Cytoplasmic Polyadenylated RNA Research Articles

Correlation between the induction of mouse interferon and the amount of its mRNA.

Injection into Xenopus oocytes of polyadenylated cytoplasmic RNA from mouse C‐243‐3 cells induced with poly(rI) · poly(rC) and DEAE‐dextran resulted in the production of biologically active interferon. The synthesis of interferon by oocytes is linearly related to the amount of mRNA injected. Thus a titre of 160 reference units/oocyte was obtained by injecting 250 ng of total cytoplasmic poly(A)‐containing RNA.The kinetics of interferon production in mouse C‐243‐3 cells induced with poly(rI) · poly(rC) and DEAE‐dextran are characterized by an initial lag of 3–4 h and a burst of interferon secretion between 5–8 h with a maximum accumulation at 7 h. Comparison of the relative amount of interferon mRNA at various time intervals of induction with the cellular synthesis of interferon showed a general correlation. At 9 h after induction, i.e. during the shut off phase of interferon production, the cells contained no detectable amount of interferon mRNA (< 20 units/ml).Attempts to superinduce interferon production in mouse C‐243‐3 cells by using the cycloheximide/actinomycin D protocol as in human fibroblast cells were not successful. α‐Amanitin at concentrations similar to those required to inhibit cellular DNA‐dependent RNA polymerase form II completely inhibited the interferon production when applied during the first 3 h of induction. Addition of α‐amanitin at 3, 5 or 7 h after induction stimulated the subsequent interferon production by 2–4‐fold.

Limited action of micrococcal nuclease on trout testis nuclei generates two mononucleosome subsets enriched in transcribed DNA sequences.

Hybridization experiments show that DNA extracted from two distinct subsets of mononucleosomes (MNI and MN2) generated by a limited action of micrococcal nuclease on trout testis nuclei is enriched approximately 7-fold in sequences that are transcribed into cytoplasmic polyadenylated RNA in trout testis cells. Both subsets of mononucleosomes contain eight core histones, but MNI also possesses one or two molecules of a small, basic, high-mobility-group (HMG) protein H6 [Levy W., B., Connor, W. & Dixon, G. H. (1979) J. Biol. Chem. 254, 609-620], bound to a DNA fragment of 140 base pairs. In contrast, MN2 contains 1 molecule of H1 but no H6, and its DNA length is somewhat longer at 140-190 base pairs. The preferential release of these two subsets of mononucleosomes is correlated with the presence of a second larger HMG protein, HMG-T, in the linker regions flanking both types of mononucleosomes. The HMG-T-containing linker regions appear to be considerably more susceptible to attack by micrococcal nuclease than H1-containing linkers. Cross-reassociation reactions between the DNA from MN1 and MN2 subsets indicate that they share a significant extent of sequence overlap but also that each subset contains specific sequences that are absent in the other subset.

Metabolism of polyadenylated mRNA in growing human lymphocytes

The kinetics of degradation of newly synthesized, cytoplasmic polyadenylated RNA have been examined in normal human lymphocytes stimulated to grow with phytohemagglutinin. A single class of poly(A)-bearing RNA was identified with a half-life of approximately 50 h. In the presence of actinomycin D, the half-life was 5 to 6 h, and virtually no decay of pulselabeled material was detectable after 6 h of chase incubation with cordycepin. These findings contrast sharply with data obtained from other growing human cells used as controls: polyadenylated mRNA in MOLT-4 cells, a cultured line of T lymphocytes, had a half-life of 2 h in the presence of actinomycin D. The stability of poly(A)-containing RNA in stimulated lymphocytes from normal donors is therefore not simply a manifestation of cell proliferation. In normal resting lymphocytes, Berger and Cooper [(1975) Proc. Natl. Acad. Sci. U.S. 72, 3873–3877] reported the existence of 2 classes of polyadenylated mRNA with half-lives of under an hour and greater than 20 h, respectively. Since short-lived poly(A)-bearing mRNA is absent from mitogen-stimulated lymphocytes, the data suggest that stabilization of previously labile poly(A)-bearing RNA is one of many carefully regulated processes accompanying growth induction in normal lymphoid cells.

