Oligonucleotides complementary to the alpha-sarcin domain of 28S rRNA inhibit cell-free protein synthesis

Oligodeoxynucleotides complementary to the alpha-sarcin domain of rat 28S rRNA inhibit cell-free protein synthesis. The poly(U) translation system containing Artemia salina ribosomes was more sensitive to inhibition than the system containing rat liver ribosomes. The 21-mer, which was the most effective of the 7 oligonucleotides tested, hybridized with naked 28S rRNA. Hybridization with whole ribosomes, assayed by S1 nuclease protection, occurred only at high ionic strength or with ribosomes actively engaged in protein synthesis.     

Probing the structure of mouse Ehrlich ascites cell 5.8S, 18S and 28S ribosomal RNA in situ

The secondary structure of mouse Ehrlich ascites 18S, 5.8S and 28S ribosomal RNA in situ was investigated by chemical modification using dimethyl sulphate and 1-cyclohexyl-3-(morpholinoethyl) carbodiimide metho-p-toluene sulphonate. These reagents specifically modify unpaired bases in the RNA. The reactive bases were localized by primer extension followed by gel electrophoresis. The three rRNA species were equally accessible for modification i.e. approximately 10% of the nucleotides were reactive. The experimental data support the theoretical secondary structure models proposed for 18S and 5.8/28S rRNA as almost all modified bases were located in putative single-strand regions of the rRNAs or in helical regions that could be expected to undergo dynamic breathing. However, deviations from the suggested models were found in both 18S and 28S rRNA. In 18S rRNA some putative helices in the 5'-domain were extensively modified by the single-strand specific reagents as was one of the suggested helices in domain III of 28S rRNA. Of the four eukaryote specific expansion segments present in mouse Ehrlich ascites cell 28S rRNA, segments I and III were only partly available for modification while segments II and IV showed average to high modification.     

A photolabile oligodeoxyribonucleotide probe of the decoding site in the small subunit of the Escherichia coli ribosome: identification of neighboring ribosomal components

In this work we report the synthesis of a radioactive, photolabile oligodeoxyribonucleotide probe complementary to 16S rRNA nucleotides 1397-1405 and its exploitation in identifying 30S ribosomal subunit components neighboring its target site in 16S rRNA. Nucleotides 1397-1405 lie within a single-stranded sequence that has been linked to the decoding region of Escherichia coli ribosomes. On photolysis in the presence of activated 30S subunits, the photolabile oligodeoxyribonucleotide probe site-specifically incorporates into proteins S1, S7, S18, and S21 (identified by SDS-PAGE, RP-HPLC, and antibody affinity chromatography) and into three separate 16S rRNA regions, specifically, nucleotides A-1396, G-1405-A-1408, and A-1492 and A-1493. These results provide clear evidence that G-1405 in 16S rRNA is within 24 A (the distance between G-1405 and the photogenerated nitrene) of proteins S1, S7, S18, and S21 and each of the other nucleotides mentioned above, consistent with other studies of 30S internal structure. Although the probe binds to inactive 30S subunits about as well as to activated 30S subunits, photolysis of the inactive 30S.probe complex leads to a very different pattern of protein labeling, providing strong evidence, at the protein level, that the inactive to activated transition is accompanied by conformational change in the 1400 region of 16S rRNA.     

Inhibition of HeLa cell messenger RNA translation by 7-methylguanosine 5'-monophosphate

