Polyadenylated RNA of Volvox: isolation and partial characterization

Polyadenylated RNA from Volvox carteri has been isolated and partially characterized. Electrophoretic profiles of total cellular poly(A)-associated RNA of Volvox spheroids indicate a hetero-disperse distribution of size classes with the range extending from an apparent sedimentation value of approximately 10S to greater than 38S. The radioactive labelling kinetics of this material are typical for rapidly-turning-over RNA. The profiles of poly(A) RNA from different cell types show marked differences in average migration rate. Terminally-differentiated somatic cells contain a greater proportion of material of higher molecular weight than either gonidia (germ cells) or cleaving embryos. The poly(A) segments associated with cellular RNA, obtained by selective RNase digestion are heterogeneous in size as determined by gel electrophoresis with the largest tracts estimated to be 75-80 nucleotides long. Gonidia and embryos display the greatest degree of size heterogeneity, while somatic cells show predominantly the largest classes of poly(A) tract. It is apparent that gross changes in poly(A) RNA metabolism accompany development and cellular differentiation in Volvox.     

RNA metabolism and poly(A) distribution in mouse liver following administration of dimethylnitrosamine

Dimethylnitrosamine (DMNA) strongly inhibited RNA synthesis in mouse liver under conditions when the nucleotide pattern, rate of nucleotide synthesis and phosphorylation ratio were unaffected. (An unidentified, probably non-nucleotide, component in the acid-soluble liver fraction was selectively reduced.) The inhibition of RNA synthesis was associated with a decrease in the RNA polymerase activity of isolated liver nuclei, well established already 45 min after DMNA administration. The reduced activity included both Mg2+- and Mn2+/(NH4)2SO4-stimulated polymerase functions. The inhibition in vivo involved the whole complement of RNA, including poly (A)-containing RNA and isolated poly(A) sequences. The transfer of labelled RNA from the nucleus to the cytoplasm was not impaired. There was no detachment of poly(A)-containing RNA from the microsomes, and the proportion of tightly membrane-bound microsomal RNA and poly(A) sequences was not reduced as determined by use of a flotation technique. No breakage or shortening of the poly(A) chains was indicated by sedimentation analysis.     

Comparison of poly(A)-containing RNAs in different cell types of the lower eukaryote Schizophyllum commune

Poly(A)-containing RNAs were isolated from morphologically different cells of the fungus Schizophyllum commune. Using mRNA markers the number-average length of poly(A)-containing RNA in total RNA and in purified poly(A)-containing RNA was estimated as 1100 nucleotides. Number-average length of poly(A)-tracts was 33 nucleotides. 2.5% of total RNA is poly(A)-containing RNA and probably up to 7.5% are non-polyadenylated polydisperse RNA sequences. Saturation hybridization of poly(A)-containing RNA to gap-translated [3H]DNA resulted in 16% of the reactive single-copy DNA to become S1 nuclease resistant. It was found that purified poly(A)-containing RNA represented the entire RNA complexity, i.e. 10 000 different RNA sequences in S. commune. RNA sequences isolated from morphologically different mycelia and from fruiting and non-fruiting mycelia were identical for at least 90%.     

Structure and function of rat liver polysome populations. I. Complexity, frequency distribution, and degree of uniqueness of free and membrane-bound polysomal polyadenylate-containing RNA populations

Free and membrane-bound polysomes were isolated from rat liver in high yields with minimal degradation, cross-contamination, or contamination by nuclear or nonpolysomal cytoplasmic ribonucleoprotein. Poly(A)+ RNA fractions isolated from free and bound polysomal RNA (poly(A)+ RNAfree and poly(A)+ RNAbound) by oligo(dT) cellulose chromatography exhibited number-average lengths of 1,600 and 1,200 nucleotides, respectively, on formamide sucrose gradients. Poly(A)+ RNAfree and poly(A)+ RNAbound contain 9.1 +/- 0.55 and 10.7 +/- 0.50% poly(A) as measured by hybridization to [3H]poly(U) and comprise 2.37 and 1.22% of their respective polysomal RNA populations. Homologous poly(A)+ RNA-cDNA hybridizations revealed that greater than 95% of the mass of poly(A)+ RNAfree and poly(A)+ RNAbound contain nucleotide complexities of about 3.4 x 10(7) and 6.0 x 10(6), respectively. This represents about 20,000 and 5,000 poly(A)+ RNA species of average sizes. Heterologous hybridizations suggested that considerable overlap exists between poly(A)+ RNAfree and poly(A)+ RNAbound sequences that cannot be attributed to cross-contamination. This was confirmed by conducting heterologous reactions using kinetically enriched cDNA populations. Heterologous hybridizations involving poly(A)+ RNA derived from tightly bound polysomes and cDNAfree indicated tha most of the overlapping sequences are not contributed by loosely bound (high-salt releasable) polysomes. The ramifications of these findings are discussed.     

