Stretches Of RNA Research Articles

Antisense regulation of oncogenes in human cancer.

Gene transfer or manipulation of genes for the treatment of cancer is a rapidly expanding field. In recent years, much attention has been focused on manipulating cancer genes and applying antisense technology in therapeutic ways. Consequently, antisense RNA control is now recognized as a specific means of regulating gene expression at the posttranscriptional level. Defects in vital genes occur in many human diseases, including cancer, defects that may be due to an accumulation of mutations in the genes that leads to the production of faulty proteins. Although the biological significance of such mutant proteins still remains in question, recent experiments have demonstrated that genes overproducing faulty proteins are often associated with increased tumor cell growth. Moreover, using a stretch of antisense RNA to block the production of such defective proteins can effectively silence their genes; as a result, tumor cells stop dividing rapidly and revert to a more normal phenotype. Therefore, antisense RNA technology could have a significant impact on cancer gene therapy. Here, we have tried to give comprehensive coverage to some major cases of antisense RNA control of cancer-related genes highlighting the biological systems involved, the efficacy of the antisense RNA in altering target gene function, and how such antisense control affects the malignant phenotype. Furthermore, the therapeutic potential of the antisense technique depends on the in-depth understanding of the target gene function and its role in carcinogenesis.

Secondary Structure Model of the Last Two Domains of Single-stranded RNA Phage Qβ

We have determined the nucleotide sequence of three positive single-stranded RNA coliphages and have used this information, together with the known sequences of the related phages Qβ and SP, to construct a secondary structure model for the two distal domains of Qβ RNA. The 3′ terminal domain, which is about 100 nucleotides long, contains most of the 3′ untranslated region and folds into four short, regular hairpins. The adjacent 3′ replicase domain contains about 1100 nucleotides. Hairpins in this protein-coding domain are much longer and more irregular than in the 3′ untranslated region. Both domains are defined by long-distance interactions. The secondary structure is not a collection of hairpin structures connected by single-stranded regions. Rather, the RNA stretches between the stem-loop structures are all involved in an extensive array of long-distance interactions that contract the molecule to a rigid structure in which all hairpins are predicted to have a fixed position with respect to each other. A general feature of the model is that helices tend to be organized in four-way junctions with little or no unpaired nucleotides between them. As a result, there is a potential for coaxial stacking of adjacent stems. The essential features of the model are supported by the SInuclease cleavage pattern.Viral RNA sequences are strongly constrained by their coding function. As a result, structural evolution by simple base-pair substitutionis not always possible, as this usually requires the juxtaposition of the codon wobble positions in stems. Rather, we often observe co-ordinate base substitutions that maintain the stem, but tend to change the position at which bulges or internal loops are found. Structures that differ in this way are apparently equally fit. Also, the relative position of hairpin loops can shift several nucleotides through an alignment based on maximal sequence identity i.e. amino acid homology. The fact that these structural irregularities do not occur at the 3′ untranslated region suggests indeed that the coding function of the RNA constrains the secondary structure. Hairpins with the stable tetraloop motif GNRA and UNCG or their complement are over-represented. This suggests their involvement in segregation of plus and minus strand. The genome of the coliphages contains a well-defined high affinity binding site for the coat protein, which serves to suppress replicase translation and also acts as a nucleation point in capsid formation. Close to the 3′ end we find an additional conserved helix that meets the described consensus criteria for coat-protein binding.

Poliovirus subviral particles associated with progeny RNA in the replication complex

The poliovirus replication complex (RC), the site of genomic 36S RNA synthesis, was previously shown to contain subviral particles of 5S protomer and 14S pentamer antigenicity. The present investigation demonstrates that 5S/14S antigenic subviral particles can be cross-linked to viral RNA by UV irradiation of a subcellular fraction containing the poliovirus RC. Each capsid protein of the subviral particles, i.e. VP0, VP1 and VP3, was cross-linked to viral RNA. SDS-PAGE analysis of the cross-linked capsid proteins revealed a bandshift for VP1, whereas VP0 migrated in several bands, which were interpreted to be multimers of VP0 linked by short stretches of RNA. It was found that 36S RNA rather than replicative intermediate RNA was cross-linked to capsid proteins. Our results indicate that encapsidation of poliovirus RNA starts in the RC and is initiated by 14S pentamers.

Scanning and competition between AGs are involved in 3' splice site selection in mammalian introns.