Translation of HeLa cell RNA containing poly(A) in a wheat germ cell-free system is markedly but incompletely inhibited by 7-methylguanosine 5'-monophosphate (m7G5'p). We have analyzed the translation products synthesized in the presence of different concentrations of m7G5'p and find that translation of all mRNAs is equally inhibited. To demonstrate the specificity of the inhibitor for RNAs with 5'-terminal m7G5' ppp... we show that specific translation products of satellite tobacco necrosis virus RNA, which does not have this 5' terminus, are synthesized in the presence of m7G5' p. Protein synthesis programmed by endogenous mRNA in a HeLa cell-free system is inhibited after a 10-min lag by m7G5' p. Other guanosine nucleotides without the 7-methyl group or with the phosphate in a different position are not inhibitor. We show that translation of all mRNAs is inhibited to a similar extent by m7G5'p in the HeLa cell-free system, by synthesizing 35S-labeled proteins in the presence of different inhibitory concentrations of this nucleotide and analyzing the translation products by electrophoresis and autoradiography. Translation of encephalomyocarditis virus RNA added to the HeLa cell-free system is not inhibited by m7"g5p; this viral RNA does not have this nucleotide at the 5' terminus. This indicates that m7G5'p specifically inhibits translation of mRNAs with the 5' terminus m7G5'ppp... and suggests that initiation of translation of picornavirus RNA may proceed via a mechanism different from that of cellular mRNAs.     

A novel allosterically trans-activated ribozyme, the maxizyme, with exceptional specificity in vitro and in vivo

Gut endocrine cells possess the capacity to take up and decarboxylate biogenic amine precursors. Decarboxylation is mediated by aromatic L-amino acid decarboxylase (AADC) which is encoded by mRNAs differing in their 5' untranslated regions (UTR) depending on the usage of alternative first exons, 1a and 1b, each with its own acceptor site (-13 and -8 bases relative to the translation start site, respectively). We describe here a novel splice variant of exon 1a-AADC mRNA in rat antral mucosa. Both exon 1a and 1b mRNAs were expressed in rat antral mucosa, but the 1a form was spliced into the acceptor site usually associated with exon 1b (-8). An enteroendocrine cell line (STC-1) expressed exon 1a or exon 1b mRNAs spliced into the -8 acceptor site of exon 2. Transient transfection of a range of cell lines with reporter constructs revealed that all three 5' UTRs efficiently supported expression of the Luciferase reporter. There is therefore a novel, functional 5' UTR of AADC mRNA in rat antral mucosa; alternative AADC splice variants could provide the capacity for control at the level of mRNA translation.     

Rotavirus RNA replication requires a single-stranded 3' end for efficient minus-strand synthesis

The segmented double-stranded (ds) RNA genome of the rotaviruses is replicated asymmetrically, with viral mRNA serving as the template for the synthesis of minus-strand RNA. Previous studies with cell-free replication systems have shown that the highly conserved termini of rotavirus gene 8 and 9 mRNAs contain cis-acting signals that promote the synthesis of dsRNA. Based on the location of the cis-acting signals and computer modeling of their secondary structure, the ends of the gene 8 or 9 mRNAs are proposed to interact in cis to form a modified panhandle structure that promotes the synthesis of dsRNA. In this structure, the last 11 to 12 nucleotides of the RNA, including the cis-acting signal that is essential for RNA replication, extend as a single-stranded tail from the panhandled region, and the 5' untranslated region folds to form a stem-loop motif. To understand the importance of the predicted secondary structure in minus-strand synthesis, mutations were introduced into viral RNAs which affected the 3' tail and the 5' stem-loop. Analysis of the RNAs with a cell-free replication system showed that, in contrast to mutations which altered the structure of the 5' stem-loop, mutations which caused complete or near-complete complementarity between the 5' end and the 3' tail significantly inhibited (>/=10-fold) minus-strand synthesis. Likewise, incubation of wild-type RNAs with oligonucleotides which were complementary to the 3' tail inhibited replication. Despite their replication-defective phenotype, mutant RNAs with complementary 5' and 3' termini were shown to competitively interfere with the replication of wild-type mRNA and to bind the viral RNA polymerase VP1 as efficiently as wild-type RNA. These results indicate that the single-strand nature of the 3' end of rotavirus mRNA is essential for efficient dsRNA synthesis and that the specific binding of the RNA polymerase to the mRNA template is required but not sufficient for the synthesis of minus-strand RNA.     