Study of the double-stranded fragments in the poly(A)-containing cytoplasmic RNA of the rat liver

The double-stranded sequences in the poly(A)-containing cytoplasmic RNA (c-dsRNA) hybridize mainly with the repetitive DNA. 70-80% of double-stranded regions in the cytoplasmic poly (A)-RNA are identical to double-stranded regions of heterogeneous nuclear RNA in normal as well as in cortisone-treated rats. The thermal stability of cytoplasmic double-stranded regions is higher in the presence of polyadenylate sequences than in their absence. It is suggested that the double-stranded sequences in the poly (A) +RNA interact with poly (A) stretches and form higher order structures. The thermal stability of c-dsRNA isolated from cortisone-treated rats is higher than that from control rats.     

Complex population of nonpolyadenylated messenger RNA in mouse brain

The complexity of nonadenylated mRNA [poly(A)-mRNA] has been determined by hybridization with single-copy DNA (scDNA) and cDNA. Our results show that poly(A)- and poly(A)+ mRNA are essentially nonoverlapping (nonhomologous) sequence populations of similar complexity. The sum of the complexities of poly(A)+ mRNA and poly(A)- mRNA is equal to that of total polysomal RNA or total mRNA, or the equivalent of approximately 1.7 x 10(5) different sequences 1.5 kb in length. Poly(A)- mRNA, isolated from polysomal RNA by benzoylated cellulose chromatography, hybridized with 3.6% of the scDNA, corresponding to a complexity of 7.8 x 10(4) different 1.5 kb sequences. The equivalent of only one adenosine tract of approximately 20 nucleotides per 100 poly(A)- mRNA molecules 1.5 kb in size was observed by hybridization with poly(U). cDNA was transcribed from poly(A)- mRNA using random oligonucleotides as primers. Only 1-2% of the single-copy fraction of this cDNA was hybridized using poly(A)+ mRNA as a driver. These results show that poly(A)- mRNA shares few sequences with poly(A)+ mRNA and thus constitutes a separate, complex class of messenger RNA. These measurements preclude the presence of a complex class of bimorphic mRNAs [that is, species present in both poly(A)+ and poly(A)- forms] in brain polysomes.     

Isolation and translation in vitro of poly(A)+RNA from the free-living nematode Panagrellus silusiae.

Polysomal RNA was isolated from the free-living nematode Panagrellus silusiae. Passage of this RNA through a cellulose column resulted in the fractionation of the input RNA into poly(A)-RNA (ca. 97.5% of the total) and poly(A)+ RNA (ca. 2.5% of the total). RNase digestion, followed by polyacrylamide gel electrophoresis, revealed that the poly(A)+ RNA contained poly(A) tracts that ranged from 75 to 104 nucleotides in length with a mean value of about 90 residues. There was no evidence of poly(A) sequences in the poly(A)- RNA fraction. Poly(A)+ RNA gave a 25- to 50-fold stimulation (over background) of amino acid incorporation in the wheat germ cell-free protein-synthesizing system. At least 26 proteins were evident after electrophoresis in cylindrical sodium dodecyl sulfate-polyacrylamide gels. Poly(A)-RNA was capable of stimulating protein synthesis in vitro with about five discrete proteins being produced. In summary, the properties of mRNA from a simple organism such as P. silusiae are very similar to those of more complex eukaryotes.     

The putative 15 S precursor of globin mRNA contains a poly (A) sequence

[3H] Uridine or [3H] adenosine pulse-labelled nuclear RNA was isolated from chicken immature red blood cells and separated on denaturing formamide sucrose gradients. RNA of each gradient fraction was hybridized with unlabelled globin DNA complementary to mRNA (cDNA) and subsequently digested by RNAase A and RNAase T1. The experiments revealed two RNA species with globin coding sequences sedimenting 9 S and approx. 15 S, the latter probably representing a precursor of 9 S globin mRNA. A poly (A) sequence was demonstrated in this RNA by two different approaches. Nuclear RNA pulse-labelled with [3H] uridine was fractionated by chromatography on poly (U)-Sepharose. Part of the 15 S precursor was found in the poly(A)-containing RNA. In the second approach 15 S RNA pulse-labelled with [3H]adenosine was hybridized with globin cDNA, incubated with RNAase A and RNAase T1 and subjected to chromatography on hydroxyapatite. The hybrids were isolated and after separation of the strands degraded with DNAase I, RNAase A and RNAase T1. By this procedure poly(A) sequences of approximately 100 nucleotides could be isolated from the 15 S RNA with globin coding sequences. The poly(A) sequence was completely degraded by RNAase T2.     

