Cap-Assisted Internal Initiation of Translation of Histone H4 Franck Martin, Sharief Barends, Sophie Jaeger, Laure Schaeffer, Lydia Prongidi-Fix, Gilbert Eriani  Molecular Cell  Volume 41, Issue 2, Pages 197-209 (January 2011) DOI: 10.1016/j.molcel.2010.12.019 Copyright © 2011 Elsevier Inc. Terms and Conditions

Molecular Cell 2011 41, 197-209DOI: (10.1016/j.molcel.2010.12.019) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 1 UTRs' Role and m7G Cap Dependence during H4 mRNA Translation (A) Diagram representing the different truncated H4 transcripts used in the study. (B) Translation products formed in WGE and RRL separated by 15% SDS-PAGE and [35S]-labeled H4 quantification. The graph represents relative expressions, and error bars denote the standard deviations from three independent experiments. (C and D) Translation of H4 mRNA (250 nM) is inhibited in the presence of increasing concentrations of the cap analog m7GpppG (0.48–2.5–12.5–62.5–300 μM). Excess of free ApppG does not impede H4 translation, while H4 could not be translated from an A-capped H4 mRNA (D). Error bars indicate the standard deviations from three independent experiments (see also Figure S1). Molecular Cell 2011 41, 197-209DOI: (10.1016/j.molcel.2010.12.019) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 2 H4 mRNA Recruits Ribosomes in the Absence of m7G Cap (A) Toe-print analysis performed on wild-type (Wt) or Met1Thr (M1T) H4 mRNAs carrying a standard m7G- or an A cap modification. Initiation complexes were assembled in RRL extracts (lane T) and fractionated into pellet (P) and supernatant (S) in order to discriminate between RRL-dependent (nonspecific RNA-binding proteins) and ribosome-specific RT stops. Cycloheximide (1 mg/ml) was used to stall the initiation complexes. Toe-prints were found at position +19 downstream the AUG codon. The +19 toe-print position detected with Moloney murine leukemia virus (MMLV) reverse transcriptase is equivalent to a +17 toe-print detected with avian myeloblastosis virus (AMV) reverse transcriptase (see Figure S2). The toe-print shift from +20 to +19 after ultracentrifugation results from the loss of the aminoacyl-tRNA molecule from the ribosomal A site (Joseph and Noller, 1998). (B) Ribosome assembly was studied on sucrose-gradient with RRL programmed with 3′ end radiolabeled m7G- or A-capped H4 mRNA. Translation extracts were separated on 7%–47% sucrose gradients, and the different complexes were detected by Cerenkov counting. Radioactivity in the different fractions is expressed as a percentage of the total counts (see also Figure S2). Molecular Cell 2011 41, 197-209DOI: (10.1016/j.molcel.2010.12.019) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 3 EMSA Analysis of eIF4E Binding to H4 mRNA (A and B) (A) Binding of eIF4E (2 μM) to 3′ end radiolabeled m7G- or A-capped H4 mRNAs was investigated by EMSA, and the effect of adding m7GpppG or ApppG cap analogs (500 μM both) on eIF4E binding to H4 mRNA was tested (B). (C) Uncapped H4 mRNA (0.4 μM) is an efficient competitor for eIF4E binding, whereas uncapped-GFP mRNA (0.4 μM) does not compete. (D) eIF4E variants were constructed and tested for H4 binding by EMSA. Both mutants (2 μM) were able to bind H4 mRNA. (E) Deletion of the first 27 or 36 aa residues of eIF4E (2 μM) abolishes the interaction with H4 mRNA. Δ symbol refers to the amino acid deletion from the amino-terminal end. The effect of the triple Ala-mutagenesis of the amino-terminal peptide from eIF4E was evaluated by EMSA at 2 μM. The sequence of the 30 first amino acids residues from the human eIF4E is shown as well as the Ala mutations introduced (see also Figure S3). Molecular Cell 2011 41, 197-209DOI: (10.1016/j.molcel.2010.12.019) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 4 Characterization of the 4E-SE Moiety of H4 mRNA (A) Progressive deletions of H4 mRNA were evaluated by EMSA to define the minimal eIF4E-binding site. Δ symbol refers to the nucleotide deletion from the 5′ or 3′ end. (B) Shown is in vitro translation in RRL in the presence of H4 mRNA fragments. Increasing concentrations from 0 to 30 μM of the Δ27-5′ and Δ154-5′ Δ126-3′ fragment (marked with ∗) were added to