Polyribosomal Poly Research Articles

The nuclear MicroSpherule protein 58 is a novel RNA-binding protein that interacts with fragile X mental retardation protein in polyribosomal mRNPs from neurons

The fragile X syndrome, the leading cause of inherited mental retardation, is due to the inactivation of the fragile mental retardation 1 gene (FMR1) and the subsequent absence of its gene product FMRP. This RNA-binding protein is thought to control mRNA translation and its absence in fragile X cells leads to alteration in protein synthesis. In neurons, FMRP is thought to repress specific mRNAs during their transport as silent ribonucleoparticles (mRNPs) from the cell body to the distant synapses which are the sites of local synthesis of neuro-specific proteins. The mechanism by which FMRP sorts out its different mRNAs targets might be tuned by the intervention of different proteins. Using a yeast two-hybrid system, we identified MicroSpherule Protein 58 (MSP58) as a novel FMRP-cellular partner. In cell cultures, we found that MSP58 is predominantly present in the nucleus where it interacts with the nuclear isoform of FMRP. However, in neurons but not in glial cells, MSP58 is also present in the cytoplasmic compartment, as well as in neurites, where it co-localizes with FMRP. Biochemical evidence is given that MSP58 is associated with polyribosomal poly(A)+ mRNPs. We also show that MSP58, similar to FMRP, is present on polyribosomes prepared from synaptoneurosomes and that it behaves as an RNA-binding protein with a high affinity to the G-quartet structure. We propose that this novel cellular partner for FMRP escorts FMRP-containing mRNP from the nucleus and nucleolus to the somato-dendritic compartment where it might participate in neuronal translation regulation.

A novel gene is transcribed in the chicken alpha-globin gene domain in the direction opposite to the globin genes.

A novel gene transcribed in the direction opposite to that of the globin genes was found in the chicken alpha-globin gene domain. Northern hybridisation with single-stranded riboprobes revealed that a 4.5-kb poly(A)+ RNA is transcribed in antisense polarity with respect to the globin genes. The transcription unit encoding this RNA seems to overlap the entire cluster of alpha-globin genes and extends at least 15 kb upstream from pi, the first of the alpha-globin genes. This new transcript shows partial sequence homology with that encoded by the human "-14" gene. An oligonucleotide based on part of a restriction fragment of chicken DNA that is 80% homologous to exon 4 of the human "-14" gene hybridises with a 4.5-kb RNA molecule. In situ hybridisation of globin-antisense probes, that detect polyribosomal mRNAs of 1.7 and 2.5 kb on Northern blots, shows these "antisense" transcripts to be present in the cytoplasm. The 4.5-kb RNA is absent in polyribosomal poly(A)+ RNA and may, hence, represent a nuclear pre-mRNA transcribed from the chicken gene that is homologous to the human "-14" gene. The expression of this gene is not specific to erythroid cells; analogous transcripts were also detected in poly(A)+ RNA extracted from a chicken lymphoblastoma cell line (HP50). Taken together, these data allow us to postulate the existence in the chicken genome of a novel gene, for which we suggest the name "ggPRX" in analogy to the murine mProx1, a gene identified in the upstream region of the alpha-globin gene domain in mice.

Transcriptional activity at the 3′ end of the actin gene at 5C on the X chromosome of Drosophila melanogaster

Polyribosomal poly(A) + RNA from stage-specific embryos of Drosophila melanogaster was analyzed by Northern hybridizations to determine the transcription at the 5C actin gene (act5C) and to establish whether these mRNAs are used in translation. A 3′ end probe, which contains 30 nucleotides of the actin coding sequence as well as 3′-end-adjacent sequence, hybridizes in addition to the act5C transcript to a smaller transcript 450 nucleotides long. Hybridizations with the 3′ end strand-specific probes show that this transcript has the same orientation as the actin gene. This transcript is temporally coexpressed with the act5C gene over most of the developmental stages examined. S 1 nuclease mapping and primer extension experiments place its 5′ end about 20 nucleotides upstream from the actin translation termination signal. Furthermore, the transcript appears to translate in an in vitro translation system into a protein of 7.4 kDa, as expected from the size of the open reading frame. The data suggest, but do not prove, that the 3′-end-transcribed but untranslated region of act5C may be involved in actin gene expression.

Microheterogeneity in a cytochrome P-450 multigene family

The cytochrome P-450-mediated mixed-function monooxygenase system is one of the major enzyme systems involved in the detoxification of foreign compounds, including many drugs and carcinogens (Hietanen, 1982). In contrast, some xenobiotics can be activated by cytochrome P450 to compounds that can bind covalently to cellular macromolecules resulting in deleterious effects such as toxicity, mutation and neoplasia (Gelboin, 1980). Any one organism usually possesses a multiplicity of cytochromes P450, which differ from each other in molecular weight, amino acid composition, substrate and reaction specificity, and immunochemical properties (Nebert & Negishi, 1982). The cytochromes P-450 are inducible by a wide variety of foreign compounds, the specific variant induced depending on the compound. We have shown that PB induces a rat-liver cytochrome P450 variant (PB P-450) of M , 52000 by approximately 40fold (Phillips et al., 1981, 19830). The induction is mediated by a 20-fold increase in the translatable mRNA coding for PB P-450 (Shephard et al., 1982). In order to investigate further the mechanism of the induction process we have cloned cDNAs coding for PB P450 (Phillips et al . , 19836). A cDNA library representative of total liver polyribosomal poly(A)+ RNA isolated from PB-treated rats was established. The library was screened by differential colony hybridization to 32P-labelled cDNAs synthesized from poly(A)+ RNA extracted from the livers of PBor NF-treated or untreated rats. Colonies giving a stronger signal with 'PB cDNA' than with either 'NF cDNA' or 'control cDNA' were subjected to hybridselected translation and immunoprecipitational analysis with anti-(PB P-450) serum. A plasmid, pP-450(1), coding for PB P-450, was isolated and used as a molecular hybridization probe. Northern blot and solution hybridization analysis showed that PB P-450 is encoded by a single mRNA size-class of 2 I00 nucleotides that is induced 20-fold by phenobarbital treatment and decreased 3-fold by P-naphthoflavone (Phillips et al . , 19836,~).