Heterogeneity and 5′-terminal structures of the late RNAs of simian virus 40

We have investigated the 5′-terminal structures of the late lytic RNAs of simian virus 40 (SV40) by binding the plus strand of small DNA fragments labeled in the 5′-terminal position with 32P to specific regions of cytoplasmic polyadenylated late RNA, extending these “primer” fragments in a 3′ direction with reverse transcriptase, fractionating the extended products on denaturing polyacrylamide gels and performing DNA sequence analyses on the extended products. Extension of a primer bound to the 5′ terminus of the body of 19 S RNA revealed a multiplicity of extended products. RNAs from which these products are derived fall into four classes, the first containing sequences colinear with SV40 DNA and the latter three containing splices which fuse the 5′ terminus of the 19 S body at residue 476 (0.765 map units) to residues 444, 291 and 212, respectively. Of 15 extended products we have analyzed, eight have 3′ termini with the sequence T-A(A). These termini lie at positions 243, 182, 110, 55, and 5189 on the SV40 genome. A number of lines of evidence, including nucleotide sequence analysis of late RNA labeled in vivo and the fact that the principal sequence in SV40 late mRNAs adjacent to capped structures is A-U(U), suggest that the termini at positions 243 and 182 correspond to the 5′ termini of discrete in vivo 19 S RNAs and that stops at the shorter positions may also correspond to the 5′ termini of specific in vivo species of 19 S RNA. An additional extended product was observed with a sequence colinear with SV40 DNA and a 3′ terminus of T-A-A at residue 548 (0.779 map units). It contains sequences complementary to the VP3 but not the VP2 initiation codons of 19 S RNA. For each of the three different gaps we have found in the late 19 S RNAs, as well as four additional gaps we have analyzed in the late 16 S and early 19 S RNAs of SV40, identical di-, tri-or tetranucleotide sequences lie on the ungapped precursor at sites which undergo splicing; these sequences may be involved in determining the specificity of the splicing reaction.

Growth-related fluctuation in messenger RNA utilization in animal cells.

Monkey fibroblasts maintained in culture regulate their levels of intracellular protein throughout the growth cycle by means of variations in the rate of protein biosynthesis. Cytoplasmic mRNA in stationary phase cells was compared to that in exponential phase cells. In stationary phase cells 56% of the cytoplasmic polyadenylated RNA was found in the 40--90S postpolysomal region of sucrose sedimentation gradients, while only 23% was found in this region in exponential phase cells. Analysis of electron micrographs of sectioned exponential and stationary phase cells revealed that this shift in polyadenylated RNA location is accompanied by a loss of polysome-like aggregates of ribosomes. Most if not all of this species of postpolysomal polyadenylated RNA is not being translated by single ribosomes since no detectable amounts of nascent peptide were present in this region. This nonpolysomal polyadenylated RNA is comparable in size to polysomal polyadenylated RNA. The length of the 3'-poly(A) tract was also comparable for these two species. The extent of capping of poly(A)-containing molecules was also comparable for these two species. The template activity of nonpolysomal RNA in a wheat germ extract was comparable to that of polysomal RNA. The peptides produced by these two preparations were of a similar large size. Furthermore, most of the nonpolysomal polyadenylated RNA of stationary phase cells was driven into polysomes in the presence of a low dose of cycloheximide. Therefore, we conclude that the untranslated mRNA that accumulates in stationary phase cells is structurally intact, is fully capable of being translated, and is not being translated due to the operation of a translational initiation block.

Stability of polyadenylated RNA in differentiating myogenic cells.

Three independent methods of measurement showed that cytoplasmic polyadenylated RNA from the differentiating myogenic cell line L8 consists of two main populations with regard to stability, one with a half-life of less than 4 h and the other with a half-life of 17--54 h. Similar results were obtained in the presence and absence of actinomycin D. During the fusion of mononucleated myoblasts into multinucleated fibers, there was an increase in both the steady-state pool of the more stable polyadenylated RNA and the proportion of stable polyadenylated RNA synthesized in pulse labelling.

The capacity of polyadenylated RNA from myogenic cells treated with actinomycin D to direct protein synthesis in a cell-free system.

Cytoplasmic polyadenylated RNA of myogenic cells was shown to decay with biphasic kinetics, suggesting the existence of two main populations of mRNA with respect to stability. In the present study, the stability of mRNA extracted from actinomycin-D-treated cultures of a myogenic cell line was tested by its capacity to direct protein synthesis in the wheat germ cell-free system. The products were analyzed by dodecylsulphate/polyacrylamide gel electrophoresis. All major radioactive bands found in gels used for analyzing the products of the cell-free system directed by polyadenylated RNA extracted from untreated cultures were also found in similar gels containing products of RNA extracted after many hours of application of actinomycin D. The capacity to code for specific protein bands decays with a half-life ranging between 11 and 40 h. No fast-decaying translatable mRNA could be detected by this method. Instead, it was found that during the first 4--6 h following application of actinomycin D, the capacity of RNA to stimulate incorporation of amino acids into total acid-insoluble material increased by 20--30%. The synthesis of specific products increased by up to 100%. The possibility that the fast-decaying polyadenylated RNA or part of it is nontranslatable RNA is discussed.

