Viral and chloroplastic signals essential for initiation and efficiency of translation in Agrobacterium tumefaciens

The construction of high-level protein expression vectors using the CaMV 35S promoter in concert with highly efficient translation initiation signals for Agrobacterium tumefaciens is a relatively less explored field compared to that of Escherichia coli. In the current study, we experimentally investigated the capacity of the CaMV 35S promoter to direct GFP gene expression in A. tumefaciens in the context of different viral and chloroplastic translation initiation signals. GFP expression and concomitant translational efficiency was monitored by confocal microscopy and Western blot analysis. Among all of the constructs, the highest level of translation was observed for the construct containing the phage T7 translation initiation region followed by the chloroplastic Rubisco Large Subunit (rbcL) 58-nucleotide 5′ leader region including its SD-like sequence (GGGAGGG). Replacing the SD-like (GGGAGGG) with non SD-like (TTTATTT) or replacing the remaining 52 nucleotides of rbcL with nonspecific sequence completely abolished translation. In addition, this 58 nucleotide region of rbcL serves as a translational enhancer in plants when located within the 5′ UTR of mRNA corresponding to GFP. Other constructs, including those containing sequences upstream of the coat proteins of Alfalfa Mosaic Virus, or the GAGG sequence of T4 phage or the chloroplastic atpI and/or PsbA 5′ UTR sequence, supported low levels of GFP expression or none at all. From these studies, we propose that we have created high expression vectors in A. tumefaciens and/or plants which contain the CaMV 35S promoter, followed by the translationally strong T7 SD plus RBS translation initiation region or the rbcL 58-nucleotide 5′ leader region upstream of the gene of interest.     

The 5' untranslated region of the maize alcohol dehydrogenase gene provides efficient translation of mRNA in plants under stress conditions

Reduced level of expression of most cell proteins under stress conditions is determined by low efficiency of cap-dependent translation of corresponding mRNAs. The maize gene encoding alcohol dehydrogenase, adh1, is an example of a gene which mRNA is efficiently translated under hypoxia. Using reporter gene assay we showed that the leader sequence of adh1 mRNA, provides efficient translation of reporter gene gfp in Nicotiana benthamiana cells under hypoxia and heat shock. The presence of this leader sequence in 5' UTR of mRNA does not change the level of expression in aerobic conditions, but under hypoxia and heat shock the levels of reporter gfp expression were reduced about 5-10 fold in the absence of leader and remained unaffected in its presence in 5'UTR. We found that this leader sequence does not change the level of mRNA stability and does not exhibit promoter activity. Consequently, leader sequence acts as translational enhancer providing efficient translation of mRNA in plant cells under stress conditions. Introduction of this sequence into standard expression cassettes may be used for development of new systems of expression of target proteins in plants, efficient under stress conditions.     

小麦花粉特异性表达的cDNA的分离及表达特性

应用抑制差示杂交和5′/3′RACE PCR方法分离了小麦(Triticum aestivum L.)花粉特异性表达的全长cDNA(TaPSG719,GenBank:AY451238)),该基因全长1172 bp,5′非编码区序列长达329 bp,包含多个上游可译框架(uORF);该基因编码188个氨基酸的蛋白质,大小约20 kD,等电点为12.1。Northern杂交和RT-PCR分析表明该基因在成熟花粉特异表达,而在小孢子、叶片、根和未成熟的种子、幼茎和子房等组织几乎检测不到。进一步研究小麦花粉发育过程的表达水平表明,TaPSG719在单核和双核小孢子阶段不表达,在开花前5d(已完成有丝分裂)开始表达并迅速增强达到高峰,但随着花粉的成熟表达水平逐渐下降。表明TaPSG719是一个花粉中晚期特异性表达基因。经BLAST同源性分析表明,与目前已登录的基因没有显著的同源性。Southern杂交表明TaPSG719可能为一个多拷贝基因。为研究TaPSG719 cDNA 5′非编码区序列的uORF对可译框架的翻译的影响,构建不同缺失或突变的表达载体,采用麦胚体外翻译系统,结果显示含uORF的5′非编码区序列能显著抑制蛋白质的翻译水平,表明TaPSG719基因表达至少部分是在翻译水平上调控。     

