Specific hydrolysis of rabbit globin messenger RNA by S1 nuclease

S1 nuclease isolated from Aspergillus oryzae has been used to investigate the secondary structure of rabbit globin messenger RNA (mRNA). The enzyme, which is specific for single stranded nucleotides, digests globin mRNA to a limited extent, with 65-75% of the mRNA nucleotides resistant to digestion under mild conditions. This limited digestion is not due to enzyme inactivation, but rather to the normal activity of the single-strand nuclease. The reaction was studied as a function of temperature, salt and enzyme concentration. Analysis of the products of digestion on 20% acrylamide- 7M urea slab gels reveals a stable pattern of unique fragments ranging in size from 9 to 71 nucleotides. Separated alpha and beta globin mRNAs show similar, but not identical gel patterns, indicating strong structural similarities between the two species. The high degree of nuclease resistance, along with the fragment patterns seen on polyacrylamide gels, gives evidence to support a model of rabbit globin mRNA which contain specific, rather than random, helical structure.     

Poly (A)-rich ribonucleoprotein complexes from HeLa cell messenger RNA

Polyribosomal messenger RNA from HeLa cells contain 3'-OH-terminal polyadenylate sequences approximately 133 nucleotides in length (weight average). When analyzed at the ribonucleoprotein level of organization these poly(A)-rich sequences are found to contain tightly bound proteins. These proteins remain associated with the poly(A)-rich RNA during affinity chromatography of RNase A and T1-digested polyribosomes on poly(U)-Sepharose in 0.5 M NaCl, and co-elute from the column with the RNA at 50% formamide. Controls establish that the co-purification of the proteins with poly(A) on poly(U)-Sepharose requires the molecular integrity of the poly(A). Polyacrylamide gel electrophoresis resolves the poly(A)-specific proteins into two components of 74,000 and 62,000 molecular weight. The larger protein is the same size as that previously reported to be associated with poly(A)-rich sequences in HeLa heterogeneous nuclear RNA (Kish, V.M., and Pederson, T. (1975), J. Mol. Biol. 95, 227-238). It is concluded that both HeLa nuclear and polyribosomal poly(A) sequences have a protein (62,000 molecular weight) associated with poly(A) appears to be confined only to messenger RNA.     

3' non-coding region sequences in eukaryotic messenger RNA

The sequence A-A-U-A-A-A is present in six different purified messenger RNA molecules (specifically the alpha-and beta-globulin mRNAs of rabbit and human, the immunoglobulin light chain mRNA of mouse (MOPC 21) and the ovalbumin mRNA of chicken) about 20 residues away from the 3'-terminal poly (A) sequence. In addition, a large selection of the 3' non-coding regions of rabbit and human globulin mRNAs (both the alpha and beta globin mRNAs) are 85% homologous, demonstrating that this region is significantly conserved in evolution.     

Partial purification of a progesterone-inducible messenger RNA (avidin) from hen oviduct

The messenger RNA (mRNA) for avidin, which represents less than 0.05% of the total cellular proteins, was partially purified from hen oviduct, and the presence of avidin mRNA was shown to depend upon prior stimulation by progesterone. A total nucleic acid extract was subjected to oligo (dT)-cellulose chromatography, followed by Sepharose 4B chromatography, preparative agarose gel electrophoresis, and sucrose gradient centrifugation. The relative purity of each preparation was assessed by translation in a wheat-germ system; avidin messenger RNA activity was measured by specific immunoprecipitation of synthesized proteins. Avidin mRNA was separated from the bulk of the total messenger RNA activity of the oviduct and from all ribosomal RNAs to produce greater than a 1000-fold enrichment of avidin mRNA activity compared with total cellular RNA. Based on the translation assay, the most highly purified fraction contained about 2.5% avidin messenger RNA. Avidin mRNA activity was absent in partially purified mRNA obtained from estrogen-stimulated chick oviducts, but was detected in oviducts following progesterone administration.     