Partial purification of transcriptionally active nucleosomes from trout testis cells

Mononucleosomes (MN1) enriched in structural non-histone proteins and transcribed DNA sequences were obtained by limited digestion of trout testis nuclei with micrococcal nuclease followed by selective solubilization in 0.1 M NaCl. These monosomes consist of the four inner histones plus stoichiometric amounts of the non-histone protein H6, of the HMG group, complexed with 140 base pairs of DNA. Hybridization experiments indicate that MN1 DNA is enriched in sequences complementary to cytoplasmic polyadenylated RNA.

Gaps and duplicated sequences in the leaders of SV40 16S RNA.

In order to investigate the 5' terminal structural heterogeneity of the 16S size class of SV40 late RNA, we have bound an SV40 DNA fragment labeled at its 5' termini with P32 to the .939-.945 map unit region of late lytic cytoplasmic polyadenylated RNA, used reverse transcriptase to prepare cDNA copies of the 5' termini of this RNA, separated the cDNA products on an 8% polyacrylamide-7 M urea gel and subjected these products to nucleic acid sequence analysis. A number of discrete cDNAs were obtained. Analysis of these cDNAs has suggested the presence of three categories of 16S species all containing the same body extending from residues 1381-2592 (.939-.170 m.u.) but differring in the structure of their leader segments. Members of the first category contain leaders which are colinear with SV40 DNA, have a common 3' terminus at residue 444 and extend varying distances in a 5' direction. The most abundant 16S species contains a leader of 203 nucleotides and is a member of this group. RNAs of the second category contain leaders with an internal gap between residues 211-352. The single RNA comprising the third category contains a leader with a tandem repetition of nucleotides 351-443 at the 3' terminus of its leader.

The dynamics of maternal poly(A)-containing mRNA in fertilized sea urchin eggs

Cytoplasmic polyadenylated RNA with the characteristics of sequestered mRNA exists in the unfertilized sea urchin egg. Following egg activation, the amount of poly(A) doubles, but total RNA content stays constant. Chromatography of the RNA on poly(U)-Sepharose shows that the amount of RNA that bears a poly(A) tract increases slightly (approximately 20–30%) during the 2 hr after fertilization. When a cDNA transcript of the poly(A) + mRNA from 2 hr zygotes is reacted against poly(A) + RNA from either eggs or zygotes, the kinetics of reassociation of the two preparations seem identical; hence the RNA sequences bearing poly(A) are the same in eggs and zygotes. Measurement of the length of the poly(A) tract in eggs and zygotes shows an increase in number average length from about 45 bases to 60 bases. Measurement of tract length of poly(A) in two cell zygotes by adenosine/AMP ratios of radioactive RNA shows that the poly(A) tract of the zygote is solely accounted for by radioactive RNA, indicating extensive turnover of the poly(A). It in concluded that the poly(A) tract in these cells is subject to both lengthening and shortening, with the former predominating in this instance. The increase in poly(A) does not involve polyadenylation of different sequences, but is due to an increase in the number of polyadenylated sequences and the length of the poly(A) tracts that they bear.

Decay of cytoplasmic polyadenylated RNA's labeled with [3H]orotate in rat liver and the effect of the re-utilization of labeled precursor on such decay.

ADVERTISEMENT RETURN TO ISSUEPREVArticleDecay of cytoplasmic polyadenylated RNAs labeled with [3H]orotate in rat liver and the effect of the re-utilization of labeled precursor on such decayRobert E. Moore, James A. Cardelli, and Henry C. PitotCite this: Biochemistry 1977, 16, 13, 3037–3044Publication Date (Print):June 1, 1977Publication History Published online1 May 2002Published inissue 1 June 1977https://doi.org/10.1021/bi00632a035RIGHTS & PERMISSIONSArticle Views13Altmetric-Citations6LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (957 KB) Get e-Alerts Get e-Alerts

Distribution of polyadenylated RNA sequences in the cytoplasm of Drosophila cells

Complementary DNA (cDNA) was synthesized by reverse transcriptase, on a template of total cytoplasmic polyadenylated RNA from Drosophila cells. This cDNA was hybridized to an excess of total cytoplasmic polyadenylated RNA, polysomal polyadenylated RNA and non-polysomal cytoplasmic polyadenylated RNA. Our results show that a significant fraction of the total cytoplasmic polyadenylated RNA sequences are not bound to polysomes. However, the majority of these sequences are present in the population of non-polysomal polyadenylated cytoplasmic RNA molecules.