In mammalian intron splicing, the mechanism by which the 3' splice site AG is accurately and efficiently identified has remained unresolved. We have previously proposed that the 3' splice site in mammalian introns is located by a scanning mechanism for the first AG downstream of the branch point-polypyrimidine tract. We now present experiments that lend further support to this model while identifying conditions under which competition can occur between adjacent AGs. The data show that the 3' splice site is identified as the first AG downstream from the branch point by a mechanism that has all the characteristics expected of a 5'-to-3' scanning process that starts from the branch point rather than the pyrimidine tract. Failure to recognize the proximal AG may arise, however, from extreme proximity to the branch point or sequestration within a hairpin. Once an AG has been encountered, the spliceosome can still see a limited stretch of downstream RNA within which an AG more competitive than the proximal one may be selected. Proximity to the branch point is a major determinant of competition, although steric effects render an AG less competitive in close proximity (approximately 12 nucleotides). In addition, the nucleotide preceding the AG has a striking influence upon competition between closely spaced AGs. The order of competitiveness, CAG congruent to UAG > AAG > GAG, is similar to the nucleotide preference at this position in wild-type 3' splice sites. Thus, 3' splice site selection displays properties of both a scanning process and competition between AGs based on immediate sequence context. This refined scanning model, incorporating elements of competition, is the simplest interpretation that is consistent with all of the available data.

Scanning and competition between AGs are involved in 3' splice site selection in mammalian introns.

In mammalian intron splicing, the mechanism by which the 3' splice site AG is accurately and efficiently identified has remained unresolved. We have previously proposed that the 3' splice site in mammalian introns is located by a scanning mechanism for the first AG downstream of the branch point-polypyrimidine tract. We now present experiments that lend further support to this model while identifying conditions under which competition can occur between adjacent AGs. The data show that the 3' splice site is identified as the first AG downstream from the branch point by a mechanism that has all the characteristics expected of a 5'-to-3' scanning process that starts from the branch point rather than the pyrimidine tract. Failure to recognize the proximal AG may arise, however, from extreme proximity to the branch point or sequestration within a hairpin. Once an AG has been encountered, the spliceosome can still see a limited stretch of downstream RNA within which an AG more competitive than the proximal one may be selected. Proximity to the branch point is a major determinant of competition, although steric effects render an AG less competitive in close proximity (approximately 12 nucleotides). In addition, the nucleotide preceding the AG has a striking influence upon competition between closely spaced AGs. The order of competitiveness, CAG congruent to UAG > AAG > GAG, is similar to the nucleotide preference at this position in wild-type 3' splice sites. Thus, 3' splice site selection displays properties of both a scanning process and competition between AGs based on immediate sequence context. This refined scanning model, incorporating elements of competition, is the simplest interpretation that is consistent with all of the available data.

Translational Repression by the Bacteriophage T4 Gene 32 Protein Involves Specific Recognition of an RNA Pseudoknot Structure

An RNA pseudoknot has been shown to form the 5′-end of bacteriophage T4 gene 32 mRNA that is essential to autoregulation of gene 32 (g3g) mRNA by gene product 32 (gp32), a single-stranded nucleic acid binding protein. Structure-mapping of RNA oligonucleotides with structure-specific RNases indicate that two stem regions consisting of nucleotides -67 to -64 base-paired to -52 to -55 (stem-1) and nucleotides -62 to -56 base paired to -40 to -46 (stem-2) can fold into a "pseudoknotted" structure that may be analogous to the semi-continuous a-helical pseudoknot. Our results suggest that the g32 mRNA pseudoknot can form under conditions where specific autoregulation by gp32 is observed. Although the g32 mRNA pseudoknot is stabilized by Mg 2+, it exists in equilibrium with a 3′-hairpin structure. Gel mobility studies carried out with defined length oligonucleotides indicate the gp32 does, in fact, bind tightly to the pseudoknot. These studies agree with the proposal of McPheeters et al., that the pseudoknot represents a nucleation site essential for autogenous gp32 translation regulation. Although disruption of tertiary structure interactions in this pseudoknot (with EDTA) significantly reduces the ability of gp32 to specifically recognize its own mRNA, in vitro mutagenesis studies suggest the sequence of stem-2 and of the loop region (nucleotides -47 to -50) also represent important determinants for specific gp32 autoregulation. Based on a competition assay relying on gel mobility shifts, the order of importance of the major elements of the pseudoknot are stem-1 > sequence of stem-2 or loop-2 > stem-2. In this assay, disruption of stem-1 decreased the ability of the resulting structure to compete for gp32 binding by approximately fourfold. Both stem-1 and stem-2 appear to be essential to maintain high-level expression from gp32 mRNA in an in vitro transcription/translation system. Taken together, these results support the translational control model in which the pseudoknot region is a nucleation point for cooperative gp32 binding, which then proceeds in a 3′ direction through a long stretch of single-stranded RNA that includes the initiation codon for gene 32.