Interaction of poly(rC) binding protein 2 with the 5' noncoding region of hepatitis A virus RNA and its effects on translation

Utilization of internal ribosome entry segment (IRES) structures in the 5' noncoding region (5'NCR) of picornavirus RNAs for initiation of translation requires a number of host cell factors whose distribution may vary in different cells and whose requirement may vary for different picornaviruses. We have examined the requirement of the cellular protein poly(rC) binding protein 2 (PCBP2) for hepatitis A virus (HAV) RNA translation. PCBP2 has recently been identified as a factor required for translation and replication of poliovirus (PV) RNA. PCBP2 was shown to be present in FRhK-4 cells, which are permissive for growth of HAV, as it is in HeLa cells, which support translation of HAV RNA but which have not been reported to host replication of the virus. Competition RNA mobility shift assays showed that the 5'NCR of HAV RNA competed for binding of PCBP2 with a probe representing stem-loop IV of the PV 5'NCR. The binding site on HAV RNA was mapped to nucleotides 1 to 157, which includes a pyrimidine-rich sequence. HeLa cell extracts that had been depleted of PCBP2 by passage over a PV stem-loop IV RNA affinity column supported only low levels of HAV RNA translation. Translation activity was restored upon addition of recombinant PCBP2 to the depleted extract. Removal of the 5'-terminal 138 nucleotides of the HAV RNA, or removal of the entire IRES, eliminated the dependence of HAV RNA translation on PCBP2.     

Age-dependent silencing of globin transgenes in the mouse

Variegation of transgene expression, a heterocellular or mosaic pattern of expression seen in all mice in a given transgenic line, is a frequently observed but unexplained phenomenon. We have encountered variegation with globin transgenes; when lacZ expression is driven by globin control elements a proportion of erythrocytes express beta-galactosidase (beta-gal), while the remaining erythrocytes express none. The percentage of expressing cells is constant within each line (at any particular developmental stage), but varies between lines. Such variation may account for much of the line-to-line variability which has been reported in the expression of a transgene construct. We have now extended these observations by studying expression of several globin/lacZ transgenes with increasing age. Expression of beta-gal is variegated in all lines in adult mice, including those made with a beta-globin promoter and locus control region driving lacZ. The extent of variegation differs widely between lines, but in all lines there is a marked decline in the number of erythrocytes expressing beta-gal with increasing age. Progression of silencing continues long past the point at which globin switching is complete, suggesting that it is not related to this process. We observe that age-dependent silencing is most severe in high copy number animals. Increasing variegation of transgene expression with ageing of mice is likely to complicate interpretation of the developmental regulation of transgenes. We speculate that it reflects a general mechanism of epigenetic regulation.     

Identification of the coding region for a second poly(A) polymerase in Escherichia coli

We had earlier identified the pcnB locus as the gene for the major Escherichia coli poly(A) polymerase (PAP I). In this report, we describe the disruption and identification of a candidate gene for a second poly(A) polymerase (PAP II) by an experimental strategy which was based on the assumption that the viability of E. coli depends on the presence of either PAP I or PAP II. The coding region thus identified is the open reading frame f310, located at about 87 min on the E. coli chromosome. The following lines of evidence support f310 as the gene for PAP II: (i) the deduced peptide encoded by f310 has a molecular weight of 36,300, similar to the molecular weight of 35,000 estimated by gel filtration of PAP II; (ii) the deduced f310 product is a relatively hydrophobic polypeptide with a pI of 9.4, consistent with the properties of partially purified PAP II; (iii) overexpression of f310 leads to the formation of inclusion bodies whose solubilization and renaturation yields poly(A) polymerase activity that corresponds to a 35-kDa protein as shown by enzyme blotting; and (iv) expression of a f310 fusion construct with hexahistidine at the N-terminus of the coding region allowed purification of a poly(A) polymerase fraction whose major component is a 36-kDa protein. E. coli PAP II has no significant sequence homology either to PAP I or to the viral and eukaryotic poly(A) polymerases, suggesting that the bacterial poly(A) polymerases have evolved independently. An interesting feature of the PAP II sequence is the presence of sets of two paired cysteine and histidine residues that resemble the RNA binding motifs seen in some other proteins.     

Heat shock alters poly(A) tail length of Dictyostelium discoideum hsp32 RNA

BACKGROUND: Primary leiomyosarcoma of bone is a very rare malignant tumor with uncertain pathogenicity. METHODS: The authors studied five cases of surgically treated primary leiomyosarcoma of bone. Clinical histories and radiographic findings were recorded. Regular clinical and radiographic controls were obtained postoperatively. In all cases, immunohistochemical studies were used to confirm the diagnosis. Molecular biologic examinations, using the polymerase chain reaction technique with microsatellite DNA markers from regions of tumor-relevant genes, were performed to determine the stability of the genome or to detect some typical genomic changes. RESULTS: The study included three women and two men, with an average age of 42 years. The tumor was located in the pelvis in two patients, in the femur in two patients, and in the proximal tibia in one patient. All tumors were classified as high-grade tumors (four stage IIB, one stage IIA). Radiographically, all tumors appear as purely osteolytic lesions, with a geographic or moth-eaten appearance and without any sclerotic margin. Three patients underwent limb salvage surgery followed by endoprosthetic replacement. The other two patients required amputation. The mean follow-up was 19 months (range, 8-29 months). Three patients died of disease, with a mean postoperative survival period of 18 months (range, 6-27 months). Four patients developed diffuse pulmonary metastases after an average of 10.5 months. One of those patients responded well to chemotherapy. In all cases, immunohistochemistry showed strong reactivity of the tumor cells for (alpha-SMA and vimentin. Molecular biologic investigations revealed a high rate of genomic instabilities in all of the stage IIB tumors. CONCLUSION: Clinical follow-up suggests that primary osseous leiomyosarcoma has an aggressive biologic behavior. The immunohistochemical studies are useful tools and suggest that osseous leiomyosarcoma arise from the vascular smooth muscle cells within the bone. The molecular biologic findings of a high rate of genomic instability confirm the hypothesis that this rare entity is of an aggressive nature.     