Changes in the Sequence Diversity of Polyadenylated Cytoplasmic RNA during Testis Differentiation in Rainbow Trout (Salmo gairdnerii)

We have compared the sequence diversity of polyadenylated cytoplasmic RNA derived from naturally maturing trout testis at three different stages of spermatogenesis, by performing hybridization experiments between cDNA synthesized on a template of polyadenylated RNA and a vast excess of polyadenylated RNA. Polyadenylated RNA from early testis has a base sequence complexity of 7.2x10(9) daltons. As testis maturation proceeds, there is a decrease in the complexity of polyadenylated mRNA sequences in testis cells and the relative abundance of individual mRNAs varies over a more narrow range. Heterologous hybridization reactions demonstrate that a substantial fraction of polyadenylated RNA sequences present in early testis cytoplasm is absent from late testis cytoplasm. Nevertheless, all the sequences of late testis mRNA are represented in the population of early testis mRNA molecules.

Content of N-6 methyl adenylic acid in heterogeneous nuclear and messenger RNA of HeLa cells

With the aid of a suitable thin layer chromatographic procedure, the N-6 methyl adenylic acid (m6A), content of a variety of 32P labeled RNA species from HeLa cells has been measured. Poly(A)-containing (poly(A)+) cytoplasmic RNA has on the average one m6Ap per 800 to 900 nucleotides. This value is independent of the length of the molecules. The proportion of m6Ap in poly(A)+ cytoplasmic RNA does not change between 4 and 18 hours of labeling with 32P, suggesting that the majority of the messenger RNA molecules may have a similar level of internal methylation regardless of their half-life. The non-polyadenylated, non-ribosomal cytoplasmic RNA fraction sedimenting from 10S TO 28S is less methylated with approximately one m6A per 2,700 nucleotides. Heterogeneous nuclear RNA molecules (DMSO treated) which sediment from 28S to 45S have approximately one m6Ap per 3,000 nucleotides. The hnRNA molecules sedimenting from 10S to 28S have one m6Ap per 1,800 nucleotides. Poly(A)+ nuclear RNA is enriched in m6A, containing 1 residue of m6A per 700 to 800 nucleotides, a value close to that obtained for the polyadenylated cytoplasmic RNA.

Renaturation kinetics of cDNA complementary to cytoplamic polyadenylated RNA from rainbow trout testis. Accessibility of transcribed genes to pancreatic DNase.

We have determined the fraction of polyadenylated cytoplasmic RNA from trout testis complementary to unique and repetitive DNA. Some 21% of the cDNA probe representative of this RNA population renatures with rapid kinetics, characteristics of repetitive sequences. The major proportion of the cDNA renatures with unique sequence DNA. Experiments with fractionated cDNA probes allow us to conclude that, in trout testis, the most abundant polyadenylated mRNAs are not preferentially transcribed from repetitive DNA, as it has shown to be the case in two eukaryotic cell lines. Treatment of trout testis nuclei with DNase I, under conditions in which 10% of the total DNA is digested, preferentially depletes the DNA of sequences being transcribed into polyadenylated mRNA. These data confirm the results of H. Weintraub and M. Groundine [(1976) Science 193, 848-856] and those of A. Garel and R. Axel [(1976) Proc. Natl. Acad. Sci. U.S.A. 73, 3966-3970] and suggest that the conformation of DNA in the active genes of chromatin is such that it is more susceptible to digestion by DNaseI.

Localization of sequences specifying messenger RNA to light-staining G-bands of human chromosomes

Total cytoplasmic polyadenylated RNA was isolated from the human lymphocyte cell line Wil2 by oligo(dT)-cellulose chromatography. Tritiated complementary DNA (cDNA) was transcribed from the RNA and used as a probe for in situ hybridization to metaphase chromosomes. The majority of G-negative or lightly staining bands were found to be preferential sites of hybridization.