Different conformational forms of Escherichia coli and rat liver 5S rRNA revealed by Pb(II)‐induced hydrolysis

Different stable forms of Escherichia coli and rat liver 5S rRNA have been probed by Pb(II)-induced hydrolysis. In the native A forms of 5S rRNA, Pb2+ reveal single-stranded RNA stretches and regions of increased conformational flexibility or distorted by the presence of bulged nucleotides. Hydrolysis of urea/EDTA-treated E. coli 5S rRNA (B form) shows the presence of two strong helical domains; helix A retained from the A form and a helix composed of RNA regions G33-C42 and G79-C88. Other RNA regions resistant to hydrolysis may be involved in alternative base pairing, causing conformational heterogeneity of that form. Pb(II)-induced hydrolysis distinguishes two different forms of rat liver 5S rRNA; the native A form and the form obtained by renaturation of 5S rRNA in the presence of EDTA. Pb(II)-hydrolysis data suggest that both forms are highly structured. In the latter form, the orientation of the bulged C66 is changed with respect to helix B. At the same time, a new helical segment is possibly formed, composed of nucleotides from helix C and loop c on one side and from helix E and loop d' on the other.

Comparison of Partial 16S rRNA Sequences of Different Helicobacter pylori Strains, Helicobacter mustelae and a Gastric Campylobacter-like Organism (GCLO)

Partial 16S rRNA sequences of seven Helicobacter pylori strains from three different continents were compared. High homogeneity (99.5%–100%) of 16S rRNA between H. pylori strains was demonstrated, when a 397 nucleotides long stretch of RNA was compared. The obtained consensus sequence was practically identical with one of the two published sequences, 99.4% similarity ( Romaniuk et al., J. Bact. 169 , 2137–2141 (1987)), whereas with the other published sequence the identity was lower, 95.8% similarity ( Thompson et al., Int. J. System. Bact. 38 , 190–200 (1988)). Comparison of the consensus H. pylori sequence with the sequence of Helicobacter mustelae , the spiral organism isolated from ferrets, revealed 93.8% similarity. H. mustelae was also compared with published sequences of Wollinella succinogenes , and Campylobacter jejuni . The percentual sequence similarities were 96.0 and 83.7, respectively. A gastric campylobacter-like organism (GCLO, NCTC 11847), isolated from the gastric antrum has been demonstrated to resemble C. jejuni biochemically. By 16S rRNA sequences we demonstrate that the GCLO is in fact a C. jejuni strain.

Pseudo-templated transcription in prokaryotic and eukaryotic organisms.

Pseudo-templated transcription describes a process that results in the production of a homopolymeric stretch of RNA directed by a shorter stretch (usually 3-7) of complementary nucleotides on the template (either RNA or DNA). The effects of pseudo-templated transcription are very diverse. In some cases, strictly homopolymeric RNAs are made; in others, pseudo-templated nucleotides are added at either the 5' or 3' ends of otherwise normally templated transcripts; and finally, sometines pseudo-templated nucleotides are inserted at a specific internal site during elongation within an otherwise normally templated RNA

Complete cDNA sequence of a South American isolate of potato virus X

The complete cDNA sequence corresponding to the genome RNA of a South American strain of potato virus X (PVXc) is reported. The sequence (6432 nucleotides) contains five open reading frames coding for polypeptides with molecular weights of 165.3, 24.3, 12.3 7.6 and 25.0 and displays an overall homology of 77.4% with those previously reported for two European isolates. Comparison of amino acid sequences shows an average homology of 87%. Two major domains of variability, located between amino acids 476–615 of ORF 1 and 64–100 of ORF 5, are identified. Sequence similarities between RNA stretches lying upstream of ORFs 2, 4 and 5, and at the 3'-non coding regions of PVX and other plus-strand RNA viruses are described.

A rev/β-galactosidase fusion protein binds in vitro transcripts spanning the rev-responsive element of human immunodeficiency virus type 1 (HIV-1)

The rev protein of human immunodeficiency virus type 1 (HIV-1), a phosphoprotein of 20 kDa apparent molecular mass, is essential to target the mRNA for virion polypeptides into the cytoplasm. This effect is mediated by a specific RNA stretch (rev-responsive element = RRE) localized within a 3′-terminal segment of the mRNA encoding virion proteins. We present evidence that rev expressed as a β-galactosidase fusion protein in E. coli forms a complex with in vitro transcripts containing the RRE; it can be precipitated by monoclonal antibodies with rev or β-galactosidasespecificity. In addition, specific binding of rev protein to RNA could be demonstrated by Northwestern blotting.