Polyadenylated RNA from Vicia faba meristematic root cells. Localization and size estimation of the poly (A) segment.

After incubating root apices from two-day-old bean seedlings with [3H] adenine the RNA was extracted from whole cells or polysomes, and the poly (A) sequences were isolated by nuclease digestion followed by poly(U)-Sepharose chromatography. The alterations of the RNA molecules due to the various treatments were monitored by sucrose density gradients. It was found that sequential extraction first at pH 7.6 then at pH 9.0 did not result in a separation between RNA poor in poly(A) sequences and poly(A)-rich RNA. Furthermore chromatography analysis of hydrolysates from nuclease-resistant RNA extracted either at pH 7.6 or pH 9.0 revealed that AMP constituted nearly 95% of the bases and that the poly(A) sequences, about 200 bases, were located at the 3' terminus of the polyadenylated RNA. No size difference was found for the poly(A) segment between the pH-7.6-extracted RNA and that extracted at pH 9.0.     

The poly(A)(+)RNA sequence complexity is also represented in poly(A)(-)RNA in sea-urchin embryos

The extent to which the poly(A)(+)RNA sequence complexity from sea-urchin embryos is also represented in poly(A)(-)RNA was determined by cDNA cross-hybridization. Eighty percent or more of both the cytoplasmic poly(A)(+)RNA and polysomal poly(A)(+)RNA sequences appeared in a poly(A)(-) form. In both cases, the cellular concentrations of the poly(A)(-)RNA molecules that reacted with the cDNA were similar to the concentrations of the homologous poly(A)(+) sequences. Additionally, few, if any, abundant poly(A)(+)mRNA molecules were quantitatively discriminated by polyadenylation, since the abundant poly(A)(+)sequences were also abundant in poly(A)(-)RNA. Neither degradation nor inefficient binding to oligo (dT)-cellulose can account for the observed cross-reactivity. These data indicate that, in sea-urchin embryos, the poly(A) does not regulate the utilization of mRNA by demarcating an mRNA subset that is specifically and completely polyadenylated.     

Polyadenylic acid-containing RNA and its polyadenylic acid sequences newly synthesized in isolated embryonic cells of Xenopus laevis.

Newly synthesized polyriboadenylic acid [poly(A)]-containing RNA and its poly(A) sequences were isolated and characterized in Xenopus embryonic cells. Upon sedimentation analysis, the poly(A)-containing RNA labeled for 30 min showed a very heterogeneous size distribution ranging from 9 to >40 S. After 5 hr of labeling, the profile became much less heterogeneous and the main component was distributed in the 9–28 S region. The average molecular weight of 6.5–7.0 × 10⁵ daltons was calculated for the 5-hr labeled RNA. This poly(A)-containing RNA, comprising about 10% of the total labeled RNA, was metabolically stable and accumulated linearly for 5 hr. Gel electrophoresis of the RNA revealed the presence of little or no free poly(A) sequences. Most of the poly(A) sequences, which were isolated from 30-min labeled poly(A)-containing RNA migrated as a single discrete component approximately 150 nucleotides long. In contrast, they were slightly smaller (130 nucleotides long) and more heterogeneous, when obtained from the poly(A)-containing RNA labeled for 5 hr. From these results, it may be likely that the embryonic poly(A)-containing RNA is similar in size to the steady-state population of the poly(A)-containing RNA reported to occur in vitellogenic oocytes and cultured kidney cells of the same species.     

Differential accumulation of poly(A)+ RNA between virulent and double-stranded RNA-induced hypovirulent strains of Cryphonectria (Endothia) parasitica.

The double-stranded RNA responsible for transmissible hypovirulence in Cryphonectria (Endothia) parasitica was found to affect the accumulation of specific poly(A)+ RNA. Using differential hybridization techniques, two genes were isolated, Vir1 and Vir2, which were specifically expressed as poly(A)+ RNAs in the virulent cells. The highly expressed RNA sequences from these genes were not found in total RNA isolated from either American or European hypovirulent strains, although the genes were present in their genomes. Other virulence- and hypovirulence-specific RNA sequences were also detected. One isolated hypovirulence-specific RNA sequence was expressed in both virulent and hypovirulent cells, but in a two- to fourfold-higher concentration in the hypovirulent cells. The results show that hypovirulence is associated with concurrent changes in a few highly expressed poly(A)+ RNAs, which suggests a specific effect of the double-stranded RNA on fungal gene expression.     

