Comparison of cytoplasmic and nuclear poly(A)-containing RNA sequences in Xenopus liver cells.

Poly(A)-containing RNAs from cytoplasm and nuclei of adult Xenopus liver cells are compared. After denaturation of the RNA by dimethysulfoxide the average molecule of nuclear poly(A)-containing RNA has a sedimentation value of 28 S whereas the cytoplasmic poly(A)-containing RNA sediments slightly ahead of 18 S. To compare the complexity of cytoplasmic and nuclear poly(A)-containing RNA, complementary DNA (cDNA) transcribed on either cytoplasmic or nuclear RNA is hybridized to the RNA used as a template. The hybridization kinetics suggest a higher complexity of the nuclear RNA compared to the cytoplasmic fraction. Direct evidence of a higher complexity of nuclear poly(A)-containing RNA is shown by the fact that 30% of the nuclear cDNA fails to hybridize with cytoplasmic poly(A)-containing RNA. An attempt to isolate a specific probe for this nucleus-restricted poly(A)-containing RNA reveals that more than 10(4) different nuclear RNA sequences adjacent to the poly(A) do not get into the cytoplasm. We conclude that a poly(A) on a nuclear RNA does not ensure the transport of the adjacent sequence to the cytoplasm.     

Subcellular distribution and properties of poly(A)-containing RNA from cultured plant cells.

Cultured sycamore cells rapidly incorporate [3H]uridine or [32P]orthophosphate into rRNA precursors and polydisperse RNA. Mature rRNA accumulates only after a lag period of approximately 40 min. Fractionation of pulse-labelled cells and analysis of the RNA shows that after 30 min the rRNA precursors, together with some polydisperse RNA, are confined to the nucleus. In consequence radioactive polydisperse RNA can be isolated from polyribosomes in the complete absence of labelled rRNA. Approximately 40% of this RNA is retained by an oligo(dT)-cellulose column and by this criterion is judged to contain poly(A) sequences. A smaller proportion of nuclear polydisperse RNA also contains poly(A). The tendency for poly(A)-containing RNA to aggregate complicates molecular weight determinations. Denaturation of poly(A)-containing RNA in 8 M urea prior to gel electrophoresis produces a broad peak of RNA with an average Mr = 10(6). Analysis of the nucleotide composition of total cell poly(A)-containing RNA shows that it contains 41% AMP. Roughly 6% of this RNA is resistant to digestion by ribonuclease A and T1. AMP is the only nucleotide detectable in these fragments. From their mobility during electrophoresis in 8 M urea at 60 degrees C with 5.8-S, 5-S and tRNA as molecular weight markers it is concluded that the poly(A) regions contain an average of 160 nucleotides.     

Purification and tissue-specific expression of casein kinase from the lactating guinea-pig mammary gland

A serine-specific casein kinase, an integral membrane protein of the lactating guinea-pig mammary gland, has been purified from a Golgi-enriched membrane fraction, using a combination of sucrose gradient centrifugation and chromatography on ATP-agarose. The enzyme comprises a polypeptide of estimated Mr 74 000 as judged by sodium dodecyl sulphate/polyacrylamide gel electrophoresis, compared with a monomer Mr of 50 000 as determined by sucrose gradient centrifugation in the presence of 500 mM NaCl and 0.1% Triton X-100. Kinetic studies show that the purified enzyme exhibits kinetic constants distinctly different from the rabbit reticulocyte casein kinases I and II, whilst polyclonal antisera raised against the mammary gland enzyme did not cross-react with soluble liver or reticulocyte protein kinase activities. Immunoblotting and immunocytochemical analyses demonstrate the mammary gland enzyme's apparently unique location in lactating mammary gland tissue. Comparative studies with polyclonal antisera raised against bovine galactosyltransferase, show that casein kinase and galactosyltransferase have a similar intracellular localisation in the lactating mammary gland as judged by immunocytochemistry at the light level, but that casein kinase was unique to mammary gland whereas galactosyltransferase could be found in other tissues. The results extended our earlier observations which suggest a Golgi location for casein kinase, and demonstrate that future studies using this enzyme may well prove advantageous for the study of intracellular mechanisms involved in the biogenesis of organelles, in this instance the Golgi apparatus.     

Size distribution of poly(A)-containing messencer RNA from free polysomes of HeLa cells

The sedimentation properties of pulse-labeled and long-term labeled mRNA from highly purified HeLa cell free-polysomes, selected for poly(A) content by two successive passages through poly(T)-cellulose columns, were analyzed under native and denatured conditions. The sedimentation profile of the mRNA on both sodium dodecyl SO₄-sucrose gradients and formaldehyde-sucrose gradients showed a broad distribution of components with estimated molecular weights ranging from 2 × 10⁵ to 5.5 × 10⁶ daltons and a weight-average molecular weight of 8.5 × 10⁵ daltons.     

'Cap' structures in maize poly(A)-containing RNA.

