Comparative hybrid arrest by tandem antisense oligodeoxyribonucleotides or oligodeoxyribonucleoside methylphosphonates in a cell-free system.

Antisense oligonucleotides containing either anionic diester or neutral methylphosphonate internucleoside linkages were prepared by automated synthesis, and were compared for their ability to arrest translation of human dihydrofolate reductase (DHFR) mRNA in a nuclease treated rabbit reticulocyte lysate. In the case of oligodeoxyribonucleotides, tandem targeting of three 14-mers resulted in synergistic and complete selective inhibition of DHFR synthesis at a total oligomer concentration of 25 microM. Hybrid arrest by three or six tandem oligodeoxyribonucleoside methylphosphonates was dramatically less effective. This difference does not result from preferential recognition of hybrids involving oligodeoxyribonucleotides by endogenous RNaseH activity. A ribonuclease protection assay demonstrated that antisense oligodeoxyribonucleoside methylphosphonates bind selectively to target RNA sequences, but with 275 fold lower affinity than the corresponding oligodeoxyribonucleotides. This low binding affinity results in poor arrest of translation, and may be related to the stereochemistry of the methylphosphonate linkage.     

Insertion sequence IS10 anti-sense pairing initiates by an interaction between the 5' end of the target RNA and a loop in the anti-sense RNA

Transposition of insertion sequence IS10 is regulated by an anti-sense RNA which inhibits transposase expression when IS10 is present in multiple copies per cell. The anti-sense RNA (RNA-OUT) consists of a stem domain topped by a flexibly paired loop; the 5' end of the target molecule, RNA-IN, is complementary to the top of the loop, and complementarity extends for 35 base-pairs down one side of RNA-OUT. We present here genetic evidence that anti-sense pairing, both in vitro and in vivo, initiates by interaction of the 5' end of RNA-IN and the loop domain of RNA-OUT; other features of the reaction are discussed. In the context of this model, we discuss features of this anti-sense system which are important for its biological effectiveness, and suggest that IS10 provides a convenient model for design of efficient artificial anti-sense RNA molecules.     

Modulation of eucaryotic initiation factor-4E binding to 5'-capped oligoribonucleotides by modified anti-sense oligonucleotides.

The 5' cap structure of eucaryotic mRNA plays a pivotal role in mRNA metabolism. This report demonstrates that anti-sense oligonucleotides equipped with 3'-overhanging nucleotides modulate the amount of recombinant human eucaryotic initiation factor-4E that binds to a 5'-capped oligoribonucleotide. The degree of inhibition or enhancement of protein binding is dependent upon the number and sequence of overhanging nucleotides. A 45% inhibition of complexation was observed by the addition of one 3'-overhanging guanosine residue. Addition of a second residue (+2/GN) resulted in a higher degree of inhibition, 77-88%. In contrast, addition of one adenosine residue enhanced the formation of the eucaryotic initiation factor-4E-m7GpppRNA complex by 213%. Modulation of protein interactions with the 5'-cap structure is likely to effect several biological events, including pre-mRNA processing, transport of the mRNA from the nucleus to the cytoplasm and translation of the target mRNA. This targeting strategy in anti-sense chemistry may have practical applications in experimental biology and medicine.     

Effect of the tripartite leader on synthesis of a non-viral protein in an adenovirus 5 recombinant.

The EIa region of an Adenovirus 5 recombinant has been substituted by a modular gene encoding dihydrofolate reductase (DHFR). In this recombinant, the mouse DHFR cDNA was positioned behind sequences of the major late promoter and the complete tripartite leader. The leader sequences end in the normal 5' splice site (SS) of the third leader, so that RNA splicing joins the tripartite leader to a 3' splice site immediately upstream of the DHFR cDNA. At late stages of infection, high levels of DHFR mRNAs were synthesized. At early times in the late stage, this mRNA was efficiently translated; however, at later times translation of DHFR decreased probably due to poor competition with other late mRNAs. Synthesis of DHFR protein from an analogous Adenovirus 5 recombinant containing only the first late leader was studied in parallel. Equivalent levels of DHFR mRNA were expressed after infection with this recombinant virus; however, the efficiency of DHFR translation was at least 20 fold lower than that of the DHFR mRNA containing the tripartite leader. This suggests that the tripartite leader sequence is important for translation in the late stage of infection. As reported previously, the Ad5 recombinant containing only the first leader vastly overexpresses polypeptide IX from a novel mRNA, formed by the splicing of the first leader in the modular DHFR gene to the 3' splice site in the EIb region. Cells infected with this recombinant synthesize very little normal mRNA from the EIb region. Here, we demonstrated that coinfection of 293 cells with this recombinant and wild type Adenovirus 5 also results in decreased EIb mRNA synthesis. We propose that the overproduction of polypeptide IX suppresses mRNA expression from the EIb and IX promoter sites, probably by an autoregulation loop active during lytic growth.     

Competition between ribosome and SecA binding promotes Escherichia coli secA translational regulation.

SecA protein, the protein translocation ATPase of Escherichia coli, autogenously regulates its translation during normal protein secretion by binding to a secretion-responsive element located near the 5' end of its gene on geneX-secA mRNA. In order to characterize this autoregulation further, RNA footprinting and primerextension inhibition (toeprinting) studies were carried out with a segment of geneX-secA RNA, 30S ribosomal subunits and tRNAfMet along with purified SecA protein. The results show that ribosome and SecA-binding sites overlap, indicating that a simple competition for binding of geneX-secA mRNA presumably governs the translation initiation step. Further analysis showed that SecA protein was able to specifically dissociate a preformed 30S-tRNAfMet-geneX-secA RNA ternary complex as indicated by the disappearance of its characteristic toeprint after SecA addition. These findings are consistent with secA autoregulation, and they suggest a novel mechanism for the autoregulatory behavior of this complex protein.     