Kinetic analysis of Escherichia coli RNase H using DNA-RNA-DNA/DNA substrates.

The kinetic properties of Escherichia coli ribonuclease H (RNase H) were investigated using oligonucleotide substrates that consist of a short stretch of RNA, flanked on either side by DNA (DNA-RNA-DNA). In the presence of a complementary DNA strand, RNase H cleavage is restricted to the short ribonucleotide stretch of the DNA/RNA heteroduplex. The DNA-RNA-DNA substrate utilized for kinetic studies: (formula; see text) is cleaved at a single site (decreases) in the presence of a complementary DNA strand, to generate (dT)7-(rA)2-OH and p-(rA)2-(dT)9. Anion exchange high performance liquid chromatography was used to separate and quantitate the cleavage products. Under these conditions, RNase H-specific and nonspecific degradation products could be resolved. Kinetic parameters were measured under conditions of 100% hybrid formation (1.2-1.5 molar excess of complementary DNA, T much less than Tm). A linear double reciprocal plot was obtained, yielding a Km of 4.2 microM and a turnover number of 7.1 cleavages per s per RNase H monomer. The kinetic properties of substrate analogs containing varying lengths of RNA (n = 3-5) and 2'-O-methyl modifications were also investigated. Maximal turnover was observed with DNA-RNA-DNA substrates containing a minimum of four RNA residues. Kcat for the rA3 derivative was decreased by more than 100-fold. The Km appeared to decrease with the size of the internal RNA stretch (n = 3-5). No significant difference in turnover number of Km was observed when the flanking DNA was replaced with 2'-O-methyl RNA, suggesting that RNase H does not interact with this region of the heteroduplex.

Structure of secondary cleavage sites of E. coli RNAaseIII in A3t RNA from bacteriophage T7.

Five 'secondary' cleavage sites of E. coli RNAaseIII within the initiator RNA A3t (141 nucleotides) from bacteriophage T7 are described. Cleavage takes place apparEntly within or at the bottom of a short double-stranded stretch of RNA. Three secondary sites are efficiently cleaved in the presence of magnesium 2+ ions, two additional sites are cleaved in the presence of manganese 2+ ions at low monovalent salt concentrations (less than 0.05 M).

Occurence and loss of alkali-labile sites in newly synthesized DNA of UV-irradiated Escherichia coli K12

In pulse-labelled DNA of ultraviolet-irradiated E. coli, alkali-labile sites were detected. They do not occur in undamaged cells. These sites are produced in wild-type cells as well as in uvrA, uvrB and recA derivatives. Restoration of the synthesis of DNA molecules free of alkali-labile sites requires recA products and involves also uvrA and uvrB products. The chemical nature of alkali-labile sites and their biological function are obscure. They might be stretches of RNA that traverse the lesions, blocking DNA replication and priming recA-dependent DNA replication.

Subgenomic mRNA in OK10 defective leukemia virus-transformed cells.

OK10, a defective leukemia virus, is produced as a defective particle by so-called nonproducer transformed quail fibroblasts. OK10 defective viral particles contain an 8-kilobases (kb)-long genomic RNA, lack any detectable reverse transcriptase activity, and are not infectious. We studied the genetic content of OK10 RNA extracted from both virions and infected cells. As shown by RNA-cDNA hybridizations in stringent conditions, about 77% (6.4 kb) of the OK10 8.0kb RNA was related to avian leukosis viruses in the three structural genes gag, pol, and env, as well as in the c region. The remainder of the OK10 genome-encoding capacity (</=1.6 kb) was homologous to the MC29-specific transforming sequence myc(m) and therefore has been named myc(o). EcoRI restriction analysis of the OK10 integrated proviral DNA with different probes indicated the presence of only one provirus in the OK10 QB5 clone, which agreed with the gene order: 5'-gag-Deltapol-myc(o)-Deltaenv-c- 3'. Heteroduplex molecules formed between the viral OK10 8.0-kb RNA and the 6.8-kb SacI DNA fragment of the Prague A strain of Rous sarcoma virus confirmed that structure and indicated that the myc(o) sequence formed a continuous RNA stretch of 1.4 to 1.6 kb long between Deltapol and Deltaenv. We also examined the myc(o)-containing mRNA's transcribed in OK10-transformed cells. OK10-transformed quail fibroblasts (OK10 QB5) transcribed two mRNA species of 8.0 and 3.6 kb containing the myc(o) sequence. The genetic content of the 3.6-kb species made it a possible maturation product of the genome size 8-kb species by splicing out the gag and pol sequences. In OK10-transformed bone marrow cells (OK10 BM), a stable bone marrow-derived cell line producing OK10, the myc(o) sequence was found in four RNA species of 11.0, 8.0, 7.0, and 3.6 kb. Again, the genetic content of these mRNA's indicated that (i) the 3.6-kb species could be spliced out of the 8.0-kb-genome size mRNA and (ii) the 11.0-kb-long mRNA could represent a read-through of the OK10 provirus, the corresponding maturation product being, then, a 7.0-kb mRNA. The 7.0- and 3.6- kb mRNA's both contained the myc(o) sequence, but no sequences related to the gag or pol gene. In conclusion, whereas the myc sequences have been generally thought to be expressed through a gag-onc fusion protein, as for MC29 and CMII viruses, our experiments indicate that they could also be expressed as a non-gag-related product made from a subgenomic mRNA in the OK10-transformed cells.

