Destabilization of messenger RNA/complementary DNA duplexes by the elongating 80 S ribosome

In a previous study, we demonstrated that the ability of a cDNA fragment to hybrid-arrest the translation of its complementary mRNA in rabbit reticulocyte lysate depends on the position of the mRNA/cDNA duplex within the mRNA molecule. In the present report, we further characterize the mechanisms involved in the destabilization and subsequent translation of mRNA/cDNA hybrids by mapping in detail the positional dependence of hybrid-arrested translation of the human alpha- and beta-globin mRNAs and by directly assessing the stability of mRNA/cDNA duplexes in reticulocyte lysate under a variety of translational conditions. The mapping studies in this report demonstrate that the translation of a hybridized mRNA requires exposure of the 5' nontranslated region and the AUG initiation codon, as well as those bases 3' to the AUG which are typically protected by an initiating 80 S ribosome. The translation of these mRNA/cDNA hybrids is associated with the complete removal of cDNA from the mRNA coding region; this disruption of the mRNA/cDNA duplex is blocked by inhibitors of translational initiation and elongation. cDNAs which extend into the 3' nontranslated region remain associated with the mRNA during normal translation but are completely removed from the mRNA during translation if translational termination is suppressed. Taken together, these findings demonstrate that the disruption of mRNA/cDNA duplexes in rabbit reticulocyte lysate is tightly linked to the assembly and migration of 80 S ribosomes.     

Target-specific arrest of mRNA translation by antisense 2'-O-alkyloligoribonucleotides.

We describe a novel experimental approach to investigate mRNA translation. Antisense 2'-O-allyl oligoribonucleotides (oligos) efficiently arrest translation of targeted mRNAs in rabbit reticulocyte lysate and wheat germ extract while displaying minimal non-specific effects on translation. Oligo/mRNA-hybrids positioned anywhere within the 5' UTR or the first approximately 20 nucleotides of the open reading frame block cap-dependent translation initiation with high specificity. The thermodynamic stability of hybrids between 2'-O-alkyl oligos and RNA permits translational inhibition with oligos as short as 10 nucleotides. This inhibition is independent of RNase H cleavage or modifications which render the mRNA untranslatable. We show that 2'-O-alkyl oligos can also be employed to interfere with cap-independent internal initiation of translation and to arrest translation elongation. The latter is accomplished by UV-crosslinking of psoralen-tagged 2'-O-methyloligoribonucleotides to the mRNA within the open reading frame. The utility of 2'-O-alkyloligoribonucleotides to arrest translation from defined positions within an mRNA provides new approaches to investigate mRNA translation.     

alpha-DNA. VI: Comparative study of alpha- and beta-anomeric oligodeoxyribonucleotides in hybridization to mRNA and in cell free translation inhibition.

alpha and beta-anomeric d(G2T12G2) oligodeoxyribonucleotides were compared for their hybridization to rA12: the observed melting temperatures are 27 degrees C for beta-oligodeoxyribonucleotide/RNA hybrid and 53 degrees C for alpha-oligodeoxyribonucleotide/RNA. alpha-oligonucleotides with the four bases, complementary to natural mRNAs, were synthesized for the first time, labeled at their 5'-end with [32P] and used as probes in Northern blot experiments. In spite of these higher affinities for their target RNA's, they were unable to block translation of natural or synthetic mRNA's in rabbit reticulocyte lysate. We have studied the RNase H activity on model rA12:alpha- or beta-d(G2T12G2) hybrids or on mRNA:alpha- or beta-oligonucleotides hybrids. Specific hybridization protects RNA strech when using alpha-oligonucleotides but not beta-oligonucleotides. Thus, our results show the inability of RNase H to degrade RNA in alpha-oligodeoxyribonucleotides:RNA duplexes.     

Comparative activity of alpha- and beta-anomeric oligonucleotides on rabbit beta globin synthesis: inhibitory effect of cap targeted alpha-oligonucleotides

Alpha-anomeric oligonucleotides are resistant to nucleases and display parallel annealing to RNA complementary sequences. We compared the effect of alpha- and beta-oligonucleotides targeted against various mRNA regions on the rabbit beta globin in vitro synthesis. In order to determine the role of RNase H, experiments were performed in both rabbit reticulocyte lysate and wheat germ extract. As expected beta-oligonucleotides were found more efficient in wheat germ extract which is rich in RNase H activity and alpha-oligonucleotide targeted against the initiation codon or downstream had no effect because they do not induce mRNA cleavage by RNase H. However, we report, for the first time, a specific translation inhibition by alpha-oligonucleotides. This occurs provided they are targeted against the cap region in 5' of the mRNA.     

Inhibition of translation initiation by antisense oligonucleotides via an RNase-H independent mechanism.