小麦花粉特异性表达的cDNA的分离及表达特性

应用抑制差示杂交和5′/3′RACE PCR方法分离了小麦(Triticum aestivum L.)花粉特异性表达的全长cDNA(TaPSG719,GenBank:AY451238)),该基因全长1 172 bp,5′非编码区序列长达329 bp,包含多个上游可译框架(uORF);该基因编码1 88个氨基酸的蛋白质,大小约20 kD,等电点为12.1.Northern杂交和RT-PCR分析表明该基因在成熟花粉特异表达,而在小孢子、叶片、根和未成熟的种子、幼茎和子房等组织几乎检测不到.进一步研究小麦花粉发育过程的表达水平表明,TaPSG719在单核和双核小孢子阶段不表达,在开花前5 d(已完成有丝分裂)开始表达并迅速增强达到高峰,但随着花粉的成熟表达水平逐渐下降.表明TaPSG719是一个花粉中晚期特异性表达基因.经BLAST同源性分析表明,与目前已登录的基因没有显著的同源性.Southern杂交表明TaPSG719可能为一个多拷贝基因.为研究TaPSG719 cDNA 5′非编码区序列的uORF对可译框架的翻译的影响,构建不同缺失或突变的表达载体,采用麦胚体外翻译系统,结果显示含uORF的5′非编码区序列能显著抑制蛋白质的翻译水平,表明TaPSG719基因表达至少部分是在翻译水平上调控.     

Alternative translation of human fibroblast growth factor 2 mRNA occurs by internal entry of ribosomes.

Alternative initiations of translation of the human fibroblast growth factor 2 (FGF-2) mRNA, at three CUG start codons and one AUG start codon, result in the synthesis of four isoforms of FGF-2. This process has important consequences on the fate of FGF-2: the CUG-initiated products are nuclear and their constitutive expression is able to induce cell immortalization, whereas the AUG-initiated product, mostly cytoplasmic, can generate cell transformation. Thus, the different isoforms probably have distinct targets in the cell. We show here that translation initiation of the FGF-2 mRNA breaks the rule of the cap-dependent ribosome scanning mechanism. First, translation of the FGF-2 mRNA was shown to be cap independent in vitro. This cap-independent translation required a sequence located between nucleotides (nt) 192 and 256 from the 5' end of the 318-nt-long 5' untranslated region. Second, expression of bicistronic vectors in COS-7 cells indicated that the FGF-2 mRNA is translated through a process of internal ribosome entry mediated by the mRNA leader sequence. By introducing additional AUG codons into the RNA leader sequence, we localized an internal ribosome entry site to between nt 154 and 318 of the 5' untranslated region, just upstream of the first CUG. The presence of an internal ribosome entry site in the FGF-2 mRNA suggests that the process of internal translation initiation, by controlling the expression of a growth factor, could have a crucial role in the control of cell proliferation and differentiation.     

Dual-domain, dual-targeting organellar protein presequences in Arabidopsis can use non-AUG start codons

The processes accompanying endosymbiosis have led to a complex network of interorganellar protein traffic that originates from nuclear genes encoding mitochondrial and plastid proteins. A significant proportion of nucleus-encoded organellar proteins are dual targeted, and the process by which a protein acquires the capacity for both mitochondrial and plastid targeting may involve intergenic DNA exchange coupled with the incorporation of sequences residing upstream of the gene. We evaluated targeting and sequence alignment features of two organellar DNA polymerase genes from Arabidopsis thaliana. Within one of these two loci, protein targeting appeared to be plastidic when the 5' untranslated leader region (UTR) was deleted and translation could only initiate at the annotated ATG start codon but dual targeted when the 5' UTR was included. Introduction of stop codons at various sites within the putative UTR demonstrated that this region is translated and influences protein targeting capacity. However, no ATG start codon was found within this upstream, translated region, suggesting that translation initiates at a non-ATG start. We identified a CTG codon that likely accounts for much of this initiation. Investigation of the 5' region of other nucleus-encoded organellar genes suggests that several genes may incorporate upstream sequences to influence targeting capacity. We postulate that a combination of intergenic recombination and some relaxation of constraints on translation initiation has acted in the evolution of protein targeting specificity for those proteins capable of functioning in both plastids and mitochondria.     

Improved vectors for stable expression of foreign genes in mammalian cells by use of the untranslated leader sequence from EMC virus.

