Translation of mRNAs can vary in efficiency, so that the amount of protein produced is modulated. This is an important level of gene regulation; indeed, a correlation between mRNA and protein abundance is seen only for secreted proteins, whereas for intracellular proteins the differing rates of translation of different mRNAs removes this correlation [18
]. Features all along the mRNA can affect translation efficiency.
Structural features of the 5' UTR have a major role in the control of mRNA translation. Messenger RNAs encoding proteins involved in developmental processes, such as growth factors, transcription factors or proto-oncogenes, all of which need to be strongly and finely regulated, often have 5' UTRs that are longer than average [19
], with upstream initiation codons or open reading frames (ORFs) and stable secondary structures that hamper translation efficiency (Table ). Other specific motifs and secondary structures in the 5' UTR can also modulate translation efficiency.
Examples of genes with 5' UTRs longer than average and with upstream ORFs and/or repeat elements
Under normal conditions, following the transport of an mRNA from the nucleus to the cytoplasm, the eIF4F protein complex assembles at the cap. This complex consists of three subunits: eIF4E, the cap-binding protein; eIF4A, which has RNA helicase activity; and eIF4G, which interacts with various other proteins, including polyadenylate-binding protein. The ATP-dependent helicase activity of eIF4A, stimulated by the RNA-binding protein eIF4B, unwinds any secondary structure in the mRNA, thus creating a 'landing platform' for the small (40S) ribosomal subunit [20
]. When concentration of ribosomes or translation factor are limiting, the poly(A) tail can cooperate with 5' cap to enhance translation initiation through the intervention of a polyadenylate-binding protein that can physically interact with eIF4F complex [21
In most eukaryotic mRNAs, it is thought that translation initiates at the first AUG codon encountered by the 40S ribosomal subunit as it moves, or scans, 3' along the mRNA from the 5' m7G cap. Sequences flanking the AUG initiation codon are not random but fit a consensus sequence; in mammals, this sequence is GCCRCCaugG, and the most conserved nucleotides are the purine (R), usually A, in position -3 with respect to the AUG start codon and the guanine in position +4. The strong preference for A at position -3 and G at position +4 is also conserved in other animals and in plants and fungi. The sequence context of the first AUG codon, in particular the part located in the untranslated region, may modulate the efficiency with which it is recognized as a translation initiation codon.
It is noteworthy that a large fraction of 5' UTRs contain upstream AUGs, from 15% to nearly 50% depending on the organism (Figure ), suggesting that the 'first AUG rule' predicted by the scanning model of ribosome start-site selection is disobeyed in a large number of cases. This implies that the 40S ribosomal subunit can sometimes bypass the most upstream AUG codon, possibly because its sequence context makes it a poor initiation codon, to initiate translation at a more distal AUG. With this mechanism, called 'leaky scanning', multiple different proteins can be obtained from the same mRNA [22
]. Moreover, it has been calculated that the presence of an upstream AUG correlates with a long 5' UTR and with a 'weak' start codon context of the AUG that is usually used, whereas transcripts with an optimal start-codon context have short 5' UTRs without upstream AUGs [23
], suggesting that upstream AUGs may have a role in keeping the basal translational level of a gene low.
If an in-frame stop codon is found following the upstream AUG and before the main start codon, it creates an upstream ORF. After translation of the upstream ORF and the detachment of the large (60S) ribosomal subunit, the small ribosomal subunit has multiple alternative fates, which affect translation efficiency and mRNA stability. The 40S subunit may hold onto the mRNA, resume scanning, and reinitiate translation at a downstream AUG codon, or it may leave the mRNA, thus impairing translation of the main ORF. The ability of a ribosome to reinitiate is limited in eukaryotes by the stop codon context [24
] and by the length of the upstream ORF; if the upstream ORF is longer than around 30 codons [25
], the ribosome cannot reinitiate. This process is known to down-regulate translation of the mRNAs for the yeast transcription factors GCN4 and YAP1, which contain upstream ORFs [26
Secondary structures in 5' UTRs are also important in the regulation of translation. Experimental data suggest that moderately stable secondary structures (a change in free energy (ΔG) above -30 kcal/mol) directly involving the AUG start codon do not stall the migration of 40S ribosomal subunit; a significant decrease in the efficiency of translation is observed only when very stable structures (ΔG below -50 kcal/mol) are formed. UTR sequences with such very stable secondary structures are reported in Table . The inhibitory effects of these structures can be overcome by an increase in the level of eIF4A, the subunit of the eIF4F complex that promotes the unwinding of RNA secondary structures in cooperation with eIF4B and eIF4H [27
Examples of 5' UTR sequences with highly stable stem-loop structures
An alternative mechanism for translation initiation, which occurs independently of the 5' cap, was discovered for the first time in picornaviruses [28
]: a sequence element in the 5' UTR acts as an internal ribosome entry site (IRES). IRES elements have been found in many cellular mRNAs encoding regulatory proteins, such as proto-oncogene products like c-Myc, homeodomain proteins, growth factors (like the fibroblast growth factor FGF-2) and their receptors. The concept of IRESs has been very critically reviewed by Kozak [29
], who originally defined the importance of initiation codon context. Comparative analysis of known cellular IRESs leads to the identification of a common structural motif shared by many mRNAs, including those encoding the immunoglobulin heavy chain binding protein BiP and FGF2: a Y-shaped stem-loop just upstream of the AUG initiation codon [30
] (see Table and Figure ). It has recently been discovered that short sequence motifs complementary to the small ribosomal RNA may also act as IRESs [31
5' UTR sequences with experimentally proved IRES elements
Sequence elements that are the target of trans
-acting RNA binding proteins can also regulate translation. For example, the iron-responsive element (IRE) located in the 5' UTR of mRNAs encoding proteins involved in iron metabolism (ferritin, 5-aminolevulinate synthase and aconitase) may inhibit translation through the iron-dependent binding of iron regulatory proteins, which impede the normal scanning process of the small ribosomal subunit in translation initiation. In addition, most vertebrate mRNAs that encode ribosomal proteins and translation elongation factors analyzed to date contain a 5' terminal oligopyrimidine tract (TOP) consisting of 5-15 pyrimidines immediately adjacent to the m7G cap. This tract is required for coordinated translational repression during growth arrest, differentiation, development and certain drug treatments [32