Probing the closed-loop model of mRNA translation in living cells

Abstract

The mRNA closed-loop, formed through interactions between the cap structure, poly(A) tail, eIF4E, eIF4G and PAB, features centrally in models of eukaryotic translation initiation, although direct support for its existence in vivo is not well established. Here, we investigated the closed-loop using a combination of mRNP isolation from rapidly cross-linked cells and high-throughput qPCR. Using the interaction between these factors and the opposing ends of mRNAs as a proxy for the closed-loop, we provide evidence that it is prevalent for eIF4E/4G-bound but unexpectedly sparse for PAB1-bound mRNAs, suggesting it primarily occurs during a distinct phase of polysome assembly. We observed mRNA-specific variation in the extent of closed-loop formation, consistent with a role for polysome topology in the control of gene expression.

Keywords: cap-poly(A) synergy; eukaryotic translation; mRNA closed-loop; polysomes, ribosomal recycling.

Figure 1.

(A) Overall experimental strategy. Each yeast line used encoded a recombinant version of either PAB1, eIF4G or eIF4E bearing an affinity tag with a Protein A moiety. The corresponding proteins were affinity-purified in the presence of ribonuclease. (B) Top: qPCR primers were designed against various regions of a target transcript to test for co-purification. Bottom: Enrichment of SSC1 transcript regions with eIF4E, eIF4G and PAB1, by RT-qPCR. IP / Input was normalized to the average across all 5 qPCR assays in SSC1. Error bars represent 95% confidence intervals from 3 qPCR replicates.

Figure 2

(See previous page). (A) Quantitative isolation of protein A-tagged eIF4E (top) or PAB1 (bottom) by IP using IgG affinity chromatography, and detection of co-purifying eIF4G. Input lanes: 1×, 0.5×, 0.25×, 0.125× of lysate equivalents. FT: Flow-through, 1× equivalent. IP: Immunoprecipitated fraction, 1× equivalent. (B) Co-enrichment of mRNA extremities with eIF4E and PAB1 (average of 3 biological replicates, error bars correspond to standard error of the mean). Values were normalized to the average 3' enrichment (for PAB1) or 5' enrichment (for eIF4E) across all transcripts. Blue dashed lines: transcript extremities. (C) Collapse and restoration of polysomes after glucose starvation (10 minutes) and re-feeding (5 minutes) of eIF4E::ProA yeast cultures. Representative UV-absorbance traces (sedimentograms) of crosslinked cytoplasmic extracts fractionated through sucrose gradients. Signal to the right of the 80S monosome peak (indicated) corresponds to translated (polysomal) mRNAs. (D) Correlation between the closed-loop status (4E:3') of transcripts with or without starvation / re-feeding. Error bars denote standard error of the mean (3 biological replicates). Only TDH1 showed significant change (corrected p = 0.006).