Dicistronic mRNA expression vectors efficiently translate a 5' open reading frame (ORF) and contain a selectable marker within the 3' end which is inefficiently translated. In these vectors, the efficiency of translation of the selectable 3' ORF is reduced approximately 100-fold and is highly dependent on the particular sequences inserted into the 5' cloning site. Upon selection for expression of the selection marker gene product, deletions within the 5' ORF occur to yield more efficient translation of the selectable marker. We have generated improved dicistronic mRNA expression vectors by utilization of a putative internal ribosomal entry site isolated from encephalomyocarditis (EMC) virus. Insertion of the EMC virus leader sequence upstream of an ORF encoding either a wildtype or methotrexate resistant dihydrofolate reductase (DHFR) reduces DHFR translation up to 10-fold in a monocistronic DHFR expression vector. However, insertion of another ORF upstream of the EMC leader to produce a dicistronic mRNA does not further reduce DHFR translation. In the presence of the EMC virus leader, DHFR translation is not dependent on sequences inserted into the 5' end of the mRNA. We demonstrate that stable high level expression of inserted cDNAs may be rapidly achieved by selection for methotrexate resistance in DHFR deficient as well as DHFR containing cells. In contrast to previously described dicistronic expression vectors, these new vectors do not undergo rearrangement or deletion upon selection for amplification by propagation in increasing concentrations of methotrexate. The explanation may be either that the EMC virus leader sequence allows internal initiation of translation or that cryptic splice sites in the EMC virus sequence mediate production of monocistronic mRNAs. These vectors may be generally useful to rapidly obtain high level expression of cDNA genes in mammalian cells.     

A phylogenetically conserved sequence within viral 3'' untranslated RNA pseudoknots regulates translation.

Both the 68-base 5' leader (omega) and the 205-base 3' untranslated region (UTR) of tobacco mosaic virus (TMV) promote efficient translation. A 35-base region within omega is necessary and sufficient for the regulation. Within the 3' UTR, a 52-base region, composed of two RNA pseudoknots, is required for regulation. These pseudoknots are phylogenetically conserved among seven viruses from two different viral groups and one satellite virus. The pseudoknots contained significant conservation at the secondary and tertiary levels and at several positions at the primary sequence level. Mutational analysis of the sequences determined that the primary sequence in several conserved positions, particularly within the third pseudoknot, was essential for function. The higher-order structure of the pseudoknots was also required. Both the leader and the pseudoknot region were specifically recognized by, and competed for, the same proteins in extracts made from carrot cell suspension cells and wheat germ. Binding of the proteins is much stronger to omega than the pseudoknot region. Synergism was observed between the TMV 3' UTR and the cap and to a lesser extent between omega and the 3' UTR. The functional synergism and the protein binding data suggest that the cap, TMV 5' leader, and 3' UTR interact to establish an efficient level of translation.     

A potential mechanism for selective control of cap-independent translation by a viral RNA sequence in cis and in trans.

Highly efficient cap-independent translation initiation at the 5'-proximal AUG is facilitated by the 3' translation enhancer sequence (3'TE) located near the 3' end of barley yellow dwarf virus (BYDV) genomic RNA. The role of the 3'TE in regulating viral translation was examined. The 3'TE is required for translation and thus replication of the genomic RNA that lacks a 5' cap (Allen et al., 1999, Virology253:139-144). Here we show that the 3'TE also mediates translation of uncapped viral subgenomic mRNAs (sgRNA1 and sgRNA2). A 109-nt viral sequence is sufficient for 3'TE activity in vitro, but additional viral sequence is necessary for cap-independent translation in vivo. The 5' extremity of the sequence required in the 3' untranslated region (UTR) for cap-independent translation in vivo coincides with the 5' end of sgRNA2. Thus, sgRNA2 has the 3'TE in its 5' UTR. Competition studies using physiological ratios of viral RNAs showed that, in trans, the 109-nt 3'TE alone, or in the context of 869-nt sgRNA2, inhibited translation of genomic RNA much more than it inhibited translation of sgRNA1. The divergent 5' UTRs of genomic RNA and sgRNA1 contribute to this differential susceptibility to inhibition. We propose that sgRNA2 serves as a novel regulatory RNA to carry out the switch from early to late gene expression. Thus, this new mechanism for temporal control of translation control involves a sequence that stimulates translation in cis and acts in trans to selectively inhibit translation of viral mRNA.     