Messenger RNA sequences in nuclear ribonucleoprotein particles are complexed with protein as shown by nuclease protection

Soon after their transcription, eukaryotic messenger RNA precursors (heterogeneous nuclear RNA) are assembled into ribonucleoprotein particles (hnRNP). In the present experiments we have asked whether the mRNA-homologous components of hnRNA are complexed with proteins in hnRNP or, alternatively, exist as stretches of naked RNA in the particles. Mild digestion of HeLa cell hnRNP particles by micrococcal nuclease, pancreatic RNAase or an endogenous particle-bound nuclease, followed by hybridization of the surviving RNA with a complementary DNA probe for poly(A) + mRNA sequences reveals that messengerhomologous sequences are protected from nuclease attack in hnRNP.

Genome of avian myelocytomatosis virus MC29: analysis by heteroduplex mapping.

The virion RNA of avian myelocytoma virus MC29 was hybridized to full genome length DNA of the Prague strain of Rous sarcoma virus and analyzed by heteroduplex mapping in the electron microscopy. The results show that MC29 specific sequences for which there are no homologous counterparts in the Rous sarcoma virus genome make up a contiguous stretch of RNA about 1.8 kilobases long. These sequences are located approximately in the middle of the genome, replacing the 3' half of the gag gene, the entire pol gene, and the 5' portion of the env gene, which are absent from MC29. This MC29 specific genetic substitution may contain information for the leukemogenic transformation of the host cell.

Analysis of rous sarcoma virus (RSV) RNA structure by means of specific nucleases

The effects of Escherichia coli RNases III and IV and of Aspergillus oryzae T 1 ribonuclease and S1 nuclease on RSV 70 S and 35 S RNA have been analyzed by the fingerprinting technique. The results of such a study indicate that RSV RNA is a highly structured molecule in which the two 35 S subunits are intimately associated via a large number of specific interactions to form the 70 S complex. One of these interactions occurs in a region located at the 5′ end of the molecule and involves a double-stranded RNA stretch characterized by the presence of a large T, RNase-resistant oligonucleotide. Among the several folded RNA sequences detected, two of them are found at the initiation sites for in vitro revere transcription.

A model of post-transcriptional control in eukaryotic cells

A model is proposed which implies post-transcriptional cleavage of primary transcription products namely heterogeneous nuclear RNA. The stretches to be excised are complementary to other RNA stretches which are coded for by different transcription sequences. Once complementary sequences have hybridized, an endonuclease cuts the molecule at the junction between double-stranded and single-stranded portions. Only single-stranded portions, possibly after RNA ligase iigation, are translated, as mRNA, on polysomes.

Biogenesis of vaccinia: Specific inhibition of rapidly labeled host DNA in vaccinia inoculated cells

Because separation of the bulk and nascent (i.e., rapidly labeled) DNA fractions of HeLa cells could be effected satisfactorily it became possible to ascertain the effects of inoculating uv-irradiated vaccinia virus on in vivo host DNA synthesis. In both control and infected cells, labeled 4s fragments, formed during 1-min pulses with [ 3H]Tdr, existed as single- or double-stranded molecules covalently linked to short stretches of RNA. During the chase period, most of the rapidly labeled DNA within uninfected cells became associated with the bulk DNA component but failed to do so within inoculated cells. Host DNA ligase activity was unaffected by the infection, and there was a rapid breakdown of the 4s DNA fragments, leading to the conclusion that arrest of HeLa nuclear DNA synthesis by vaccinia is, most probably, due to the in vivo hydrolysis of nascent DNA. The present data further substantiate previous studies from this laboratory demonstrating the hydrolysis of in vitro synthesized nascent DNA by a DNAse activity originating from the virus core.