio).

The following antibodies were used: rat Anti-DYKDDDDK Tag Antibody (1:1000; Agilent, 200474), mouse Pab1p Monoclonal Antibody (1G1) (1:1000; Invitrogen, MA5-47390), IRDye 800CW Goat anti-Rat IgG Secondary Antibody (1:5000; LICORbio, 926-32219), IRDye 680RD Goat anti-Mouse IgG Secondary Antibody (1:5000; LICORbio, 926-68070).

Provided data:
The .pdf provided shows all Western blots quantified in the published manuscript (duplicate for each experiment), with the loading scheme and annotation.
The raw are .tif files for each of these are also provided. In naming, '2GE' represents withdrawal from 2% sugar to 2% glcyerol and 2% ethanol and AA indicates addition of the mitochondrial inhibitior Antimycin A.