We have used alpha-oligomers as antisense oligonucleotides complementary to three different sequences of the rabbit beta-globin mRNA: a region adjacent to the cap site, a region spanning the AUG initiation codon or a sequence in the coding region. These alpha-oligonucleotides were synthesized either with a free 5' OH group or linked to an acridine derivative. The effect of these oligonucleotides on mRNA translation was investigated in cell-free extracts and in Xenopus oocytes. In rabbit reticulocyte lysate and in wheat germ extracts oligomers targeted to the cap site and the initiation codon reduced beta-globin synthesis in a dose-dependent manner, whereas the target mRNA remained intact. The anti-cap alpha-oligomer was even more efficient that its beta-counterpart in rabbit reticulocyte lysate. In contrast, only the alpha-oligomer, linked to the acridine derivative, complementary to the cap region displayed significant antisense properties in Xenopus oocytes. Therefore initiation of translation can be arrested by oligonucleotide/RNA hybrids which are not substrates for RNase-H.     

Complementary oligodeoxynucleotide mediated inhibition of tobacco mosaic virus RNA translation in vitro.

Two different "antisense" oligodeoxynucleotides and their RNA analogues, each complementary to non-overlapping sequences of 51 bases near the 5' end of TMV RNA, inhibit in vitro translation of the genomic RNA in a rabbit reticulocyte lysate. Inhibition is dependent upon complementarity, concentration, and hybridization of the oligomers with TMV RNA. Inhibition is observed at molar ratios of TMV RNA to antisense oligomers as low as 1:1.5. A plateau of inhibition at which 10-25% of the control signal remains is achieved by molar ratios of TMV RNA:antisense DNA or RNA greater than or equal to 1:15. The extent of inhibition is not increased by the simultaneous presence of both complementary fragments. Oligodeoxynucleotides and their RNA analogues identical to the same regions of TMV RNA have no direct effect on translation, however, they can block inhibition by the antisense fragments. Translation of BMV RNA is not affected by any of the oligodeoxynucleotides. Polyacrylamide gel electrophoresis shows translation of TMV p126 is selectively inhibited. We conclude that the observed inhibition of translation is due to direct interference with ribosome function.     

Specific hybridization arrest of dihydrofolate reductase mRNA in vitro using anti-sense RNA or anti-sense oligonucleotides

Three anti-sense RNAs and ten synthetic anti-sense oligonucleotides were tested for their ability specifically to arrest translation of human dihydrofolate reductase (DHFR) mRNA in a nuclease-treated rabbit reticulocyte lysate. Quantitative hybrid arrest of DHFR mRNA by anti-sense RNA required that the RNA hybridize to the 5' end of DHFR mRNA. Oligonucleotides of length 11-20, complementary to various sites near the 5' end of DHFR mRNA, also could cause specific inhibition of DHFR mRNA translation. Oligonucleotide length and concentration were shown to be important variables in hybrid arrest of DHFR mRNA. Neither the exact oligonucleotide binding site position near the 5' end of the mRNA nor prehybridization conditions were important variables. The combination of short oligonucleotides with contiguous binding sites was shown to synergize their ability to inhibit specifically DHFR mRNA translation.     

Amplification and molecular cloning of murine adenosine deaminase gene sequences

A previously isolated mouse Cl-1D derived cell line (B-1/25) overproduces adenosine deaminase (EC 3.5.4.4) by 3200-fold. The present studies were undertaken to determine the molecular basis of this phenomenon. Rabbit reticulocyte lysate and Xenopus oocyte translation studies indicated that the B-1/25 cells also overproduced adenosine deaminase mRNA. Total poly(A+) RNA derived from B-1/25 was used to construct a cDNA library. After prehybridization with excess parental Cl-1D RNA to selectively prehybridize nonamplified sequences, 32P-labeled cDNA probe synthesized from B-1/25 total poly(A+) RNA was used to identify recombinant colonies containing amplified mRNA sequences. Positive clones containing adenosine deaminase gene sequences were identified by blot hybridization analysis and hybridization-selected translation in both rabbit reticulocyte lysate and Xenopus oocyte translation systems. Adenosine deaminase cDNA clones hybridized with three poly(A+) RNA species of 1.5, 1.7, and 5.2 kilobases in length, all of which were overproduced in the B-1/25 cell line. Dot blot hybridization analysis using an adenosine deaminase cDNA clone showed that the elevated adenosine deaminase level in the B-1/25 cells was fully accounted for by an increase in adenosine deaminase gene copy number. The adenosine deaminase cDNA probes and the cell lines with amplified adenosine deaminase genes should prove extremely useful in studying the structure and regulation of the adenosine deaminase gene.     

RNA-DNA hybrids containing damaged DNA are substrates for RNase H

During the replication of the lagging strand, RNA-DNA hybrids are formed and the RNA is subsequently degraded by the action of RNase H. Little is known about the effects of damaged DNA on lagging strand replication and subsequent RNA removal. The rates and sites of digestion by E. coli RNase H of RNA-DNA hybrids containing either a thymine glycol or urea site in the DNA strand have been examined. The cleavage patterns for duplexes containing thymine glycol or urea differ from that of a fully complementary duplex. There is one major product of the digestion of the fully complementary hybrid, but three products are formed in the reactions with the hybrids containing damaged DNAs. Cleavage is partially redirected to the position adjacent to the damaged sites. The overall rate of cleavage of these hybrids containing damaged DNA is comparable to that of the fully complementary duplex. These results indicate that the cleavage of RNA-DNA hybrids by RNase H is less selective when a damaged site is present in the DNA strand.     

