Alternative translation start sites and hidden coding potential of eukaryotic mRNAs

It is widely suggested that a eukaryotic mRNA typically contains one translation start site and encodes a single functional protein product. However, according to current points of view on translation initiation mechanisms, eukaryotic ribosomes can recognize several alternative translation start sites and the number of experimentally verified examples of alternative translation is growing rapidly. Also, the frequent occurrence of alternative translation events and their functional significance are supported by the results of computational evaluations. The functional role of alternative translation and its contribution to eukaryotic proteome complexity are discussed.     

Alternative translation start sites and their significance for eukaryotic proteomes

The review is dedicated to the current notions of translation initiation and the contextual organization of eukaryotic mRNA leader regions. A hypothesis on the frequent usage of several alternative start codons is discussed. A potential contribution of alternative translation start sites to the coding potential of eukaryotic mRNAs is considered.     

AUG_hairpin: prediction of a downstream secondary structure influencing the recognition of a translation start site

Background  

The translation start site plays an important role in the control of translation efficiency of eukaryotic mRNAs. The recognition of the start AUG codon by eukaryotic ribosomes is considered to depend on its nucleotide context. However, the fraction of eukaryotic mRNAs with the start codon in a suboptimal context is relatively large. It may be expected that mRNA should possess some features providing efficient translation, including the proper recognition of a translation start site. It has been experimentally shown that a downstream hairpin located in certain positions with respect to start codon can compensate in part for the suboptimal AUG context and also increases translation from non-AUG initiation codons. Prediction of such a compensatory hairpin may be useful in the evaluation of eukaryotic mRNA translation properties.     

uORFs, reinitiation and alternative translation start sites in human mRNAs

It is known that eukaryotic ribosomes are able to translate small ORFs and reinitiate translation at downstream start codons. However, this mechanism is widely considered to be inefficient and it is not commonly taken into account. We compiled a sample of human mRNAs containing small upstream ORFs overlapping with annotated protein coding sequences. Statistical analysis supported the hypothesis on reinitiation of translation at downstream AUG codons and functional significance of potential alternative ORFs. It may be assumed that some 5'UTR-located upstream ORFs can deliver ribosomes to alternative translation starts, and they should be taken into consideration in the prediction of human mRNA coding potential.     

Structure and functions of the translation initiation factor eIF4E and its role in cancer development and treatment

In eukaryotic cells, protein synthesis is a complex and multi-step process that has several mechanisms to start the translation including cap-dependent and cap-independent initiation. The translation control of eukaryotic gene expression occurs principally at the initiation step. In this context, it is critical that the eukaryotic translation initiation factor eIF4E bind to the 7-methylguanosine (m7G) cap present at the 5′-UTRs of most eukaryotic mRNAs. Combined with other initiation factors, eIF4E mediates the mRNA recruitment on ribosomes to start the translation. Moreover, the eIF4E nuclear bodies are involved in the export of specific mRNAs from the nucleus to the cytoplasm. In this review, we focus on the eIF4E structure and its physiological functions, and describe the role of eIF4E in cancer development and progression and the current therapeutic strategies to target eIF4E.     

Computer analyses of complete genomes suggest that some archaebacteria employ both eukaryotic and eubacterial mechanisms in translation initiation