Processivity of the Saccharomyces cerevisiae poly(A) polymerase requires interactions at the carboxyl-terminal RNA binding domain

The interaction of the Fip1 subunit of polyadenylation factor I with the Saccharomyces cerevisiae poly(A) polymerase (PAP) was assayed in vivo by two-hybrid analysis and was found to involve two separate regions on PAP, located at opposite ends of the protein sequence. In vitro, Fip1 blocks access of the RNA primer to an RNA binding site (RBS) that overlaps the Fip1 carboxy-terminal interaction region and, in doing so, shifts PAP to a distributive mode of action. Partial truncation of this RBS has the same effect, indicating that this site is required for processivity. A comparison of the utilization of ribo- and deoxyribonucleotides as substrates indicates the existence on PAP of a second RBS which recognizes the last three nucleotides at the 3' end of the primer. This site discriminates against deoxyribonucleotides at the 3' end, and interactions at this site are not affected by Fip1. Further analysis revealed that the specificity of PAP for adenosine is not simply a function of the ATP binding site but also reflects interactions with bases at the 3' end of the primer and at another contact site 14 nucleotides upstream of the 3' end. These results suggest that the unique specificity of PAP for ribose and base, and thus the extent and type of activity with different substrates, depends on interactions at multiple nucleotide binding sites.     

Negative control of the poly(A)-binding protein mRNA translation is mediated by the adenine-rich region of its 5'-untranslated region

Translation of the mRNA for the poly(A)-binding protein (PABP) may be autoregulated by the binding of PABP to the A-rich segment of its 5'-untranslated region (UTR). To test this hypothesis, we examined the effect of different fragments of the 5'-UTR from human PABP cDNA on the translation of the beta-galactosidase (beta-Gal) reporter gene. Presence of the A-rich sequence from the 5'-UTR of PABP mRNA inhibited expression of the chimeric beta-Gal gene in transfected HeLa cells. The differences in expression of beta-Gal polypeptide was due to the translational repression of beta-Gal mRNA containing the A-rich 5'-UTR of PABP mRNA. The A-rich region of the 5'-UTR located within nucleotides 58-146 of PABP mRNA was sufficient to mediate translational control of this mRNA expression. We also examined the effect of overexpression of PABP mRNA in HeLa cells. The ectopic PABP mRNA without the A-rich 5'-UTR region was translated efficiently, whereas the translation of the endogenous PABP mRNA was substantially inhibited in the transfected cells. In contrast, the ectopic PABP mRNA containing the A-rich 5'-UTR region did not show similar effect on the translation of the endogenous PABP mRNA in these cells. These results suggest that feedback control of mRNA translation is involved in regulating PABP expression in HeLa cells.     

The enzymic addition of poly(A) to the 3'-end of RNA using bacteriophage MS 2 RNA as a model system

ATP : RNA adenyltransferase, purified from Escherichia coli, was used to add a series of adenosine residues to the 3'-end of MS2RNA. Incubations of the order of a few minutes at 37 degrees C were sufficient for synthesis of a short poly(A) chain that did not appreciably alter the hydrodynamic or electrophoretic properties of MS2 RNA. The size of the poly(A) tails was estimated by gel electrophoresis after prior hydrolysis of the primer RNA with pancreatic ribonuclease. These results were in good agreement with the values calculated on the basis of the relative amount of incorporated AMP. After the addition of a short poly(A) tail, approximately 50% of the treated material binds specifically to an oligo(dT)-cellulose column. The majority of the recovered poly(a)-containing RNA was still intact, as shown by analysis on polyacrylamide gel. After incubations beyond 6 min, slowly sedimenting material, also showing reduced electrophoretic mobility, was formed. Presumably this material corresponds to RNA chains to which long poly(A) tails are linked.