RRL programmed with m7G-capped H4 mRNA. H4 synthesis was quantified and expressed relative to the assay without fragment. Error bars represent the standard deviation of three independent experiments. (C) RT-primer-extension analysis of eIF4E-H4 mRNA complexes in the presence of 0.33 and 0.66 μM of eIF4E (lanes 2 and 3). Two stops absent in the control lanes are detected, evidence that eIF4E interacts tightly at that level of the mRNA. (D) Secondary structure of nucleotides 187–219 summarizing the H4 mRNA solution-structure probing results. (E) Secondary structure comparison between the 4E-SE element of H4 and cyclin D1. The conservation of a set of residues UX2UX2A is shown in the structure (highlighted in yellow in the figure) (Culjkovic et al., 2006). Molecular Cell 2011 41, 197-209DOI: (10.1016/j.molcel.2010.12.019) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 5 Secondary Structure of the 135 First Nucleotides of Histone H4 mRNA and Interaction with the m7G Cap Structure (A) Diagram representing results of defining the solution structure of H4 mRNA as described in the Experimental Procedures. The structure contains three helices connected by a three-way helix junction followed by a stem-loop structure. The initiation codon is boxed. The two black stars show the location of the +19 and +20 ribosome toe-prints. (B) Significant differences were detected when m7G-capped and uncapped H4 mRNA were probed. (C) Crosslink experiments on m7G-capped H4 mRNA and m7s6G-capped H4 mRNA. Crosslinks were performed at 312 nm and revealed by reverse transcription. The two stops detected at positions 40 and 41 are shown in (B). (D) Crosslink experiments on m7s6G-capped H4 mRNA in the presence of 1.5 mM m7GpppG (see also Figure S4). Molecular Cell 2011 41, 197-209DOI: (10.1016/j.molcel.2010.12.019) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 6 Effects on H4 Translation of Mutations in the 4E-SE and Three-Way Helix Junction (A) Four nucleotides were simultaneously mutated in the 4E-SE in order to disrupt two of the three Watson-Crick interactions. (B) Capped mRNAs were translated in RRL and WGE, followed by protein-product separation by 15% SDS-PAGE, and quantification and data were expressed relative to the wild-type H4 mRNA translation. Stability of the mutated mRNA in extracts was tested and found to be unchanged compared to the native one (data not shown). Error bars represent the standard deviation of three independent experiments. (C) Single mutants 2, 3, and 4 are located in the three-way helix junction. (D) Mutants 2, 3, and 4 were tested as in (B). Error bars represent the standard deviation of three independent experiments. (E) Mutants of the 4E-SE and three-way helix junction were combined and tested in translation. Error bars represent the standard deviation of three independent experiments. (F) Ribosome assembly in HeLa cell extracts programmed with radiolabeled m7[32P]G-capped H4 mRNA in the presence of GMP-PNP or cycloheximide. Radioactivity was detected by Cerenkov counting. The graph represents the radioactivity in the different fractions expressed as a percentage of the total counts (see also Figure S5). Molecular Cell 2011 41, 197-209DOI: (10.1016/j.molcel.2010.12.019) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 7 Binding of eIF4E to 4E-SE Element Induces Release of the Cap from the Cap-Binding Pocket (A) Binding of eIF4E to 4E-SE was monitored by primer extension on uncapped and m7G-capped H4 mRNA. Increasing concentrations of eIF4E were added and RT stops were quantified to reveal eIF4E binding; error bars indicate the standard deviations of three independent determinations. (B) Release of the cap from the three-way helix junction domain was monitored by UV crosslinking experiments. m7s6G-capped H4 mRNA was subjected to UV312 nm exposure and crosslinks and analyzed by primer extension. eIF4E binding decreases the cap crosslinking efficiency The histogram represents the ratios ± UV; error bars indicate the standard deviations of three independent determinations (see also Figure S6). Molecular Cell 2011 41, 197-209DOI: (10.1016/j.molcel.2010.12.019) Copyright © 2011 Elsevier Inc. Terms and Conditions