The translation initiation mechanism of archaebacteria is still not clearly understood. Our previous work showed that ATG triplets before start codons have been strongly depleted in eukaryotic genomes, presumably because ribosome of eukaryotes scans mRNA from the 5' to 3' direction to find proper start codons. Extra ATG triplets before start codons would confuse the process and thus they have been negatively selected in eukaryotic genomes. In eubacterial genomes, on the other hand, ribosome binds to the Shine-Dalgarno (SD) sequence at once without mRNA scanning, and the characteristic patterns of ATG triplet depletion were not observed (Saito, R., Tomita, M., 1999. On negative selection against ATG triplets near start codons in eukaryotic and procaryotic genomes. J. Mol. Evol. 48, 213-217). The ATG triplet analysis on archaebacterial genomes revealed that Methanococcus jannaschii and Pyrococcus horikoshii show patterns similar to eukaryotes, implying that these species employ scanning of mRNA from the 5' to 3' direction in the process of translation initiation. On the other hand, our earlier study found that these archaea have SD-like sequences, which are complementary to the 3' end sequence of 16S rRNA, as in eubacterial translation initiation (Osada, Y., Saito, R., Tomita, M. Analysis of base-pairing potentials between 16S rRNA and 5' UTR for translation initiation in various procaryotes. Bioinformatics, in press). These two results combined lead us to conclude that these archaea probably use a hybrid mechanism; their ribosome scans mRNAs from the 5' to 3' direction and then 16S rRNA binds to the SD-like sequence of the 5' UTR.     

Sulfolobus solfataricus translation initiation factor 1 stimulates translation initiation complex formation

The eukaryotic translation initiation factor 1 binds to the ribosome during translation initiation. It is instrumental for initiator-tRNA and mRNA binding, and has a function in selection of the authentic start codon. Here, we show that the archaeal homolog aIF1 has analogous functions. The aIF1 protein of the archaeon Sulfolobus solfataricus is bound to the small ribosomal subunit during translation initiation and accelerates binding of initiator-tRNA and mRNA to the ribosome. Accordingly, aIF1 stimulated translation of an mRNA in a S. solfataricus in vitro translation system. Moreover, this study suggested that the C terminus of the factor is of relevance for its function.     

真核生物mRNA翻译起始机制研究进展

在真核生物中,mRNA翻译是一个复杂的多步骤过程,包括起始、延伸和终止3个阶段。其中,起始阶段的调控是影响mRNA翻译的关键。目前已经发现,mRNA翻译起始方式有多种,以最早发现的m ⁷G帽依赖性扫描机制最为经典,但当细胞处于逆境,经典起始机制受到抑制时,其他类型的起始机制会将其替代以保证翻译的顺利进行。本文对目前已发现的真核生物mRNA不同翻译起始机制特别是经典起始机制的替代机制进行了综述,旨在为深入认识真核生物基因在翻译水平上的表达调控提供参考。     

真核细胞中多重表达框mRNA的选择性翻译起始

扫描模型和遗漏扫描模型是真核生物mRNA翻译起始的两种主要机制,但其仍存在某些例外情况,如对具有多顺反子结构的mRNA,选择性翻译起始的发生机制目前仍不清楚.本研究基于GFP蛋白开放表达框(ORF)构建了一系列重组表达载体,用以转录在移码翻译顺序及同一翻译顺序下,AUG起始密码子处于不同序列背景,以及间隔不同距离的多顺反子结构mRNA.通过转染人Bel 7402细胞系,研究了这些多顺反子结构mRNA的翻译起始模式.结果表明,在移码翻译顺序下,多顺反子mRNA可翻译出对应的不同蛋白质,而在同一翻译顺序下,GFP蛋白表达框中的多个AUG密码子,仅有首位起始密码子可发挥作用,提示核糖体在从首位起始密码子开始翻译的同时,可能会有部分核糖体继续向下扫描并识别下游的起始密码子,而这种选择性的翻译起始效率,主要取决于密码子所处的序列背景及间隔距离等因素.     