Poly(A)-containing RNA was isolated from maize embryos by chromatography on columns of oligo(dT)-cellulose and exhaustively digested with ribonucleases T2, T1, and A. Fractionation of the digests by two-dimensional electrophoresis revealed the presence of three 7-methylguanosine-terminated 'cap structures' of the type m7GpppNp.     

Subcellular distribution of total poly (A) -containing RNA and ferritin-mRNA in the cytoplasm of rat liver.

Poly(A)-containing RNA was isolated from rat liver microsomes and from the post-microsomal supernatant fraction. Approximately 15% of total rat liver poly(A)-containing RNA was found to be present in the post-microsomal supernatant. The relative capacity for apoferritin synthesis of each poly(A)-containing RNA preparation was measured in a cell-free system derived from wheat germ. The post-microsomal supernatant fraction was found to be highly enriched with ferritin mRNA and accounted for 40–50% of the total ferritin-mRNA present in the cytoplasm of rat liver.     

Differential accumulation and localization of maternal poly(A)-containing RNA during early development of the ascidian,Styela

The regional distribution of poly(A)⁺ RNA was examined in sections of Styela oocytes and fertilized eggs by in situ hybridization with [³H]poly(U). The nucleus and cytoplasm of previtellogenic oocytes contain equivalent densities of [³H]poly(U) binding sites. The concentration of these sites is reduced in the cytoplasm, but not the nucleus, during vitellogenesis. Consequently, the germinal vesicle (GV) plasm of mature oocytes is characterized by an eightfold elevation in [³H]poly(U) binding activity relative to the surrounding cytoplasm. The distinctive cytoplasmic regions of the mature oocyte do not exhibit differential concentrations of [³H]poly(U) binding sites. Following fertilization which triggers GV breakdown, meiosis, and ooplasmic segregation, the high density of [³H]poly(U) binding sites characteristic of the GV plasm is conserved in the basophilic cytoplasm during its extensive migration and eventual accumulation in the animal hemisphere of the egg. The insensitivity of the [³H]poly(U) binding sites of the basophilic cytoplasm to actinomycin D suggests that they are of maternal origin. It is concluded that maternal poly(A)⁺ RNA is subject to differential accumulation in the GV plasm and its derivative ooplasm during the early development of Styela.     

Changes in the composition of double-stranded sequences of poly(A)-containing RNA in cell cytoplasm during hormonal induction

Double-stranded segments (c-ds) have been studied in the poly(A)+ cytoplasmic rat liver RNA. Duplexes about 40 base pairs long have been shown to be of intermolecular character and originate from the interaction between ss-RNA and complementary regions of the poly(A)-containing RNA molecules. Shorter ds-sequences are, mainly, of intramolecular nature. Double-stranded sequences of different length differ also in their oligonucleotide composition, according to fingerprint analysis data. Under the action of cortisone, only several kinds of double-stranded sequences have been demonstrated to increase in the population of cytoplasmic poly(A)+RNA. The function of ds-regions in the hormonal regulation of gene expression is suggested.     

Fractionation and characterization of rat liver poly(A)-containing RNA.

Three fractions of poly(A)-containing RNA were separated from total rat liver RNA using poly(U)-Sepharose 4B affinity chromatography. The poly(A)-containing RNA fractions were released by thermal elution. Fraction 1, eluted under the mildest conditions, and had poly(A) tracts of approx. 200 AMP units in length which appeared to be associated with poly(U) sequences of 20-50 UMP in length. Fraction 1 appeared to be present mainly in the nucleus and, its size distribution was similar to that of fractions 2 and 3. Fractions 2 and 3 eluted at higher temperatures and were associated mainly with polysomal and microsomal fractions. Poly(U) sequences were absent in fractions 2 and 3 while their poly(A) sequences had a size distribution characteristic of those reported in the mRNA of other organisms.     

Cytoplasmic distribution of pulse-labelled poly(A)-containing RNA,particularly 26 S RNA,during myoblast growth and differentiation

Total poly(A)-containing RNA in different polysomal and supernatant cytoplasmic fractions was analysed after pulse-labelling in dividing myoblasts and fused myotubes. In particular, the peak of 26 S RNA (putative messenger for the large subunit of myosin) is located in a light region of the gradient coinciding with the monosome-trisome fractions prior to fusion, and is found in the heavy polysomes only after fusion. These heavy polysomes are free (i.e. not membrane bound). Treatment of the light part of the polysome gradient with EDTA shows that the 26 S RNA found here does not exist as part of a polysomal complex, but is present as a ribonucleoprotein particle cosedimenting in this region. Previous experiments had indicated that in actively dividing myoblasts 26 S RNA has a relatively short half-life but that it becomes “stable” after the cessation of mitosis just prior to fusion. RNA chase experiments performed in the present study show that the “short-lived” 26 S RNA from dividing myoblasts, which is present as a ribonucleoprotein particle, does not enter the heavy polysomes. In contrast, the more stable 26 S RNA also initially present as a ribonucleoprotein, just prior to and in the early stages of fusion, can be shown by chase experiments to enter the heavy polysomes later in fusion. Hence accumulation of 26 S RNA seems to precede its activation as a messenger.     