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.     

Oligodeoxyribonucleotide length and sequence effects on intermolecular purine-purine-pyrimidine triple-helix formation.

The binding of guanosine/thymidine-rich oligodeoxyribonucleotides containing various deletions, extensions, and point mutations to polypurine DNA targets was investigated by DNase I footprinting. Intermolecular purine-purine-pyrimidine triple-helical DNA formation was best achieved using oligonucleotides 12 nucleotides in length. Longer oligonucleotides were slightly weaker in binding affinity, whereas shorter oligonucleotides were considerably weaker. Oligonucleotide extensions had a slight effect on triplex formation, while single point mutations located near the oligonucleotide ends had a greater effect. In the cases of extensions and point mutations, changes to the 3' end of the oligonucleotide had a consistently greater effect on triplex formation than changes to the 5' end. Such differences in triplex-forming ability were not caused by an intrinsic property of these oligonucleotides, since the same point mutated oligonucleotides could bind with high affinity to duplex DNAs containing complementary sites. Taken together, our data suggest that there may be an asymmetry involved in the process of purine-motif triplex formation, with interactions between the 3' end of the oligonucleotide and complementary sequences on the target duplex DNA being dominant.     

The use of single-stranded DNA and RNase H to promote quantitative ''hybrid arrest of translation'' of mRNA/DNA hybrids in reticulocyte lysate cell-free translations.

Single-stranded cDNA clones complementary to the 5' end of TMV RNA have been used to explore the conditions necessary for efficient 'hybrid arrest of translation' in the reticulocyte lysate. It is shown that incubations of 20 minutes at 60 degrees in 0.1 M KCl are sufficient to give almost complete arrest of translation using a clone complementary to the 5'-non-coding region and first 171 coding nucleotides of TMV RNA. However, hybrids with DNA complementary to regions of the mRNA downstream of the first AUG gave variable and in some cases almost no arrest of translation in the reticulocyte lysate unless they were first digested with RNase H. A simple and rapid method for giving complete and highly specific arrest of translation of particular mRNAs in complex mixtures has been developed using both cDNA clones and synthetic oligodeoxynucleotides in conjunction with RNase H digestion. Evidence is presented that suggests that 'hybrid arrest of translation' in the wheat-germ cell-free system is primarily due to the action of RNase H. When a reticulocyte lysate was doped with 20 U/ml of RNase H, its ability to translate unannealed mRNA was unaffected but it translated DNA/RNA hybrids extremely poorly.     

The eIF1A solution structure reveals a large RNA-binding surface important for scanning function

The translation initiation factor eIF1A is necessary for directing the 43S preinitiation complex from the 5' end of the mRNA to the initiation codon in a process termed scanning. We have determined the solution structure of human eIF1A, which reveals an oligonucleotide-binding (OB) fold and an additional domain. NMR titration experiments showed that eIF1A binds single-stranded RNA oligonucleotides in a site-specific, but non-sequence-specific manner, hinting at an mRNA interaction rather than specific rRNA or tRNA binding. The RNA binding surface extends over a large area covering the canonical OB fold binding site as well as a groove leading to the second domain. Site-directed mutations at multiple positions along the RNA-binding surface were defective in the ability to properly assemble preinitiation complexes at the AUG codon in vitro.     

Complete sequences of the ribosome recognition sites in vesicular stomatitis virus mRNAs: recognition by the 40S and 80S complexes.

Nucleotide sequences of the ribosome-protected translation initiation sites from the vesicular stomatitis virus (VSV) M and L protein mRNAs have been determined, completing the sequences of the sites from all the VSV mRNAs. A low level of protection at two internal AUG-containing sites in the N mRNA is also described. Small homologies are evident among some of the sites, but there are no obvious features common to all the sites other than a single AUG codon. In contrast, a large homology between the VSV M mRNA site and the alfalfa mosaic virus coat mRNA site (Koper-Zwarthoff et al., 1977) is noted. This homology suggests the existence of a common ancestral gene for these two apparently unrelated viruses. For each VSV mRNA species, the smallest sites protected in either the 40S or 80S initiation complexes are identical. These sites always contained the initiation codon, but only contained the capped 5' end in those mRNAs having the 5' end near the initiation site. If 40S ribosomes bind to the capped 5' end, either they do not protect it from nuclease digestion or the protection is only transitory in some VSV mRNAs. Consideration of the structures of the ribosome binding sites suggests that the differential effects of hypertonic shock on translation (Nuss and Koch, 1976) may be related to the distance between the 5' end of the mRNA and the initiation codon.     

Position and stability are determining factors for translation repression by an RNA G-quadruplex-forming sequence within the 5' UTR of the NRAS proto-oncogene

Nucleic acid secondary structures in the 5' untranslated regions (UTRs) of mRNAs have been shown to play a critical role in translation regulation. We recently demonstrated that a naturally occurring, conserved, and stable RNA G-quadruplex element (5'-GGGAGGGGCGGGUCUGGG-3'), located close to the 5' cap within the 5' UTR of the NRAS proto-oncogene mRNA, modulates gene expression at the translational level. Herein, we show that the translational effect of this G-quadruplex motif in NRAS 5' UTR is not uniform, but rather depends on the location of the G-quadruplex-forming sequence. The RNA G-quadruplex-forming sequence represses translation when situated relatively proximal to the 5' end, within the first 50 nt, in the 5' UTR of the NRAS proto-oncogene, whereas it has no significant effect on translation if located comparatively away from the 5' end. We have also demonstrated that the thermodynamic stability of the RNA G-quadruplex at its natural position within the NRAS 5' UTR is an important factor contributing toward its ability to repress translation.