All eukaryotic mRNAs present a 5' terminal nuclear modification, the cap structure. This structure integrates several important functions and affects RNA splicing, transport, stabilization and translation. In translation, the cap structure serves as a "molecular tag" that marks the spot where the 40S ribosomal subunit is to be recruited. Important in this recruitment process is the eIF4F complex (78). EIF4F is a 3-subunit complex composed of eIF4E, eIF4A and eIF4G. EIF4E is the cap-binding protein and is therefore obligatory for the start of cap-dependent translation initiation. EIF4A is a member of the DEA(D/H)-box RNA helicase family, a diverse group of proteins that couples ATP hydrolysis to RNA binding and duplex separation (227). EIF4A participates in the initiation of translation by unwinding secondary structure in the 5'-untranslated region of mRNAs and facilitating scanning by the 40S ribosomal subunit for the initiation codon. EIF4A alone has only weak ATPase and helicase activities, but these are stimulated by eIF4G and eIF4B (227). EIF4B, an RNA-binding protein, stimulates eIF4A helicase activity and promotes the recruitment of ribosomes to the mRNA by interacting with the 18S ribosomal RNA (rRNA) to guide the 40S ribosomal subunit to the single-stranded region of the mRNA (98). EIF4GI and eIF4GII (here generically referred to as eIF4G) serve as a scaffold for the coordinated assembly of the translation initiation complex, leading to the attachment of the template mRNA to the translation machinery at the ribosome. EIF4G brings together eIF4F, as it has two binding sites for eIF4A and one binding site for eIF4E, but more importantly, it bridges the mRNA cap (via eIF4E) and the 40S ribosomal subunit (via eIF3) (86, 97, 215) (Fig. 2). EIF4F is recognized as the key factor in selecting mRNA for translation, it is understood that the binding of eIF4F to an m7G cap commits the translational apparatus to the translation of that mRNA. The 40S ribosomal subunit is recruited to the mRNA as part of the 43S initiation complex, composed of the subunit bound to eIF2-GTP/Met-tRNAi, eIF1A and eIF3 (98, 204, 222). EIF1A and eIF1 are required for binding to the mRNA and migration of the 43S complex in a 5' to 3' direction towards the initiation codon (199). The 5' to 3' migration of the 43S complex towards the initiation codon (ribosome scanning) is a process that consumes energy in the form of ATP. EIF1A enhances eIF4F-mediated binding of the 43S complexes to mRNA, while eIF1 promotes formation of the 48S complex in which the initiator codon is base paired to the anticodon of the initiator tRNA (199). These proteins act synergistically to mediate assembly of ribosomal initiation complexes at the initiation codon and dissociate aberrant complexes from the mRNA (199). EIF1 also participates in ensuring the fidelity of initiation by acting as an inhibitor of eIF5-induced GTP hydrolysis (discussed below) (263). The ribosome stops when it binds stably at the initiation codon to form the 48S initiation complex, primarily through the RNA-RNA interaction of the AUG (mRNA), and the CAU anticodon of the bound Met-tRNAi (associated to the 40S subunit via eIF2). The initiation codon is usually the first AUG triplet in an appropriate sequence context (G/AXXAUGG, where X is any nucleotide (nt), downstream of the 5'cap (140). Once positioned on the initiation codon the eIFs bound to the 40S ribosomal subunit are displaced (98, 204). Thus, the first step in ribosomal subunit joining is hydrolysis of eIF2-bound GTP and release of eIF2-GDP from 48S complexes (49). EIF5 induces hydrolysis of eIF2-bound GTP, leading to displacement of eIF2-GDP; the inactive eIF2-GDP is recycled to the activated eIF2-GTP by eIF2B, a guanine nucleotide exchange factor (98). In the absence of eIF1, eIF5 induces rapid hydrolysis of eIF2-bound GTP in 43S complexes. However, the presence of eIF1 in 43S complexes inhibits eIF5-induced GTP hydrolysis. Interestingly, the establishment of codon-anticodon base pairing, in the 48S complexes, relieves eIF1-associated inhibition of eIF5-induced GTP hydrolysis. Thus, hydrolysis of eIF2-bound GTP in 48S complexes, assembled with eIF1, takes place (263). Therefore, eIF1 plays the role of a negative regulator, which inhibits premature GTP hydrolysis and links codon-anticodon base pairing with hydrolysis of eIF2-bound GTP. Hydrolysis of eIF2-bound GTP and release of eIF2 leads to release of eIF3 from 48S complexes assembled on AUG triplets (263). Finally, eIF5B mediates joining of a 60S subunit to the 40S subunit, resulting in formation of a protein synthesis-competent 80S ribosome in which initiator Met-tRNA is positioned in the ribosomal P site (149, 206) (Fig. 1).
The canonical scanning mechanism rules initiation of most mRNAs, but three non-classical cap-dependent initiation mechanisms have been described: leaky scanning, ribosomal shunting and termination-reinitiation (Fig. 3). These alternative means of cap-dependent translation initiation are expected to allow the scanning complex to overcome a variety of limitations imposed by the 5'UTR.
The scanning model predicts that ribosomes should initiate at the first AUG codon encountered by a scanning 40S subunit. In most mRNAs, initiation usually does indeed occur at the AUG triplet that is proximal to the 5'end of an mRNA. However, the first encountered AUG codon can be by-passed if it is present in a poor context. In this case, the 40S subunit will initiate at an AUG in a better context further downstream, in a process known as "leaky scanning" (Fig. 3A) (139). Leaky scanning is widely used in viruses, where it presumably helps economize on coding space. In HIV-1, for example, the envelope protein (ENV) is translated from an mRNA that contains an upstream ORF encoding an accessory protein Vpu in a different reading frame. To permit Env synthesis, the vpu initiation site is in a weak context (235). Similar examples exist for the hepatitis B virus (HBV) (153), the human papillomaviruses (HPV) (250), the rabies virus (36), and the simian virus 40 (238).
The scanning model also postulates that when a scanning 40S ribosomal subunit encounters a hairpin loop in the 5'UTR, it does not skip over the loop but unwinds it (141, 142, 196). Nevertheless, there are some cases when a scanning 40S ribosomal subunit encounters the structures present in the 5'UTR and skips or shunts over a large segment, bypassing intervening segments including AUG codons and strong secondary structures that normally would block the scanning process (Fig. 3B). First characterized in cauliflower mosaic virus (CaMV) 35S RNA (68) and plant-related pararetroviruses, shunting has also been observed in Sendai (147), papillomaviruses (224), and adenovirus late mRNAs (278). In ribosome shunting, ribosomes start scanning at the cap but large portions of the leader are skipped. Thereby, the secondary structure of the shunted region is preserved.
In the reinitiation mechanism, a second ORF located in the same mRNAs can be translated without the 40S subunit becoming disengaged from the mRNA after reaching the first ORF stop codon. If the 5'-proximal AUG triplet in a mammalian mRNA is followed by a short ORF, a significant fraction of ribosomes resume scanning after termination of short ORF translation and reinitiate at a downstream AUG (Fig. 3C). For example, translation of yeast GCN4 mRNA occurs by a reinitiation mechanism that is modulated by amino acid levels in the cell (99). Ribosomes that translate the first of four upstream open reading frames (uORFs) in the mRNA leader resume scanning and can reinitiate downstream. The frequency of reinitiation following uORF1 translation depends on an adequate distance to the next start codon and particular sequences surrounding the uORF1 stop codon (99).