Three forms of rat basic fibroblast growth factor are made from a single mRNA and localize to the nucleus

The molecular weight of rat basic fibroblast growth factor is predicted to be 18 kDa when the amino acid sequence is read from the single AUG initiation codon found in the cDNA. DNA sequencing upstream of this AUG codon indicated, however, that there was an extended open reading frame. In vitro translation of the rat cDNA for basic FGF gave three proteins of 18.0, 21.5, and 22.0 kDa in equal abundance. The same proteins were produced in vivo by COS cells transfected with the rat cDNA. Deletion of 81 base pairs from the reading frame upstream of the AUG codon resulted in the expression of only one protein observed at 18.0 kDa. These results indicated that the 22.0 and 21.5 kDa forms of rat basic FGF were formed when translation initiates at the alternative upstream non-AUG codons. Rat cell lines and tissues were found to express all three forms of basic FGF protein. The cDNA was used to analyze the subcellular distribution of the different forms of rat basic FGF. Subcellular fractionation and immunofluorescence of transfected COS cells showed that all three forms of the protein localized preferentially in the nucleus. Expression of a truncated cDNA from which 81 base pairs (27 amino acids) of the upstream reading frame had been deleted, showed localization of the smaller form of bFGF alone in the nucleus. These results demonstrated that although the amino acids that were deleted from the N-terminus of rat basic fibroblast growth factor have a sequence characteristic of nuclear localization motifs, they are not obligatory for the transport of the growth factor into the nucleus. Nuclear extracts taken from transfected cells also contained two smaller proteins of 16 and 12 kDa that were detected by Western blot analysis. It is possible that these are proteolytic products of bFGF.     

Translation of eukaryotic translation initiation factor 4GI (eIF4GI) proceeds from multiple mRNAs containing a novel cap-dependent internal ribosome entry site (IRES) that is active during poliovirus infection

Eukaryotic translation initiation factor 4GI (eIF4GI) is an essential scaffolding protein required to recruit the 43 S complex to the 5'-end of mRNA during translation initiation. We have previously demonstrated that eIF4GI protein expression is translationally regulated. This regulation is mediated by cis-acting RNA elements, including an upstream open reading frame and an IRES that directs synthesis of five eIF4GI protein isoforms via alternative AUG initiation codon selection. Here, we further characterize eIF4GI IRES function and show that eIF4GI is expressed from several distinct mRNAs that vary via alternate promoter use and alternate splicing. Several mRNA variants contain the IRES element. We found that IRES activity mapped to multiple regions within the eIF4GI RNA sequence, but not within the 5'-UTR per se. However, the 5'-UTR enhanced IRES activity in vivo and played a role in initiation codon selection. The eIF4GI IRES was active when transfected into cells in an RNA form, and thus, does not require nuclear processing events for its function. However, IRES activity was found to be dependent upon the presence, in cis, of a 5' m7guanosine-cap. Despite this requirement, the eIF4GI IRES was activated by 2A protease cleavage of eIF4GI, in vitro, and retained the ability to promote translation during poliovirus-mediated inhibition of cap-dependent translation. These data indicate that intact eIF4GI protein is not required for the de novo synthesis of eIF4GI, suggesting its expression can continue under stress or infection conditions where eIF4GI is cleaved.     

Translation Initiation at the CUU Codon Is Mediated by the Internal Ribosome Entry Site of an Insect Picorna-Like Virus In Vitro

AUG-unrelated translation initiation was found in an insect picorna-like virus, Plautia stali intestine virus (PSIV). The positive-strand RNA genome of the virus contains two nonoverlapping open reading frames (ORFs). The capsid protein gene is located in the 3′-proximal ORF and lacks an AUG initiation codon. We examined the translation mechanism and the initiation codon of the capsid protein gene by using various dicistronic and monocistronic RNAs in vitro. The capsid protein gene was translated cap independently in the presence of the upstream cistron, indicating that the gene is translated by internal ribosome entry. Deletion analysis showed that the internal ribosome entry site (IRES) consisted of approximately 250 bases and that its 3′ boundary extended slightly into the capsid-coding region. The initiation codon for the IRES-mediated translation was identified as the CUU codon, which is located just upstream of the 5′ terminus of the capsid-coding region by site-directed mutagenesis. In vitro translation assays of monocistronic RNAs lacking the 5′ part of the IRES showed that this CUU codon was not recognized by scanning ribosomes. This suggests that the PSIV IRES can effectively direct translation initiation without stable codon-anticodon pairing between the initiation codon and the initiator methionyl-tRNA.     