Sequences in the 5′ Nontranslated Region of Hepatitis C Virus Required for RNA Replication

Sequences in the 5' and 3' termini of plus-strand RNA viruses harbor cis-acting elements important for efficient translation and replication. In case of the hepatitis C virus (HCV), a plus-strand RNA virus of the family Flaviviridae, a 341-nucleotide-long nontranslated region (NTR) is located at the 5' end of the genome. This sequence contains an internal ribosome entry site (IRES) that is located downstream of an about 40-nucleotide-long sequence of unknown function. By using our recently developed HCV replicon system, we mapped and characterized the sequences in the 5' NTR required for RNA replication. We show that deletions introduced into the 5' terminal 40 nucleotides abolished RNA replication but only moderately affected translation. By generating a series of replicons with HCV-poliovirus (PV) chimeric 5' NTRs, we could show that the first 125 nucleotides of the HCV genome are essential and sufficient for RNA replication. However, the efficiency could be tremendously increased upon the addition of the complete HCV 5' NTR. These data show that (i) sequences upstream of the HCV IRES are essential for RNA replication, (ii) the first 125 nucleotides of the HCV 5' NTR are sufficient for RNA replication, but such replicon molecules are severely impaired for multiplication, and (iii) high-level HCV replication requires sequences located within the IRES. These data provide the first identification of signals in the 5' NTR of HCV RNA essential for replication of this virus.     

The 3'-terminal hexamer sequence of classical swine fever virus RNA plays a role in negatively regulating the IRES-mediated translation

The 3' untranslated region (UTR) is usually involved in the switch of the translation and replication for a positive-sense RNA virus. To understand the 3' UTR involved in an internal ribosome entry site (IRES)-mediated translation in Classical swine fever virus (CSFV), we first confirmed the predicted secondary structure (designated as SLI, SLII, SLIII, and SLIV) by enzymatic probing. Using a reporter assay in which the luciferase expression is under the control of CSFV 5' and 3' UTRs, we found that the 3' UTR harbors the positive and negative regulatory elements for translational control. Unlike other stem loops, SLI acts as a repressor for expression of the reporter gene. The negative cis-acting element in SLI is further mapped to the very 3'-end hexamer CGGCCC sequence. Further, the CSFV IRES-mediated translation can be enhanced by the heterologous 3'-ends such as the poly(A) or the 3' UTR of Hepatitis C virus (HCV). Interestingly, such an enhancement was repressed by flanking this hexamer to the end of poly(A) or HCV 3' UTR. After sequence comparison and alignment, we have found that this hexamer sequence could hypothetically base pair with the sequence in the IRES IIId1, the 40 S ribosomal subunit binding site for the translational initiation, located at the 5' UTR. In conclusion, we have found that the 3'-end terminal sequence can play a role in regulating the translation of CSFV.     

Double subgenomic alphaviruses expressing multiple fluorescent proteins using a Rhopalosiphum padi virus internal ribosome entry site element

Double subgenomic Sindbis virus (dsSINV) vectors are widely used for the expression of proteins, peptides, and RNA sequences. These recombinant RNA viruses permit high level expression of a heterologous sequence in a wide range of animals, tissues, and cells. However, the alphavirus genome structure and replication strategy is not readily amenable to the expression of more than one heterologous sequence. The Rhopalosiphum padi virus (RhPV) genome contains two internal ribosome entry site (IRES) elements that mediate cap-independent translation of the virus nonstructural and structural proteins. Most IRES elements that have been characterized function only in mammalian cells but previous work has shown that the IRES element present in the 5' untranslated region (UTR) of the RhPV genome functions efficiently in mammalian, insect, and plant systems. To determine if the 5' RhPV IRES element could be used to express more than one heterologous sequence from a dsSINV vector, RhPV 5' IRES sequences were placed between genes for two different fluorescent marker proteins in the dsSINV, TE/3'2J/mcs. While mammalian and insect cells infected with recombinant viruses containing the RhPV sequences expressed both fluorescent marker proteins, only single marker proteins were routinely observed in cells infected with dsSINV vectors in which the RhPV IRES had been replaced by a luciferase fragment, an antisense RhPV IRES, or no intergenic sequence. Thus, we report development of a versatile tool for the expression of multiple sequences in diverse cell types.     

