Article

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

ABSTRACT

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
    • "Interaction of eIF4G with components of the 43S complex, eIF3, eIF5, or eIF1, is thought to facilitate recruitment of the PIC to cap-bound eIF4F (for review, see Hinnebusch 2014). As might be expected, eIF4F and ATP hydrolysis are dispensable in reconstituted systems for assembly of 48S PICs at the AUG codon on synthetic mRNAs with unstructured 5 ′ UTRs but are required with native mRNAs (Pestova et al. 1998; Pestova and Kolupaeva 2002; Mitchell et al. 2010). Moreover, inactivating eIF4A in mammalian extracts decreased translation of reporter mRNAs with cap-proximal structures by impairing eIF4F binding to mRNA and reducing 43S-mRNA attachment. "
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    ABSTRACT: DEAD-box RNA helicases eIF4A and Ded1 are believed to promote translation initiation by resolving mRNA secondary structures that impede ribosome attachment at the mRNA 5′ end or subsequent scanning of the 5′ UTR, but whether they perform unique or overlapping functions in vivo is poorly understood. We compared the effects of mutations in Ded1 or eIF4A on global translational efficiencies (TEs) in budding yeast Saccharomyces cerevisiae by ribosome footprint profiling. Despite similar reductions in bulk translation, inactivation of a cold-sensitive Ded1 mutant substantially reduced the TEs of >600 mRNAs, whereas inactivation of a temperature-sensitive eIF4A variant encoded by tif1-A79V (in a strain lacking the ortholog TIF2 ) yielded <40 similarly impaired mRNAs. The broader requirement for Ded1 did not reflect more pervasive secondary structures at low temperature, as inactivation of temperature-sensitive and cold-sensitive ded1 mutants gave highly correlated results. Interestingly, Ded1-dependent mRNAs exhibit greater than average 5′ UTR length and propensity for secondary structure, implicating Ded1 in scanning through structured 5′ UTRs. Reporter assays confirmed that cap-distal stem–loop insertions increase dependence on Ded1 but not eIF4A for efficient translation. While only a small fraction of mRNAs shows a heightened requirement for eIF4A, dependence on eIF4A is correlated with requirements for Ded1 and 5′ UTR features characteristic of Ded1-dependent mRNAs. Our findings suggest that Ded1 is critically required to promote scanning through secondary structures within 5′ UTRs, and while eIF4A cooperates with Ded1 in this function, it also promotes a step of initiation common to virtually all yeast mRNAs.
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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.
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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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