Selective stimulation of translation of leaderless mRNA by initiation factor 2: evolutionary implications for translation

Translation initiation in bacteria involves a stochastic binding mechanism in which the 30S ribosomal subunit first binds either to mRNA or to initiator tRNA, fMet-tRNA(f)(Met). Leaderless lambda cI mRNA did not form a binary complex with 30S ribosomes, which argues against the view that ribosomal recruitment signals other than a 5'-terminal start codon are essential for translation initiation of these mRNAs. We show that, in Escherichia coli, translation initiation factor 2 (IF2) selectively stimulates translation of lambda cI mRNA in vivo and in vitro. These experiments suggest that the start codon of leaderless mRNAs is recognized by a 30S-fMet-tRNA(f)(Met)-IF2 complex, an intermediate equivalent to that obligatorily formed during translation initiation in eukaryotes. We further show that leaderless lambda cI mRNA is faithfully translated in vitro in both archaebacterial and eukaryotic translation systems. This suggests that translation of leaderless mRNAs reflects a fundamental capability of the translational apparatus of all three domains of life and lends support to the hypothesis that the translation initiation pathway is universally conserved.     

Comparison of the consensus sequence flanking translational start sites in Drosophila and vertebrates.

The previously presented consensus sequence for eukaryotic translation initiation sites by Kozak was derived substantially from vertebrate mRNA sequences. Drosophila nuclear genes exhibit a significantly different translation start consensus sequence. These differences probably do not represent mechanistic differences in translation initiation inasmuch as both taxa exhibit identical preferences and restrictions at the crucial -3 position. Using more conservative criteria for the assignment of consensus the following consensus sequences were derived: vertebrate--CANCAUG and Drosophila--CAAAACAUG.     

5'-poly(A) sequence as an effective leader for translation in eukaryotic cell-free systems

Poly(A) sequence of 25 adenylic residues placed immediately before the start codons of the green fluorescent protein (GFP) and firefly luciferase (Luc) mRNAs is shown to provide a high rate of translation of the heterologous messages in eukaryotic cell-free translation systems. Also the poly(A) leader is found to provide the abolition of the inhibition of translation at excess mRNA concentrations. The possibility of the practical use of the constructs with the poly(A) leader for preparative protein production is demonstrated in the wheat germ continuous-exchange cell-free (CECF) translation system.     

TISs-ST: a web server to evaluate polymorphic translation initiation sites and their reflections on the secretory targets

Background  

The nucleotide sequence flanking the translation initiation codon (start codon context) affects the translational efficiency of eukaryotic mRNAs, and may indicate the presence of an alternative translation initiation site (TIS) to produce proteins with different properties. Multi-targeting may reflect the translational variability of these other protein forms. In this paper we present a web server that performs computations to investigate the usage of alternative translation initiation sites for the synthesis of new protein variants that might have different functions.     

Prediction of translation initiation sites in human mRNA sequences with AUG start codon in weak Kozak context: A neural network approach

Translation of eukaryotic mRNAs is often regulated by nucleotides around the start codon. A purine at position −3 and a guanine at position +4 contribute significantly to enhance the translation efficiency. Algorithms to predict the translation initiation site often fail to predict the start site if the sequence context is not present. We have developed a neural network method to predict the initiation site of mRNA sequences that lack the preferred nucleotides at the positions −3 and +4 surrounding the translation initiation site. Neural networks of various architectures comprising different number of hidden layers were designed and tested for various sizes of windows of nucleotides surrounding translation initiation sites. We found that the neural network with two hidden layers showed a sensitivity of 83% and specificity of 73% indicating a vastly improved performance in successfully predicting the translation initiation site of mRNA sequences with weak Kozak context. WeakAUG server is freely available at http://bioinfo.iitk.ac.in/AUGPred/.     

Eukaryotic start and stop translation sites.

Sequences flanking translational initiation and termination sites have been compiled and statistically analyzed for various eukaryotic taxonomic groups. A few key similarities between taxonomic groups support conserved mechanisms of initiation and termination. However, a high degree of sequence variation at these sites within and between various eukaryotic groups suggest that translation may be modulated for many mRNAs. Multipositional analysis of di-, tri-, and quadrinucleotide sequences flanking start/stop sites indicate significant biases. In particular, strong tri-nucleotide biases are observed at the -3, -2, and -1 positions upstream of the start codon. These biases and the interspecific variation in nucleotide preferences at these three positions have lead us to propose a revised model of the interaction of the 18S ribosomal RNA with the mRNA at the site of translation initiation. Unusually strong biases against the CG dinucleotide immediately downstream of termination codons suggest that they may lead to faulty termination and/or failure of the ribosome to disassociate from the mRNA.     

cis-acting elements involved in the alternative translation initiation process of human basic fibroblast growth factor mRNA.