Identification of an alternative translation initiation site for the Pantoea ananatis lycopene cyclase (crtY) gene in E. coli and its evolutionary conservation

Previous sequence analyses of the lycopene cyclase gene (crt Y) from Pantoea ananatis revealed that translation of its protein product in Escherichia coli began at the ATG start codon. We found, however, that this enzyme could also be produced in E. coli without the ATG start codon present. Results of experiments using crt Y mutants revealed that a GTG (Val) sequence, located in-frame and 24 bp downstream of the ATG, could act as a potential start codon. Additionally, a point-mutated GTA (Val), replaced from alternative GTG start codon, also displayed its potential as a start codon although the strength as a translation initiation codon was considerably weak. This finding suggests that non-ATG codons, especially one base pairing with the anticodon (3'-UAC-5') in fMet-tRNA, might be also able to function as start codon in translation process. Furthermore, amino acid sequence alignment of lycopene cyclases from different sources suggested that a Val residue located within the N-terminus of these enzymes might be used as an alternative translation initiation site. In particular, presence of a conserved Asp, located in-frame and 12 bp upstream of potential start codon, supports this assumption in view of the fact that Asp (GAT or GAC) can function as part of the Shine-Dalgano sequence (AGGAGG).     

Initiation codon selection is accomplished by a scanning mechanism without crucial initiation factors in Sindbis virus subgenomic mRNA

Translation initiation of alphavirus subgenomic mRNA (sgmRNA) can occur in the absence of several initiation factors (eIFs) in infected cells; however, the precise translation mechanism is still poorly understood. In this study, we have examined the mechanism of initiation and AUG selection in Sindbis virus (SINV) sgmRNA. Our present findings suggest that sgmRNA is translated via a scanning mechanism, since the presence of a hairpin structure before the initiation codon hampers protein synthesis directed by this mRNA. In addition, translation is partially recovered when an in-frame AUG codon is placed upstream of this hairpin. This scanning process takes place without the participation of eIF4A and active eIF2. These results, combined with our findings through modifying the SINV sgmRNA leader sequence, do not support the possibility of a direct initiation from the start codon without previous scanning, or a shunting mechanism. Moreover, studies carried out with sgmRNAs containing two alternative AUG codons within a good context for translation reveal differences in AUG selection which are dependent on the cellular context and the phosphorylation state of eIF2α. Thus, initiation at the additional AUG is strictly dependent on active eIF2, whereas the genuine AUG codon can start translation following eIF2α inactivation. Collectively, our results suggest that SINV sgmRNA is translated by a scanning mechanism without the potential participation of crucial eIFs. A model is presented that explains the mechanism of initiation of mRNAs bearing two alternative initiation codons.     

The initiation codon determines the efficiency but not the site of translation initiation in Chlamydomonas chloroplasts.

To study translation initiation in Chlamydomonas chloroplasts, we mutated the initiation codon AUG to AUU, ACG, ACC, ACU, and UUC in the chloroplast petA gene, which encodes cytochrome f of the cytochrome b6/f complex. Cytochrome f accumulated to detectable levels in all mutant strains except the one with a UUC codon, but only the mutant with an AUU codon grew well at 24 degrees C under conditions that require photosynthesis. Because no cytochrome f was detectable in the UUC mutant and because each mutant that accumulated cytochrome f did so at a different level, we concluded that any residual translation probably initiates at the mutant codon. As a further demonstration that alternative initiation sites are not used in vivo, we introduced in-frame UAA stop codons immediately downstream or upstream or in place of the initiation codon. Stop codons at or downstream of the initiation codon prevented accumulation of cytochrome f, whereas the one immediately upstream of the initiation codon had no effect on the accumulation of cytochrome f. These results suggest that an AUG codon is not required to specify the site of translation initiation in chloroplasts but that the efficiency of translation initiation depends on the identity of the initiation codon.     

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.     

Non-AUG translation initiation of a fungal RING finger repressor involved in photocarotenogenesis

The RING finger protein CrgA acts as a negative regulator of light-induced carotene biosynthesis in the fungus Mucor circinelloides. Sequence analysis of the crgA coding region upstream of the first AUG codon revealed the existence of an additional non-canonical RING finger domain at the most N-terminal end of the protein. The newly identified RING finger domain is required for CrgA to regulate photocarotenogenesis, as deduced from site-directed mutagenesis experiments. The role of both RING finger domains in the stability of CrgA has been investigated in a yeast system. Wild type CrgA, but not the RING finger deleted forms, is highly unstable and is stabilized by inhibition of the proteasome function, which suggests that native CrgA is degraded by the proteasome and that active RING finger domains are required for proteasome-mediated CrgA degradation. To identify the translation start of CrgA, a mutational analysis of putative initiation codons in the 5' region of the crgA gene was accomplished. We demonstrated that a GUG codon located upstream of the first AUG is the sole initiator of CrgA translation. To our knowledge, this is the first report of a naturally occurring non-AUG start codon for a RING finger regulatory protein. A combination of suboptimal translation initiation and proteasome degradation may help to maintain the low cellular levels of CrgA observed in wild type cells, which is probably required for accurate regulation of photocarotenogenesis.     