Ribosomal protein L9 interactions with 23 S rRNA: the use of a translational bypass assay to study the effect of amino acid substitutions

During translation of bacteriophage T4 gene 60 mRNA, ribosomes bypass 50 nucleotides with high efficiency. One of the mRNA signals for bypass is a stem-loop in the first part of the coding gap. When the length of this stem-loop is extended by 36 nucleotides, bypass is reduced to 0.35% of the wild-type level. Bypass is partially restored by a mutation in the C-terminal domain of Escherichia coli large ribosomal subunit protein L9. Previous work has shown that L9 is an elongated protein with an alpha-helix that connects and orients the N and C-terminal domains that both contain a predicted RNA binding site. We have determined two binding sites of L9 on 23 S rRNA. A 778 nucleotide RNA fragment encompassing domain V (nucleotides 1999 to 2776) of the 23 S rRNA is retained on filters by L9 and contains both sites. The N and C-terminal domains of L9 were shown to interact with nucleotides just 5' to nucleotide 2231 and 2179 of the 23 S rRNA, respectively, using the toeprint assay. These L9 binding sites on 23 S rRNA suggest that L9 functions as a brace across helix 76 to position helices 77 and 78 relative to the peptidyl transferase center. In this study, bypass on a mutant gene 60 mRNA has been used as an assay to probe the importance of particular L9 amino acids for function. Amino acid substitutions in the C-terminal domain are shown to partially restore bypass. These mutant L9 proteins have reduced binding to a 23 S rRNA fragment (nucleotides 1999 to 2274) containing domain V, to which L9 binds. They partially retain both the N and C-terminal domain interactions. On the other hand, substitutions of amino acids in the N-terminal domain, which greatly reduce RNA binding, do not restore bypass. The latter mutants have completely lost the N-terminal domain interaction. Addition of an amino acid to the alpha-helix also restores gene 60 bypass. RNA binding by this mutant is similar to that observed for the C-terminal domain mutants that partially restore bypass.     

mRNA silencing in erythroid differentiation: hnRNP K and hnRNP E1 regulate 15-lipoxygenase translation from the 3' end

Although LOX mRNA accumulates early during differentiation, a differentiation control element in its 3' untranslated region confers translational silencing until late stage erythropoiesis. We have purified two proteins from rabbit reticulocytes that specifically mediate LOX silencing and identified them as hnRNPs K and E1. Transfection of hnRNP K and hnRNP E1 into HeLa cells specifically silenced the translation of reporter mRNAs bearing a differentiation control element in their 3' untranslated region. Silenced LOX mRNA in rabbit reticulocytes specifically coimmunoprecipitated with hnRNP K. In a reconstituted cell-free translation system, addition of recombinant hnRNP K and hnRNP E1 recapitulates this regulation via a specific inhibition of 80S ribosome assembly on LOX mRNA. Both proteins can control cap-dependent and internal ribosome entry site-mediated translation by binding to differentiation control elements. Our data suggest a specific cytoplasmic function for hnRNPs as translational regulatory proteins.     

Importance of initiation factor preparations in the translation of reovirus and globin mRNAs lacking a 5'-terminal 7-methylguanosine

Reovirus and globin mRNAs which lack a 5'-terminal 7-methylguanosine are translated in a fractionated cell-free protein-synthesizing system. Efficient translation occurs only at optimal concentrations of reticulocyte initiation factor preparations and does not occur at optimal concentrations of ascites initiation factor preparations or at suboptimal concentrations of reticulocyte initiation factor preparations. The translation of "uncapped" mRNA in vitro, therefore, appears to be related to the efficiency of initiation of protein synthesis. At optimal concentrations of reticulocyte initiation factors, mRNA containing a 5'-terminal 7-methylguanosine is preferentially translated in the presence of mRNA which lacks a "cap." These results indicate that the 5'-terminal 7-methylguanosine on mRNA has a facilitatory rather than obligatory role in translation.     