Location of messenger specifying sequences in mammalian chromosomes

In situ hybridization of complementary DNA (cDNA) synthesized from total cytoplasmic polyadenylated RNA isolated from Chinese hamster cells was employed to investigate the distribution of messenger specifying sequences on mammalian chromosomes. The kinetics of cDNA-nuclear DNA annealing indicate that about 85% of the cDNA represents sequences which are transcribed from non-repetitive DNA sequences. When cDNA is hybridized back to its template RNA, the reaction kinetics show that more than 60% of the poly(A) RNA is at least 104 times more complex than rabbit globin mRNA. In situ hybridization of cDNA to Chinese hamster cells fixed on slides shows no significant clustering of silver grains on interphase nuclei. On metaphase chromosomes the majority of silver grains are localized in euchromatic areas. It appears that all euchromatic segments have similar grain densities. Chromosomes 1 and 2, which have relatively little heterochromatin, do not have a higher grain density than the other chromosomes. However, the Y chromosome, which is entirely heterochromatic, contains only about 1/3 the grain density of the chromosomes 1 or 2. — When the cDNA, which anneals only to the high abundancy class of poly(A) RNA was fractionated and hybridized in situ to Chinese hamster chromosomes, the distribution of silver grains is localized in the euchromatic areas. The Y chromosome and the heterochromatic arm of the X chromosome contain less grains; telomeres of some autosomes have higher grain densities. The oligo-(dT) primer in cDNA did not affect the results of this study since no grains are found when 3H-poly(dT) was used as probe for in situ hybridization. The majority (>90%) of the grains could be blocked by competition with excess repetitive DNA in the hybridization reaction, indicating that the in situ hybridization involved predominantly repetitive sequences.

Genome location of polyadenylated transcripts of herpes simplex virus type 1 and type 2 DNA

The genome location of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) polyadenylated RNA has been studied by hybridization of herpesvirus-specific nuclear and cytoplasmic polyadenylated RNA to the Eco R, restriction enzyme fragments of herpesvirus DNA, which have been separated by agarose gel electrophoresis and transferred to nitrocellulose paper. For both HSV-1 and HSV-2 DNA, the nuclear and the cytoplasmic polyadenylated RNA are distributed on all the Eco R I fragments of the homologous DNA. In a heterologous system, total polyadenylated RNA from HSV-1-infected cells was annealed to the Eco R I fragments of HSV-2 DNA and vice versa. HSV-2 polyadenylated RNA hybridized to all fragments of HSV-1 DNA, and HSV-1 polyadenylated RNA hybridized to all but three of the fragments of HSV-2 DNA. These data indicate that the DNA base sequence homology between the two herpesvirus strains is dispersed throughout the genome and is not located in a single contiguous block.

Abundancy and Diversity of mRNA Sequences in Human Leukocytes

The messenger RNA populations in two haematopoietic tissues were compared with respect to number of different sequences present and their relative abundancies. This was accomplished by hybridising complementary DNA to the cytoplasmic polyadenylated RNA from which it was transcribed and to heterologous polyadenylated RNA. The hybridisation kinetics were essentially the same in the homologous hybridisation. However, in heterologous hybridisation some differences were observed in the high abundancy messengers.

Nucleo-cytoplasmic relationships of high-molecular-weight ribonucleic acid, including polyadenylated species, in the developing rat brain.

The metabolism of high-molecular-weight RNA in the nuclear and cytoplasmic fractions of newborn and adult rat brain was investigated after the intracranial administration of [32P]Pi. In young brain, a considerable proportion of the newly synthesized radioactive RNA is transferred to the cytoplasm, in contrast with the adult brain, where there appears to be a high intranuclear turnover. Electrophoretic analysis of the newly synthesized RNA showed that processing of the rRNA precursor to yield the 28S and 18S rRNA may be more rapid in the adult than in the young, although most of the adult rRNA in the nucleus is not transferred to the cytoplasm. In young brain, processing is probably tightly coupled to transport of rRNA into the cytoplasm, so that 28S and 18S rRNA are not subjected to possible degradation within the nucleus. Polyadenylated RNA turns over in concert with high-molecular-weight RNA in the nuclei of the adult rat brain. In the cytoplasm the polyadenylated RNA has a higher turnover rate relative to rRNA. In the young brain the polyadenylated RNA is transferred to the cytoplasm along with rRNA, although polyadenylated RNA is transported into the cytoplasm at a faster rate. The nuclear and cytoplasmic polyadenylated RNA species of young brain are larger than their corresponding adult counterparts. These results suggest that there are considerable changes in the regulation of the nucleo-cytoplasmic relationship of rRNA and polyadenylated RNA during the transition of the brain from a developing replicative phase to an adult differentiated and non-dividing state.