A simple method for electrophoretic selection and fractionation of poly(A)-containing RNA and poly(A).

A simple procedure, useful for quantitative and qualitative assays of poly(A)-containing RNA and poly(A), as well as for preparative purposes, is described. Glass-fiber filters with immobilized poly(U), a well-known technique for absorption of poly(A)-containing RNA, is combined with electrophoresis in a gel slab of agarose. In front of each of the two troughs in a gel slab, glass-fiber filters are inserted, one of which is impregnated with poly(U). Two identical RNA samples, e.g., split samples of total RNA from salivary glands of Chironomus tentans, are applied to the troughs and are moved electrophoretically across two different filters. The electrophoresis is conducted under conditions which promote the formation of duplexes between absorbed poly(U) and moving poly(A). While the passage of RNA chains across the control filter may take place essentially freely, RNA molecules that contain poly(A) hybridize with poly(U) fixed in the glass-fiber filter and become trapped there. The difference between resulting gel profiles [pattern of the total RNA minus the pattern of RNA not containing poly(A)] yields the electrophoretic distribution of poly(A)-containing RNA. In addition, poly(A)-containing RNA can be eluted from the poly(U) filter with formamide and subjected to electrophoresis without a subsequent precipitation in ethanol. No measurable quantities of ribosomal RNA or tRNA are retained on the poly(U) glass-fiber filters. The hybridization technique enables a quantitative retention of poly(A) molecules representing a wide range of chain lengths.     

Isolation of poly(A)-containing RNA on synthetic fluorophlogopite (Mica).

Synthetic fluorophlogopite, an aluminosilicate of the same structure as naturally occurring mineral mica in which potassium ions on the basal surface have been replaced by aluminum ions, has the ability to retain polynucleotides irreversibly. This property of Al³⁺-mica was used for irreversible adsorption of poly(U) and subsequent selective adsorption of poly(A)-containing RNA from rabbit reticulocyte polysomes at high salt concentration and its elution by 50% dimethylsulfoxide. The properties of RNA isolated on poly(U)-Al³⁺-mica were studied by sucrose density gradient centrifugation and by stimulation of globin synthesis in an in vitro protein synthesizing system from wheat germ and from Krebs II-ascites cells. The preparation contained 9s RNA species which corresponds to rabbit globin messenger RNA, and under optimal conditions it stimulated protein synthesis more than 100-fold. Polyacrylamide-gel electrophoresis in sodium dodecyl sulfate showed that synthesized product was identical with rabbit globin.     

Differential distribution of poly(A)-containing RNA in the embryonic cells of Oncopeltus fasciatus: Analysis by in situ hybridization with a [3H]poly(U) probe

The regional distribution of poly(A)⁺ RNA was examined in the embryonic cells of the milkweed bug, Oncopeltus fasciatus, by in situ hybridization of histological sections with a [³H]poly(U) probe. As shown by a number of control experiments, this probe interacts specifically with poly(A) sequences preserved in the sections. Using this method, it was shown that labeling of periplasmic and vitellophage nuclei increases markedly early during syncytial blastoderm formation. At this time, label also increases in the vitellophage cytoplasm but not in the cytoplasm surrounding the blastodermal nuclei. Labeling continues to increase in the blastodermal nuclei during cellularization and germ band differentiation without a concomitant accumulation in the blastodermal cell cytoplasm. At the time of germ band invagination, the region of the most intense subcellular labeing shifts from the nucleus to the cytoplasm of the invaginated cells. This shift is not evident in the blastodermal cells which remain at the surface of the egg to become the serosa. In the serosa and the vitellophage energids, labeling then decreases as histogenesis proceeds. Significant labeling of the nuclei and cytoplasm of the invaginated germ band cells continues through germ layer formation. It is concluded that poly(A)⁺ RNA, probably synthesized de novo following oviposition, is subject to differential intracellular distribution in three types of Oncopeltus embryonic cells which may reflect cell-specific patterns of mRNA or poly(A) metabolism.     

Methylated constituents of Aedes albopictus poly (A)-containing messenger RNA.

Poly (A)-containing mRNA prepared from cultured mosquito (Aedes albopictus) cells was found to contain methylated 5'-terminal "caps" as well as internal m6A residues. Both type I [m7G(5')ppp(5')Xmp] and type II [m7G(5')ppp(5')XmpYmp] caps were present, at molar ratio of ca five to one. All four common RNA bases were represented in the second position (Xm) of the caps, adenine being the most abundant and N6-methyladenine being absent. The four bases were also represented in the third position (Ym), but here uracil was the predominant base. There was approximately one internal m6A residue for every three caps. These studies demonstrate that mRNA from an invertebrate source can have a methylation pattern comparable with that of mammalian cells in it complexity.