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. 2012 Sep;40(16):7666-75.
doi: 10.1093/nar/gks511. Epub 2012 Jun 20.

eIF4E-binding protein regulation of mRNAs with differential 5'-UTR secondary structure: a polyelectrostatic model for a component of protein-mRNA interactions

Andrew Cawley  1 , Jim Warwicker
Affiliations

eIF4E-binding protein regulation of mRNAs with differential 5'-UTR secondary structure: a polyelectrostatic model for a component of protein-mRNA interactions

Andrew Cawley et al. Nucleic Acids Res. 2012 Sep.

Abstract

Control of translation in eukaryotes is complex, depending on the binding of various factors to mRNAs. Available data for subsets of mRNAs that are translationally up- and down-regulated in yeast eIF4E-binding protein (4E-BP) deletion mutants are coupled with reported mRNA secondary structure measurements to investigate whether 5'-UTR secondary structure varies between the subsets. Genes with up-regulated translational efficiencies in the caf20Δ mutant have relatively high averaged 5'-UTR secondary structure. There is no apparent wide-scale correlation of RNA-binding protein preferences with the increased 5'-UTR secondary structure, leading us to speculate that the secondary structure itself may play a role in differential partitioning of mRNAs between eIF4E/4E-BP repression and eIF4E/eIF4G translation initiation. Both Caf20p and Eap1p contain stretches of positive charge in regions of predicted disorder. Such regions are also present in eIF4G and have been reported to associate with mRNA binding. The pattern of these segments, around the canonical eIF4E-binding motif, varies between each 4E-BP and eIF4G. Analysis of gene ontology shows that yeast proteins containing predicted disordered segments, with positive charge runs, are enriched for nucleic acid binding. We propose that the 4E-BPs act, in part, as differential, flexible, polyelectrostatic scaffolds for mRNAs.

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Figures

Figure 1.
Figure 1.
Averaged PARS score profiles for 5′-UTRs. Profiles (red) are averaged over all 5′-UTRs in a subset of up/down translational regulation in 4E-BP deletion mutants. In addition, an 11 nt sliding window is used to average within a 5′-UTR, and 20 nts adjacent to the start codon are excluded from the analysis. The profile averaged over all mRNAs with PARS scores and known 5′-UTR lengths is shown (blue) in all panels. Results of resampling (1000 trials), extracting the same number of mRNAs present in a subset, from all mRNAs, are shown as standard deviation around the overall profile. Profiles are shown for 5′-UTRs in the following translationally regulated mRNA sets: (a) caf20Δ up-regulated, (b) caf20Δ down-regulated, (c) eap1Δ up-regulated and (d) eap1Δ down-regulated.
Figure 2.
Figure 2.
Effects of UTR length on averaged PARS score profiles. Data for 5′-UTRs of mRNAs translationally up-regulated in the caf20Δ mutant are calculated as in Figure 1, but with the following variations. (a) No variation, same parameters and plot, with origin at the mRNA 5′ end, as Figure 1a. (b) The 20-nt exclusion 5′ to the start codon is increased to 40 nts. (c) In addition to the 40-nt exclusion, only mRNAs pre-filtered for 5′-UTR length 100 nts or greater are included.
Figure 3.
Figure 3.
Properties of 4E-BPs and eIF4G. Plots of (windowed) predicted structural disorder and net charge, are shown for each of Caf20p, Eap1p, and eIF4G (Tif4631p). These proteins are scaled to maintain their relative sequence lengths, and aligned on their 4E-binding motifs, marked with the consensus sequence and with a vertical green bar. Charge colour-coding is blue/positive and red/negative for both the sequence plots and molecular surfaces. Predicted disorder is shown by grey vertical lines. Around the 4E-binding motif and therefore proximal to the 5′-UTR of an eIF4E-bound mRNA, a dashed green box marks a region of about 200 amino acids, within which the disorder and net charge properties differ between these 3 proteins. Structural annotation for two eIF4G regions is displayed, in complex with other eIF4F components in each case, and with the eIF4G element drawn as an electrostatic potential coded surface. Both of the structural annotations correspond to eIF4G regions largely devoid of grey blocks, consistent with structured protein. The full 1rf8 coordinates (46) are shown for eIF4G in complex with eIF4E, although the structure of the isolated termini must be uncertain. Locations of the 3D structures in the eI4FG sequence are denoted by green bars. Orange bars give the sites of the PABP-binding region and three RNA-binding segments (19,20) in eIF4G.
Figure 4.
Figure 4.
Charge runs in yeast proteins. (a) The percentages of protein coding genes containing at least one 21 amino acid window bearing the relevant net charge, positive or negative, are shown. (b) and (c) Enrichment for the GO function terms nucleic acid binding, DNA binding, RNA binding, in protein subsets that contain at least one 21 amino acid window bearing the listed net positive charge. (b) Regions of predicted disorder; P < 0.01 (Bonferroni correction applied) for all enrichments from net charge +2 to +7, inclusive. (c) Regions of predicted order; P < 0.01 for all enrichments from +3 to +10, inclusive.
Figure 5.
Figure 5.
Properties of 4E-BPs and eIF4G, with histidine charge. The charge profiles for Caf20p, Eap1p and eIF4G (Tif4631p) are drawn as in Figure 3, but with histidine sidechains bearing +1 charge. The three RNA binding segments and the PABP binding region are indicated for eIF4G, as well as the location of the eIF4E-binding motif in all three proteins. The left-hand inset shows the charge profiles for Caf20p homologues (see text). In the right-hand inset, yeast ribosomal protein L28 is drawn as a tube (blue basic, red acidic), with a transparent grey surface representing ribosomal RNA [PDB ids 3u5d, 3u5e (39)]. The amino-terminal extension has similar charge and predicted disorder properties to the peak positive region of Caf20p.
Figure 6.
Figure 6.
PARS scores and locations of a subset of RBP motifs in the 5′-UTRs of genes translationally up-regulated in the caf20Δ mutant. The lower panel shows the number of hits for the subset of RBPs with motifs derived in earlier work (27,28). The x-axis gives the upper nucleotide for each bin (e.g. 10 is 1–10). The upper panel gives the PARS scores, averaged over nucleotides within each motif hit and over all motif hits in a bin, also matching the nucleotide bin coordinate shown in the lower panel.
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