The expression of beta-glucuronidase during preimplantation development of mouse embryos

The globin mRNAs containing between 30 and 40 polyadenylate residues can be separated from thos mRNAs containing longer poly(A) regions by Millipore filter binding. The molecular weights of the alpha-and beta-globin mRNAs containing this size class of poly(A) have beed determined by lectrophoresis on 3.7% polyacrylamide gels in the presence of 99% formamide. Because the number of adenylic acid residues in these mRNAs is known, the number of non-poly(A) nucleotides can be accurately calculated. The molecular weight of the beta-globin mRNA is 235 000 +/- 28 000 (736 +/- 88 nucleotides) and that of the alpha-globin mRNA is 208 900 +/- 43 870 (653 +/- 78 nucleotides). By subtracting the number of nucleotides in the coding and poly(A) regions, the number of non-coding nucleotides in the beta-globin mRNA were calculated to be 261, 69 more than the 193 present in the alpha-globin mRNA. Comparison of size estimates of newly synthesized globin mRNAs containing longer average lengths of poly(A) shhowed that there is no comparable processin of the 5' termini of the alpha-and beta-globin mRNAs concomitant with the stepwise degradation of the poly(A) regions which occur as the mRNAs mature.     

DNA transfection to study translational control in mammalian cells

Mammalian cells respond to changes in their environment by rapid and reversible covalent modification of the translational machinery. In most cases, these modifications involve the phosphorylation and dephosphorylation of translation initiation factors (for review see Ref. 1). The modification of translation initiation factors may affect translational activity of either specific mRNAs or general cellular mRNAs. To study the effect of a particular factor or its modification on the translational capacity of an mRNA, there are a number of potential approaches that include in vitro translation reactions as well as in vivo experiments. Generally, experiments initially report a covalent modification that correlates with altered translational capacity of either a specific or a general class of mRNAs. The modification and the particular amino acid residue involved are then identified. Then mutations are made at the modified residue to prevent modification (for example, a serine-to-alanine mutation to prevent phosphorylation) and the effect of the mutant factor on the translation of a target mRNA is tested. The most convenient method for monitoring the effect of a mutant translation factor on translation is the use of transient DNA transfection. However, in certain situations it is desirable to isolate stably transfected cell lines to study the effect of overexpression, underexpression, or expression of a particular mutant translation factor. This article reviews two methods that are routinely used to study translational control that involve either transient or stable DNA transfection.     

Molecular cloning of the actin gene from yeast Saccharomyces cerevisiae

Two overlapping DNA fragments from yeast Saccharomyces cerevisiae containing the actin gene have been inserted into pBR322 and cloned in E.coli. Clones were identified by hybridization to complementary RNA from a plasmid containing a copy of Dictyostelium actin mRNA. One recombinant plasmid obtained (pYA102) contains a 3.93-kb Hindlll fragment, the other (pYA208) a 5.1-kb Pstl fragment, both share a common 2.2-kb fragment harboring part of the actin gene. Cloned yeast actin DNA was identified by R-loop formation and translation of the hybridized actin mRNA and by DNA sequence analysis. Cytoplasmic actin mRNA has been estimated to be about 1250 nucleotides long. There is only one type of the actin gene in S.cerevisiae.     

Human La protein: a stabilizer of histone mRNA

Histone mRNA is destabilized at the end of S phase and in cell-free mRNA decay reaction mixtures supplemented with histone proteins, indicating that histones might autoregulate the histone mRNA half-life. Histone mRNA destabilization in vitro requires three components: polysomes, histones, and postpolysomal supernatant (S130). Polysomes are the source of the mRNA and mRNA-degrading enzymes. To investigate the role of the S130 in autoregulation, crude S130 was fractionated by histone-agarose affinity chromatography. Two separate activities affecting the histone mRNA half-life were detected. The histone-agarose-bound fraction contained a histone mRNA destabilizer that was activated by histone proteins; the unbound fraction contained a histone mRNA stabilizer. Further chromatographic fractionation of unbound material revealed only a single protein stabilizer, which was purified to homogeneity, partially sequenced, and found to be La, a well-characterized RNA-binding protein. When purified La was added to reaction mixtures containing polysomes, a histone mRNA decay intermediate was stabilized. This intermediate corresponded to histone mRNA lacking 12 nucleotides from its 3' end and containing an intact coding region. Anti-La antibody blocked the stabilization effect. La had little or no effect on several other cell cycle-regulated mRNAs. We suggest that La prolongs the histone mRNA half-life during S phase and thereby increases histone protein production.     

