Regulation of translation initiation in eukaryotes: mechanisms and biological targets

Abstract

Translational control in eukaryotic cells is critical for gene regulation during nutrient deprivation and stress, development and differentiation, nervous system function, aging, and disease. We describe recent advances in our understanding of the molecular structures and biochemical functions of the translation initiation machinery and summarize key strategies that mediate general or gene-specific translational control, particularly in mammalian systems.

Figure 1. Eukaryotic Cap-Dependent Translation Initiation and Its Regulation by eIF2α Kinases and Other Signaling Pathways

eIFs 1, 1A, and 3 promote dissociation of 80S ribosomes and, together with eIF5 and ternary complex (eIF2-GTP-Met-tRNA_i), assemble the 43S preinitiation complex (PIC). In yeast, these eIFs form a multifactor complex (MFC), which could bind to the 40S ribosomal subunit. mRNA is activated by binding of eIF4F (eIF4E·eIF4G·eIF4A) to the cap and PABP to the poly(A) tail, circularizing the mRNA. The 43S PIC binds near the cap, facilitated by eIF3/eIF5 interactions with eIF4G/eIF4B, and scans the leader for the AUG codon in an ATP-dependent reaction, with partial hydrolysis of the eIF2-bound GTP in the ternary complex to eIF2-GDP-Pi. AUG recognition triggers eIF1 dissociation from the 40S platform (not depicted), allowing release of Pi and eIF2-GDP. Joining of the 60S subunit, with release of other eIFs, is catalyzed by eIF5B-GTP, and GTP hydrolysis triggers release of eIF5B-GDP and eIF1A to yield the final 80S initiation complex. Under stress or starvation conditions, ternary complex formation is reduced by eIF2α phosphorylation, and eIF4F assembly is blocked by 4E-BP binding to eIF4E. Phosphorylation by mTOR dissociates 4E-BP from eIF4E. mTOR also promotes eIF4G and eIF4B phosphorylation either directly or via S6Ks. Mitogens and growth factors promote all of these phosphorylation events by activating mTOR via PI3K/Akt signaling or RAS/MAPK signaling. (Not shown is that MAPK signaling also engenders phosphorylation of eIF4E by kinases Mnk1/Mnk2.) (Figure adapted from Sonenberg and Hinnebusch, 2007.)

Figure 2. The PIC Associated with mRNA Binding, Scanning, and AUG Recognition

(A) A hypothetical model showing (I) the 48S PIC in an open, scanning-conducive conformation with a non-AUG codon in the P-site and eIF1A in the A-site. GTP in the ternary complex is partially hydrolyzed in a manner stimulated by eIF5, but Pi release from eIF2-GDP-Pi is blocked by eIF1, bound near the P-site. Both eIF1 and eIF1A promote scanning. (II) Pairing of Met-tRNA_i ^Met with the AUG codon elicits a conformational change that increases the separation between the eIF1A C-terminal tail (CTT) and eIF1 and results in tighter binding of eIF1A to the PIC, mediated by the eIF1A N-terminal tail (orange) and by neutralizing an antagonistic effect of the eIF1A CTT on PIC interaction (perhaps via CTT-eIF5 interaction). (III) Dissociation of eIF1 from its location near the P-site allows release of Pi from eIF2-GDP-Pi, an irreversible step that drives GTP hydrolysis to completion and finalizes start codon selection (adapted from Fekete et al., 2007). (B) Cryo-EM reconstruction of a yeast 40S subunit alone (Apo) or bound to eIF1 and/or eIF1A (pt, platform; n, neck; *, connection between shoulder and head induced by eIF1/eIF1A binding). (Reprinted from Passmore et al., 2007.) (C) Cryo-EM model of eukaryotic 40S subunit bound by the hepatitis C virus IRES (purple) and mammalian eIF3 (pink) (from Siridechadilok et al., 2005, Science 310, 1513–1515; reprinted with permission from AAAS). (D) Model for the scanning PIC, based on new findings on the topology of the eIF4A/4G/4H helicase complex and spatial arrangement of its RNA-binding surfaces, which places eIF4A on the 3′ side of the PIC (reprinted from Marintchev et al., 2009).

Figure 3. Structures of the Bacterial Initiation Complex and CrPV IRES

(A) Cryo-EM model of the 30S initiation complex from the bacterium Thermus thermophilus with small ribosomal subunit, mRNA, fMet-tRNA^fMet, IF1, and GTP-bound IF2 (reprinted with permission from Macmillan Publishers Ltd: Simonetti et al., Nature 455, 416–420, 2008, copyright 2008). (B) Structures of Dicistroviridae intergenic region IRESs. (Left) Secondary structure of Plautia stali intestine virus (PSIV) intergenic region, containing 3 pseudoknots (PKs), 2 conserved stem loops (SLs), and the non-AUG start codon as important components of the IRES. (Right) Crystal structures of domain 3 of cricket paralysis virus (CrPV) IRES (boxed) and the P-site tRNA-mRNA interaction in the bacterial 70S complex, with anticodon loop in red and mRNA codon in blue (reprinted from Kieft, 2008).

Figure 4. Translational Control via the 3′ UTR

(A) Models for cap-dependent translational repression by soluble or tethered eIF4E-binding proteins. Cap-dependent initiation requires interaction of eIF4E with the mRNA 5′ cap structure, which forms the eIF4F complex together with RNA helicase eIF4A and eIF4G. By binding to both eIF4E and PABP, eIF4G mediates circularization of mRNA. A general mechanism of translational repression involves the 4E-binding proteins (4E-BPs), which compete with eIF4G for interaction with eIF4E. Other repression mechanisms are more mRNA specific. Translation of mRNAs containing a cytoplasmic polyadenylation element (CPE) is repressed by displacement of eIF4G by Maskin/4E-T, recruited to the mRNA by CPE-binding protein (CPEB). The latter model works for specific mRNAs using different modules. Translation of Drosophila oskar mRNA is inhibited by tethering eIF4E to the Bruno response element (BRE) via Bruno and Cup. A variation on the theme is presented by Bicoid, which inhibits Drosophila caudal mRNA translation by binding simultaneously to the 3′UTR Bicoid-binding region (BBR) and the eIF4E-homologous protein 4E-HP (adapted from Sonenberg and Hinnebusch, 2007). (B) Translational repression of ceruloplasmin mRNA upon interferon-γ treatment involves formation of the GAIT complex from Glu-Pro-tRNA synthetase, NS-associated protein 1, GAPDH, and 60S ribosomal protein L13a (released by phosphorylation from the 60S subunit). The complex binds to the GAIT element in the 3′UTR and blocks interaction of eIF4G with eIF3 in the 43S PIC to prevent 48S PIC assembly on ceruloplasmin mRNA (adapted from Kapasi et al., 2007). (C) Dual repression of male-specific lethal 2 (msl-2) mRNA translation by sex lethal (SXL) protein. Bound to the 3′UTR, SXL recruits UNR (upstream of N-ras) protein to block 43S PIC binding to the 5′ end of the mRNA. SXL also targets scanning ribosomes in the 5′UTR for a backup repression mechanism (figure kindly provided by Matthias Hentze and colleagues).