Alternative splicing within the elk-1 5' untranslated region serves to modulate initiation events downstream of the highly conserved upstream open reading frame 2

The 5' untranslated region (UTR) plays a central role in the regulation of mammalian translation initiation. Key components include RNA structure, upstream AUGs (uAUGs), upstream open reading frames (uORFs), and internal ribosome entry site elements that can interact to modulate the readout. We previously reported the characterization of two alternatively spliced 5' UTR isoforms of the human elk-1 gene. Both contain two uAUGs and a stable RNA stem-loop, but the long form (5' UTR(L)) was more repressive than the short form (5' UTR(S)) for initiation at the ELK-1 AUG. We now demonstrate that ELK-1 expression arises by a combination of leaky scanning and reinitiation, with the latter mediated by the small uORF2 conserved in both spliced isoforms. In HEK293T cells, a considerable fraction of ribosomes scans beyond the ELK-1 AUG in a reinitiation mode. These are sequestered by a series of out-of-frame AUG codons that serve to prevent access to a second in-frame AUG start site used to express short ELK-1 (sELK-1), an N-terminally truncated form of ELK-1 that has been observed only in neuronal cells. We present evidence that all these events are fine-tuned by the nature of the 5' UTR and the activity of the α subunit of eukaryotic initiation factor 2 and provide insights into the neuronal specificity of sELK-1 expression.     

Leaky scanning is the predominant mechanism for translation of human papillomavirus type 16 E7 oncoprotein from E6/E7 bicistronic mRNA

Human papillomaviruses (HPV) are unique in that they generate mRNAs that apparently can express multiple proteins from tandemly arranged open reading frames. The mechanisms by which this is achieved are uncertain and are at odds with the basic predictions of the scanning model for translation initiation. We investigated the unorthodox mechanism by which the E6 and E7 oncoproteins from human papillomavirus type 16 (HPV-16) can be translated from a single, bicistronic mRNA. The short E6 5' untranslated region (UTR) was shown to promote translation as efficiently as a UTR from Xenopus beta-globin. Insertion of a secondary structural element into the UTR inhibited both E6 and E7 expression, suggesting that E7 expression depends on ribosomal scanning from the 5' end of the mRNA. E7 translation was found to be cap dependent, but E6 was more dependent on capping and eIF4F activity than E7. Insertion of secondary structural elements at various points in the region upstream of E7 profoundly inhibited translation, indicating that scanning was probably continuous. Insertion of the E6 region between Renilla and firefly luciferase genes revealed little or no internal ribosomal entry site activity. However when E6 was located at the 5' end of the mRNA, it permitted over 100-fold-higher levels of downstream cistron translation than did the Renilla open reading frame. Internal AUGs in the E6 region with strong or intermediate Kozak sequence contexts were unable to inhibit E7 translation, but initiation at the E7 AUG was efficient and accurate. These data support a model in which E7 translation is facilitated by an extreme degree of leaky scanning, requiring the negotiation of 13 upstream AUGs. Ribosomal initiation complexes which fail to initiate at the E6 start codon can scan through to the E7 AUG without initiating translation, but competence to initiate is achieved once the E7 AUG is reached. These findings suggest that the E6 region of HPV-16 comprises features that sponsor both translation of the E6 protein and enhancement of translation at a downstream site.     

Characterization of an internal ribosomal entry segment in the 5' leader of murine leukemia virus env RNA

The 5' untranslated region, also called the leader, of oncoretroviruses and lentiviruses is long and is formed of several structured domains critically important in virus replication. The 5' leader of murine leukemia virus (MLV) RNA contains an internal ribosomal entry segment (IRES) which promotes synthesis of Gag and glyco-Gag polyprotein precursors. In the present study we investigated the translational features of the 5' leader of MLV subgenomic RNA (env RNA) encoding the Env polyprotein precursor. When the env leader was inserted between two genes, such as lacZ and the neomycin resistance cassette, in a dicistronic vector, it allowed IRES-dependent translation of the 3' cistron in the rabbit reticulocyte lysate system and in murine cells. The drug rapamycin and the foot-and-mouth disease virus L protease, known to inhibit cap-dependent translation, caused an enhancement of the translation driven by the env leader sequence, consistent with an IRES activity promoting Env expression. Analysis of several deletion mutants led us to localize the minimal env IRES between the splice junction and the env AUG start codon.