鸡基因组差异表达基因翻译起始密码子上下游序列的比较研究

翻译起始调控是基因表达调控的一个关键步骤之一。本文以鸡为研究材料,比较研究了鸡基因组高表达基因和低表达基因翻译起始密码子上下游的碱基序列差异,旨在寻找影响鸡基因表达水平的特异性调控位点。全部3 020个单剪接基因完整的mRNA序列及有详细注释的5＇UTRs序列从Ensembl下载。编写计算机程序,读取每个基因mRNA起始密码子上下游各位点的碱基。研究发现,起始密码子上游-3、-2位点可能是鸡基因组基因表达起始密码子正确识别的关键位点。起始密码子上下游的碱基组成分析发现,高表达基因和低表达基因起始密码子的上游均倾向使用（G＋C）,高表达基因的使用偏倚尤为强烈。序列差异比较发现,高表达基因在-9、-6、-3、＋4位点显著偏向G,在-1、-2、-4、-5位点显著偏向C。低表达基因起始密码子上游使用A、U的频率显著高于低表达基因。在-19位点强烈偏向A,在＋1、＋11、＋14位点强烈偏向U。     

Functional analysis of a mutation occurring between the two in-frame AUG codons of human angiotensinogen

Angiotensinogen (ANG) is the specific substrate of the renin-angiotensin system, a major participant in blood pressure control. We have identified a natural mutation at the -30 amino acid position of the angiotensinogen signal peptide, in which an arginine is replaced by a proline (R-30P). Heterozygous individuals with R-30P showed a tendency to lowered plasma angiotensinogen level (1563 ng of ANG I/ml (range 1129-1941)) compared with normal individuals in the family (1892 ng of ANG I/ml (range 1603-2072)). Human angiotensinogen mRNA has two in-phase translation initiation codons (AUG) starting upstream 39 and 66 nucleotides from the cap site. R-30P occurs in a cluster of basic residues adjacent to the first AUG codon that may affect intracellular sorting of the nascent protein. Pulse-chase experiments in transiently transfected cultured cells revealed that the R-30P mutation was associated with reduced amounts of both intra- and extracellular protein. In a cell-free system, we found that two forms of native angiotensinogen were generated by alternative initiation of translation at either AUG codon. Alteration of either the first or second AUG codons abolished the synthesis of the longer and the shorter form of native angiotensinogen, respectively. Furthermore, the rate of secretion of the shorter form was lower than that of the longer form. By transplanting angiotensinogen signal peptide onto green fluorescence protein, however, we found that both forms of the signal peptide could target green fluorescence protein, normally localized in the cytoplasm, to the secretory pathway. Although the R-30P mutation may not affect intracellular sorting of angiotensinogen in a qualitative manner, it leads to a quantitative reduction in the net secretion of mature angiotensinogen through decreased translocation or increased residence time in the endoplasmic reticulum.     

MicroReview Control of translation initiation in Saccharomyces cerevisiae

The first observations regarding the control of translation initiation in the yeast Saccharomyces cerevisiae were made by Fred Sherman and his colleagues in 1971. Elegant genetic studies of the CYC1 gene resulted in the formulation of 'Sherman's Rules' for translation initiation as follows: (i) AUG is the only initiator codon. (ii) the most proximal AUG from the 5' end of a message will serve as the start site of translation; and (iii) if the upstream AUG codon is mutated then initiation begins at the next available AUG in the message. Hidden within these rules is the mechanism of eukaryotic translation initiation, as these very same rules were later shown to apply to higher eukaryotic organisms and were formulated into the scanning model. However, only in the past five years has yeast been taken seriously as an organism for studying the mechanism of eukaryotic translation initiation. The basis for this is that the yeast genes for at least four mammalian translation initiation factor homologues have been identified and the number is growing. Similar factors suggest similar mechanisms for translation initiation between yeast and mammals. For some translation initiation factors, the genetics of yeast has provided new insights into their function. A mechanism for regulating translation initiation in mammalian cells is now evident in yeast. It seems clear that the molecular genetics of yeast coupled with the available in vitro translation system will provide a wealth of information in the future regarding translational control and regulatory mechanisms. The purpose of this review is to summarize what is known about translational control in S. cerevisiae.     

Identification of compounds that decrease the fidelity of start codon recognition by the eukaryotic translational machinery

Translation initiation in eukaryotes involves more than a dozen protein factors. Alterations in six factors have been found to reduce the fidelity of start codon recognition by the ribosomal preinitiation complex in yeast, a phenotype referred to as Sui(-). No small molecules are known that affect the fidelity of start codon recognition. Such compounds would be useful tools for probing the molecular mechanics of translation initiation and its regulation. To find compounds with this effect, we set up a high-throughput screen using a dual luciferase assay in S. cerevisiae. Screening of over 55,000 compounds revealed two structurally related molecules that decrease the fidelity of start codon selection by approximately twofold in the dual luciferase assay. This effect was confirmed using additional in vivo assays that monitor translation from non-AUG start codons. Both compounds increase translation of a natural upstream open reading frame previously shown to initiate translation at a UUG. The compounds were also found to exacerbate increased use of UUG as a start codon (Sui(-) phenotype) conferred by haploinsufficiency of wild-type eukaryotic initiation factor (eIF) 1, or by mutation in eIF1. Furthermore, the effects of the compounds are suppressed by overexpressing eIF1, which is known to restore the fidelity of start codon selection in strains harboring Sui(-) mutations in various other initiation factors. Together, these data strongly suggest that the compounds affect the translational machinery itself to reduce the accuracy of selecting AUG as the start codon.     

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.