The 5′-7-Methylguanosine Cap on Eukaryotic mRNAs Serves Both to Stimulate Canonical Translation Initiation and to Block an Alternative Pathway

Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Molecular cell (Impact Factor: 14.02). 09/2010; 39(6):950-62. DOI: 10.1016/j.molcel.2010.08.021
Source: PubMed


Translational control is frequently exerted at the stage of mRNA recruitment to the initiating ribosome. We have reconstituted mRNA recruitment to the 43S preinitiation complex (PIC) using purified S. cerevisiae components. We show that eIF3 and the eIF4 factors not only stabilize binding of mRNA to the PIC, they also dramatically increase the rate of recruitment. Although capped mRNAs require eIF3 and the eIF4 factors for efficient recruitment to the PIC, uncapped mRNAs can be recruited in the presence of eIF3 alone. The cap strongly inhibits this alternative recruitment pathway, imposing a requirement for the eIF4 factors for rapid and stable binding of natural mRNA. Our data suggest that the 5' cap serves as both a positive and negative element in mRNA recruitment, promoting initiation in the presence of the canonical group of mRNA handling factors while preventing binding to the ribosome via an aberrant, alternative pathway requiring only eIF3.

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Available from: Sarah E Walker, May 08, 2014
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    • "Yeast also demonstrate a less pronounced dependence on eIF4G for mRNA recruitment in vitro, with eIF3 playing the major role (Jivotovskaya et al., 2006; Mitchell et al., 2010). However, the importance of eIF4G for increasing the rate and extent of mRNA recruitment in yeast is nevertheless in no doubt (Mitchell et al., 2010). "
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    ABSTRACT: Abstract The conventional paradigm of translation initiation in eukaryotes states that the cap-binding protein complex eIF4F (consisting of eIF4E, eIF4G and eIF4A) plays a central role in the recruitment of capped mRNAs to ribosomes. However, a growing body of evidence indicates that this paradigm should be revised. This review summarizes the data which have been mostly accumulated in a post-genomic era owing to revolutionary techniques of transcriptome-wide analysis. Unexpectedly, these techniques have uncovered remarkable diversity in the recruitment of cellular mRNAs to eukaryotic ribosomes. These data enable a preliminary classification of mRNAs into several groups based on their requirement for particular components of eIF4F. They challenge the widely accepted concept which relates eIF4E-dependence to the extent of secondary structure in the 5' untranslated regions of mRNAs. Moreover, some mRNA species presumably recruit ribosomes to their 5' ends without the involvement of either the 5' m(7)G-cap or eIF4F but instead utilize eIF4G or eIF4G-like auxiliary factors. The long-standing concept of internal ribosome entry site (IRES)-elements in cellular mRNAs is also discussed.
    Critical Reviews in Biochemistry and Molecular Biology 02/2014; 49(2). DOI:10.3109/10409238.2014.887051 · 7.71 Impact Factor
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    • "The dependence of the observed association rate on eIF4G-4EBD concentration was fit by a hyperbolic function, which we interpret as reporting on the equilibrium abundance of the free Cy5-eIF4E,eIF4G- 4EBD complex in solution. We estimated a value of 20 ± 13 nM for the K D of the Cy5-eIF4E,eIF4G-4EBD protein-protein interaction based on the half-height of the hyperbola, at least 1.3 times larger than the value of <15 nM measured for the equilibrium binding of full-length eIF4G1 to eIF4E by equilibrium fluorescence titration (Mitchell et al., 2010). Extrapolation of the observed association rate to saturating eIF4G-4EBD concentrations provided an estimate for the rate of binding of the "
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    ABSTRACT: Recognition of the mRNA 5' m(7)G(5')ppp(5')N cap is key to translation initiation for most eukaryotic mRNAs. The cap is bound by the eIF4F complex, consisting of a cap-binding protein (eIF4E), a "scaffold" protein (eIF4G), and an RNA helicase (eIF4A). As a central early step in initiation, regulation of eIF4F is crucial for cellular viability. Although the structure and function of eIF4E have been defined, a dynamic mechanistic picture of its activity at the molecular level in the eIF4F⋅mRNA complex is still unavailable. Here, using single-molecule fluorescence, we measured the effects of Saccharomyces cerevisiae eIF4F factors, mRNA secondary structure, and the poly(A)-binding protein Pab1p on eIF4E-mRNA binding dynamics. Our data provide an integrated picture of how eIF4G and mRNA structure modulate eIF4E-mRNA interaction, and uncover an eIF4G- and poly(A)-independent activity of poly(A)-binding protein that prolongs the eIF4E⋅mRNA complex lifetime.
    Structure 10/2013; 21(12). DOI:10.1016/j.str.2013.09.016 · 5.62 Impact Factor
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    • "TLC (as described previously; Eyler and Green, 2011), and the fractional yields of the peptides and free [ 35 S]Met in each reaction at different times were quantified and fit to the single exponential equation y = Y max (1Àexp [Àk obs 3 t]), where Y max is the maximum fraction of peptide formed and k obs is the observed rate constant. Toeprinting assays were performed as described (Mitchell et al., 2010) with minor variations. Further information is provided in the Supplemental Experimental Procedures. "
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    ABSTRACT: Translation factor eIF5A, containing the unique amino acid hypusine, was originally shown to stimulate Met-puromycin synthesis, a model assay for peptide bond formation. More recently, eIF5A was shown to promote translation elongation; however, its precise requirement in protein synthesis remains elusive. We use in vivo assays in yeast and in vitro reconstituted translation assays to reveal a specific requirement for eIF5A to promote peptide bond formation between consecutive Pro residues. Addition of eIF5A relieves ribosomal stalling during translation of three consecutive Pro residues in vitro, and loss of eIF5A function impairs translation of polyproline-containing proteins in vivo. Hydroxyl radical probing experiments localized eIF5A near the E site of the ribosome with its hypusine residue adjacent to the acceptor stem of the P site tRNA. Thus, eIF5A, like its bacterial ortholog EFP, is proposed to stimulate the peptidyl transferase activity of the ribosome and facilitate the reactivity of poor substrates like Pro.
    Molecular cell 05/2013; 51(1). DOI:10.1016/j.molcel.2013.04.021 · 14.02 Impact Factor
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