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.     

Construction and identification by positive hybridization-translation of a bacterial plasmid containing a rat growth hormone structural gene sequence.

The construction, identification, and use of a recombinant DNA clone containing a growth hormone structural gene sequence is described. A cDNA copy of partially purified pregrowth hormone mRNA from cultured rat pituitary tumor (GC) cells was employed in the construction of a hybrid plasmid, designated pBR322-GH1. The cloned DNA sequence was positively identified by a hybridization-translation procedure which should be applicable to any cloned structural gene sequence. This procedure involved hybridization of cytoplasmic poly(A)-containing RNA from GC cells to the cloned DNA immobilized on nitrocellulose filters, followed by elution of the hybridized RNA and translation in a mRNA-depleted rabbit reticulocyte lysate system. Physical and immunological criteria were employed to show that the translation products were enriched for pregrowth hormone. Hybridization to excess plasmid DNA of [3H]uridine-labeled, size fractionated GC cell cytoplasmic RNA was used to show that all growth hormone-specific RNA sequences are the same size as functional pregrowth hormone mRNA.     

Isolation of cDNA clones for human adenosine deaminase

Clones encoding human adenosine deaminase (ADA) were isolated from a cDNA library made from the lymphoblastoid cell line MOLT-4. The isolation procedure was based on the selection of clones hybridizing with a radioactive probe complementary to an RNA preparation, which had been highly enriched in ADA-specific mRNA. The latter RNA preparation was obtained by size-fractionating MOLT-4 RNA and selecting fractions that were translatable into ADA. The assay for the presence of ADA in the in vitro translation products, was based on immunoprecipitation with a specific anti-ADA serum. The antiserum used was shown to precipitate a 42-kDal protein with the properties of ADA. Positive clones were further screened by means of hybrid-released in vitro translation assays. Two clones were obtained which were able to select mRNA that could be translated into a 42-kDal protein immunoprecipitable with the ADA-antiserum. By use of Southern blots containing DNA from somatic cell hybrids, one of these ADA cDNA clones was assigned to the human chromosome 20 known to contain the ADA gene.     

Evidence for the regulation of protein synthesis by a wheat germ phosphoprotein factor

A 48-kilodalton phosphoprotein, termed T-protein or pT, isolated from wheat germ and purified to homogeneity is found to inhibit the translation of tobacco mosaic virus (TMV) RNA in both wheat germ and reticulocyte lysates. The translation of TMV RNA in both systems was inhibited over 80% by 8 microM pT. There was no evidence to indicate that the reticulocyte lysate also contained a pT-like protein. pT was rapidly phosphorylated in the wheat germ and reticulocyte lysates. Although the relationship between pT phosphorylation and inhibition of protein synthesis is not known, there is evidence to indicate that complete phosphorylation of pT is not required for inhibition. Furthermore, no significant differences in the kinetics of inhibition of protein synthesis between prephosphorylated and unmodified pT were observed. Investigation of the mechanism of inhibition indicated that neither the aminoacylation of tRNA nor the elongation of nascent polypeptide chains was affected by pT. On the other hand, pT was found to prevent the formation of the 80S initiation complex. This action of pT was not due to the binding of pT to the ribosomes. However, the effect of pT was found to vary with the concentrations and types of mRNA used in the translational system. These results suggest that pT may interact with specific region(s) of the mRNA and prevent its translation. Alternatively, pT could block the translation of mRNA by binding to one or more of the initiation factors that interact with mRNA to facilitate mRNA binding to the 43S preinitiation complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of cloned complementary DNA covering more than 6000 nucleotides (97%) of avian vitellogenin mRNA

The messenger RNA coding for chicken vitellogenin, a precursor of the egg-yolk proteins lipovitellin and phosvitin, is synthesized in the liver following estrogen injection. This mRNA is 6600 nucleotides long. We have previously reported the cloning and preliminary characterization of some cDNA fragments representing portions of the vitellogenin mRNA [Biochem. Biophys. Acta, 606, 34--46 (1980)]. In this paper we report the full characterization of a larger series of such clones, representing almost the entire length of the mRNA, by restriction endonuclease mapping, R-loop mapping, RNA-DNA hybridization and by translation in vitro of the RNA which hybridizes to the cloned DNA. From the results we conclude that the chicken vitellogenin mRNA, unlike that of Xenopus laevis, does not vary in sequence over most of its length, although some variations in the cDNA sequences were detected particularly in clones derived from the 3' terminus of the RNA. All sequence variants appear to be present in RNA prepared from single animals. The possible origins of these minor species are discussed. Furthermore, we describe a cDNA clone complementary to an mRNA which is about the same size as vitellogenin mRNA and which codes for an egg yolk protein antigenically related to lipovitellin. This mRNA is synthesized constitutively.