Four forms of basic fibroblast growth factor (bFGF) are synthesized from the same mRNA, resulting from alternative initiations of translation at three CUG start codons and one AUG start codon. The CUG- and AUG-initiated forms have distinct intracellular localizations and can modify cell phenotypes differently, indicating that control of the alternative expression of the different forms of bFGF has an important impact on the cell. In this study, we investigated the roles of the mRNA 5' untranslated region and the alternatively translated region located between the CUG and AUG codons in the regulation of alternative translation of the different forms of bFGF. Deletions and site-directed mutagenesis were carried out in bFGF mRNA leader, and translation was studied in vitro and in vivo. The results enabled us to identify five cis-acting RNA elements (two in the 5' untranslated region and three in the alternatively translated region) involved in the regulation of either global or alternative initiation of translation. Each of these elements had a specific effect on the level of synthesis of the different forms of bFGF. Furthermore, we showed that the 5' untranslated region regulatory elements had different effects on bFGF translation, depending on the translation system used. These results suggest that bFGF translation is modulated by cis-acting elements corresponding to secondary or tertiary RNA structures, which could be the targets of cell-specific trans-acting factors.     

Communication between eukaryotic translation initiation factors 5 and 1A within the ribosomal pre-initiation complex plays a role in start site selection

During eukaryotic translation initiation, the 43 S ribosomal pre-initiation complex scans the mRNA in search of an AUG codon at which to begin translation. Start codon recognition halts scanning and triggers a number of events that commit the complex to beginning translation at that point on the mRNA. Previous studies in vitro and in vivo have indicated that eukaryotic initiation factors (eIFs) 1, 2 and 5 play key roles in these events. In addition, it was reported recently that the C-terminal domain of eIF1A is involved in maintaining the fidelity of start codon recognition. The molecular mechanisms by which these factors work together to ensure fidelity of start site selection remain poorly understood. Here, we report the quantitative characterization of energetic interactions between eIF1A, eIF5 and the AUG codon in an in vitro reconstituted yeast translation initiation system. Our results show that recognition of an AUG codon by the 43 S complex triggers an interaction between eIF5 and eIF1A, resulting in a shift in the equilibrium between two states of the pre-initiation complex. This AUG-dependent change may be a reorganization from a scanning-competent state to a scanning-incompetent state. Mutations in both eIF1A and eIF5 that increase initiation at non-AUG codons in vivo weaken the interaction between the two factors upon AUG recognition, while specifically strengthening it in response to a UUG codon. These data suggest strongly that the interaction between eIF1A and eIF5 is involved in maintaining the fidelity of start codon recognition in vivo.     

AUG codons at the beginning of protein coding sequences are frequent in eukaryotic mRNAs with a suboptimal start codon context

MOTIVATION: The translation start site plays an important role in the control of translation efficiency of eukaryotic mRNAs. However, mRNAs with a suboptimal context of start AUG codon are relatively abundant. It is likely that at least some mRNAs with suboptimal start codon context contain the other signals providing additional information for efficient AUG recognition. RESULTS: Frequency of AUG codons at the beginning of the coding part of eukaryotic mRNAs was analyzed in relation to the context of translation start codon. It was found that the observed downstream AUG content in the mRNAs with optimal start codon context was close to the expected value, whereas it was significantly higher in the mRNAs with a suboptimal context. It is likely that downstream AUG codons can often be utilized as additional start sites to increase translation rate of mRNAs with a suboptimal context of the annotated start codon and many eukaryotic proteins can be characterized by some N-end heterogeneity.