Multiple sequence elements and a maternal mRNA product control cdk2 RNA polyadenylation and translation during early Xenopus development.

Cytoplasmic poly(A) elongation is one mechanism that regulates translational recruitment of maternal mRNA in early development. In Xenopus laevis, poly(A) elongation is controlled by two cis elements in the 3' untranslated regions of responsive mRNAs: the hexanucleotide AAUAAA and a U-rich structure with the general sequence UUUUUAAU, which is referred to as the cytoplasmic polyadenylation element (CPE). B4 RNA, which contains these sequences, is polyadenylated during oocyte maturation and maintains a poly(A) tail in early embryos. However, cdk2 RNA, which also contains these sequences, is polyadenylated during maturation but deadenylated after fertilization. This suggests that cis-acting elements in cdk2 RNA signal the removal of the poly(A) tail at this time. By using poly(A) RNA-injected eggs, we showed that two elements which reside 5' of the CPE and 3' of the hexanucleotide act synergistically to promote embryonic deadenylation of this RNA. When an identical RNA lacking a poly(A) tail was injected, these sequences also prevented poly(A) addition. When fused to CAT RNA, the cdk2 3' untranslated region, which contains these elements, as well as the CPE and the hexanucleotide, promoted poly(A) addition and enhanced chloramphenicol acetyltransferase activity during maturation, as well as repression of these events after fertilization. Incubation of fertilized eggs with cycloheximide prevented the embryonic inhibition of cdk2 RNA polyadenylation but did not affect the robust polyadenylation of B4 RNA. This suggests that a maternal mRNA, whose translation occurs only after fertilization, is necessary for the cdk2 deadenylation or inhibition of RNA polyadenylation. This was further suggested when poly(A)+ RNA isolated from two-cell embryos was injected into oocytes that were then allowed to mature. Such oocytes became deficient for cdk2 RNA polyadenylation but remained proficient for B4 RNA polyadenylation. These data show that CPE function is developmentally regulated by multiple sequences and factors.     

The relationship between polyribosomal and latent membrane-bound messenger ribonucleoprotein particles in Artemia embryos

Polysomal messenger ribonucleoprotein (mRNP) particles from developing Artemia cysts were isolated, characterized and compared with latent membrane-bound mRNP particles isolated from dormant cysts. The polyribosomal mRNP particles sedimented between 25-35 S in a sucrose gradient and had a buoyant density of 1.33 g/cm3 in Cs2So4. Latent particles had a higher sedimentation coefficient and lower buoyant density. The poly(A) + RNA in the two kinds of particles was comparable in size, 10-20 S. The protein composition of the particles, as determined by electrophoresis, was different. Polyribosomal particles contained 9 major and 6 minor proteins; a 72 k poly(A)-associated protein was present. Latent particles were characterized by a complex protein pattern ranging in apparent mol. wt between 14,000-140,000. Some proteins with similar molecular weight and isoelectric point were probably common to both kinds of particles.     

The putative 15 S precursor of globin mRNA contains a poly(A) sequence

[³H]Uridine or [³H]adenosine pulse-labelled nuclear RNA was isolated from chicken immature red blood cells and separated on denaturing formamide sucrose gradients. RNA of each gradient fraction was hybridized with unlabelled globin DNA complementary to mRNA (cDNA) and subsequently digested by RNAase A and RNAase T₁. The experiments revealed two RNA species with globin coding sequences sedimenting at 9 S and approx. 15 S, the latter probably representing a precursor of 9 S globin mRNA.A poly(A) sequence was demonstrated in this RNA by two different approaches. Nuclear RNA pulse-labelled with [³H]uridine was fractionated by chromatography on poly(U)-Sepharose. Part of the 15 S precursor was found in the poly(A)-containing RNA. In the second approach 15 S RNA pulse-labelled with [³H]adenosine was hybridized with globin cDNA, incubated with RNAase A and RNAase T₁ and subjected to chromatography on hydroxyapatite. The hybrids were isolated and after separation of the strands degraded with DNAase I, RNAase A and RNAase T₁. By this procedure poly(A) sequences of approximately 100 nucleotides could be isolated from the 15 S RNA with globin coding sequences. The poly(A) sequence was completely degraded by RNAase T₂.