Conserved boxes C and D are essential nucleolar localization elements of U14 and U8 snoRNAs

Sequences necessary for nucleolar targeting were identified in Box C/D small nucleolar RNAs (snoRNAs) by fluorescence microscopy. Nucleolar preparations were examined after injecting fluorescein-labelled wild-type and mutated U14 or U8 snoRNA into Xenopus oocyte nuclei. Regions in U14 snoRNA that are complementary to 18S rRNA and necessary for rRNA processing and methylation are not required for nucleolar localization. Truncated U14 molecules containing Boxes C and D with or without the terminal stem localized efficiently. Nucleolar localization was abolished upon mutating just one or two nucleotides within Boxes C and D. Moreover, the spatial position of Boxes C or D in the molecule is essential. Mutations in Box C/D of U8 snoRNA also impaired nucleolar localization, suggesting the general importance of Boxes C and D as nucleolar localization sequences for Box C/D snoRNAs. U14 snoRNA is shown to be required for 18S rRNA production in vertebrates.     

Possible protective and pathogenic roles of gamma delta T lymphocytes in HIV-infections (Review)

The ribosomal environment of the N-terminus of the nascent polypeptide chain has been investigated using peptides of different lengths, synthesized in situ on Escherichia coli ribosomes; the peptides each carry a photoreactive diazirine moiety at their N-terminus, so as to generate cross-links to neighbouring ribosomal components. Our previous studies [Choi, K. M. & Brimacombe, R. (1998) Nucleic Acids Res. 26, 887-895] with three independent families of peptides, derived from the E. coli ompA protein gene, the tetracycline-resistance gene and the bacteriophage T4 gene 60, identified a series of sites within the 23S rRNA to which the peptides became cross-linked. The distribution of these cross-links indicated that the nascent peptide is very flexible within the 50S subunit. Here, we demonstrate that the N-termini of the ompA and gene-60 peptides can, in addition, even become concomitantly cross-linked to the 30S subunit. The cross-linking is predominantly to 30S ribosomal proteins S1, S2, S4 and (to a lesser extent) S3, which form a cluster near to the decoding region. This result is discussed in terms of the flexibility of the nascent peptide during the co-translational folding process, and in terms of the 'ribosomal bypass' phenomenon which is known to occur during translation of the gene 60 mRNA.     

Binding of a phosphoprotein to the 3' untranslated region of the mouse protamine 2 mRNA temporally represses its translation

The synthesis of the protamines, the predominant nuclear proteins of mammalian spermatozoa, is regulated during germ cell development by mRNA storage for about 7 days in the cytoplasm of differentiating spermatids. Two highly conserved sequences, the Y and H elements present in the 3' untranslated regions (UTRs) of all known mammalian protamine mRNAs, form RNA-protein complexes and specifically bind a protein of 18 kDa. Here, we show that translation of fusion mRNAs was markedly repressed in reticulocyte lysates supplemented with a mouse testis extract enriched for the 18-kDa protein when the mRNAs contained the 3' UTR of mouse protamine 2 (mP2) or the Y and H elements of mP2. No significant decrease was seen when the fusion mRNAs contained the 3' UTR of human growth hormone. The 18-kDa protein is developmentally regulated in male germ cells, requires phosphorylation for RNA binding, and is found in the ribonucleoprotein particle fractions of a testicular postmitochondrial supernatant. We propose that a phosphorylated 18-kDa protein plays a primary role in repressing translation of mP2 mRNA by interaction with the highly conserved Y and H elements. At a later stage of male gamete differentiation, the 18-kDa protein no longer binds to the mRNA, likely as a result of dephosphorylation, enabling the protamine mRNA to be translated.