By Jessica Lu
Although the most common start codon is AUG, translation initiation can also occur at other codons with a much lower efficiency. Usually, alternative start codons only differ from AUG by one nucleotide (e.g. CUG, GUG and UUG) (Kearse & Wilusz, 2017). Alternative start codons are used by both prokaryotes and eukaryotes, though it is more common in prokaryotes (Asano, 2014). Though the codon may otherwise code for a different amino acid, alternative start codons are still generally translated as methionine in eukaryotes (Kochetov, 2008), and N-formylmethionine in prokaryotes (Belinky, Rogozin & Koonin, 2017).
In eukaryotes, translation usually occurs when 40S ribosomal subunits are recruited to the 5’ cap of mRNA and scan the mRNA in a 5’ to 3’ direction for the AUG start codon. However, the successful recognition of the AUG triplet depends on the nucleotides which surround it (i.e. its nucleotide context) (Kochetov, 2008). The optimal context is called the Kozak motif, and in mammals this is GCCRCCAUGG (AUG start codon in bold). For non-AUG translation to occur, a good Kozak context around the alternative start codon is critical. Additionally, the success of non-AUG translation is also greatly increased when there is a strong RNA secondary structure approximately 15 nt downstream of the initiation site (Ivanov et al., 2011).
In prokaryotes, alternative start codons may be used up to 20% of the time to initiate translation. However, genes starting with AUG are expressed at significantly higher levels compared to other start codons. The AUG start codon is actively maintained through selection, though the purifying selection of start codons is still significantly weaker than the selection of codons in coding sequences. The next most frequently used start codon is GUG, followed by UUG. (Belinky, Rogozin & Koonin, 2017). AUG, GUG and UUG may be thought of as the canonical start codons, but in fact translation initiation has been detected from 47 of the 64 triplet codons in E. coli (Hecht et al., 2017). Unlike the scanning model of translation in eukaryotes, in prokaryotes the ribosome binds directly to the Shine Dalgarno sequence on the mRNA, an important translation initiation signal. When an alternative start codon is used, this tends to be compensated by mutations in the Shine Dalgarno sequence that result in a stronger translation initiation signal (Belinky, Rogozin & Koonin, 2017). A well-known example of a coding region with a non-AUG start codon is lacI in the E. coli lac operon, which starts with GUG (Farabaugh, 1978).
Although alternative start codon use is less common in eukaryotes than in prokaryotes, it still plays an important biological role. The use of alternative start codons can allow the production of several different proteins from a single gene, each with a different N-terminal domain. This contributes to protein diversity. Different isoforms produced using alternative start codons have distinct biological functions. As the N-terminal domain of a protein often contains signal peptides, using alternative start codons is one way to direct proteins to different compartments (Touriol et al., 2003). Up-regulation of non-AUG translation occurs during development and when cells undergo stress. In addition, misregulation of non-AUG translation contributes to the development of cancer and numerous neurological diseases (Kearse & Wilusz, 2017).
Cancer growth can either be promoted or inhibited by non-AUG translation. An example where cancer growth is promoted is the non-AUG translation of fibroblast growth factor 2 (FGF2). If the AUG start codon is used, an 18-kDa protein isoform is formed which is mostly present in the cytosol or secreted. However, there are four upstream CUG codons which can act as alternative start codons. The protein isoforms produced from these CUG codons are localised to the nucleus. These CUG-isoforms have been shown to promote cell immortalisation in culture and had tumorigenic properties when injected into mice. It is still poorly understood how alternative start codon use is activated in cancer, but it is possibly due to misregulation of the eukaryotic initiation factors (eIFs) in cancers. (Kearse & Wilusz, 2017).
In addition to cancer, non-AUG translation also plays a role in a number of neurological diseases, called nucleotide repeat disorders. This includes Huntington’s disease and fragile X disorders. These diseases are caused by mutations that increase the number of nucleotide repeats in a gene above a certain threshold. For example, in fragile X disorders affected patients have >55 CGG repeats in the 5’ region of the FMR1 gene, whilst those who are unaffected have a mean number of 30 repeats. The increased number of repeats can induce non-AUG translation, causing what is called repeat-associated non-AUG (RAN) translation. The resulting protein products are toxic to cells, resulting in the neurological disease. It is still unclear why RAN is induced by the number of nucleotide repeats (Kearse & Wilusz, 2017).
Overall, non-AUG translation has wide implications not only for genome annotation in prokaryotes, but also for protein diversity, disease and cell function in eukaryotes. Yet more research needs to be done; in prokaryotes to check for unannotated open reading frames with non-canonical start codons and in eukaryotes to elucidate how the use of alternative start codons is induced or repressed.
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