Construction and characterization of a plasmid containing complementary DNA to mRNA encoding the N-terminal amino acid sequence of the rat glutathione transferase Ya subunit.

Free polyribosomal poly(A)-containing RNA isolated from normal rat liver was used to prepare a complementary DNA plasmid library in the Pst1 site of the plasmid pAT153 . A plasmid pGSTr155 complementary to mRNA coding for a glutathione transferase Ya subunit was selected by differential hybridization in situ and preliminary characterization was performed by hybrid-selected mRNA translation, immunoprecipitation and polyacrylamide-gel electrophoresis of the product synthesized in vitro. The nucleotide sequence of the complementary DNA contained within pGSTr155 was determined and shown to contain a single open reading frame corresponding to the first 129 amino acids of the N-terminus of the Ya subunit and a further 63 nucleotides upstream of the initiating methionine codon.

Cloning and sequence analysis of a rat liver cDNA coding for a phenobarbital-inducible microhetero-geneous cytochrome P-450 variant: regulation of its messenger level by xenobiotics

A rat liver cDNA library was prepared from total polyribosomal poly(A)+RNA extracted from phenobarbital-treated animals. A cDNA clone coding for a phenobarbital-inducible cytochrome P-450 (PB P-450) was identified by differential colony hybridization to cDNAs synthesized from liver poly(A)+ RNAs isolated from phenobarbital-treated rats for positive selection and cDNAs from either untreated rats or β-naphthoflavone-treated rats as negative controls, followed by hybrid-selected translation and analysis of the translation products by immunoprecipitation. As the cloning and screening strategies involve no prior enrichment for specific mRNAs, they also permit the identification of sequences coding for phenobarbital-induced proteins other than cytochromes P-450. This relatively straightforward approach is generally applicable to the molecular cloning of sequences coding for other inducible cytochromes P-450. Nucleic acid sequencing data indicated that the cloned PB P-450 cDNA codes for a cytochrome P-450 variant [designated P-450e(U.C.)] that is very similar, but not identical, to P-450e. Sequence analysis of the section of cDNA specifying the 3'-non-coding region of the mRNA revealed that it lacked the usual poly(A) addition site signal sequence but contained three inverted repeat structures. Solution hybridization analysis demonstrated that PB P-450 mRNA is increased 20-fold by phenobarbital treatment and decreased 3-fold by β-naphthoflavone treatment.

Transcriptional and post-transcriptional regulation in duck erythroblasts

In order to study the relative importance of transcriptional and post-transcriptional regulation in duck erythroblasts, unique sequence pre-mRNA and mRNA complexity was assayed at four levels. Hybridization and Cr 0t analysis of total nuclear RNA with nick-translated unique sequence genomic DNA showed that of the order of 10% of the duck genome is transcribed; about one-half of the spectrum of RNA sequence formed decays within 40 min in the nucleus. The full-sequence complexity of cytoplasmic mRNA amounts to less than 0.15% of that of the genomic DNA. To assay for cytoplasmic controls, messenger RNA was isolated from polyribosomes and from repressed mRNP free of ribosomes; these two compartments contain 88 and 12%, respectively, of the polyadenylated mRNA. Both mRNA populations were either transcribed into cDNA by RNA-dependent DNA-polymerase, or translated in the nuclease-treated reticulocyte lysate. The kinetic complexity of the polyribosomal cDNA hybridizing back to its template shows the presence of about 200 mRNA species in three abundancy classes. The α- and β-globin mRNAs (70% in mass of polyribosomal mRNA) are represented by about 3800 copies/cell, 7 other mRNA types by about 90 copies/cell, and 202 mRNA by 2 copies/cell. The repressed free cytoplasmic mRNP contains 1400 different mRNAs, also in three abundancy classes. The α- and β-globin mRNAs (44% in mass) are represented by about 285 copies, there are 19 mRNA types with 7 copies, and 1380 mRNAs represented by less than 1 copy per cell. These data indicate that about 1200 mRNA species are not represented at all in polyribosomal mRNA. Heterologous cross-hybridization of cDNA transcribed from free mRNP message with polyribosomal mRNA suggests that the intermediate frequency mRNA classes are very differently represented in the two compartments. The qualitative difference of these mRNA populations is also demonstrated by the analysis of the products of in vitro translation by two-dimensional electrophoresis. Among the almost fully repressed mRNA is found, e.g., the mRNA for the polyribosomal poly(A)-binding protein. In conclusion the transcribed unique sequence DNA is of a complexity more than 100 times larger than that of total cytoplasmic mRNA; at an estimated mRNA/IVS ratio in pre-mRNA of 1:5 this amounts to a selection for transport to the cytoplasm of at best 1 mRNA molecule in 20. The repressed mRNA population in the cytoplasm is at least sevenfold more complex than the translated one. Individual equilibria of activity exist for each mRNA which allow almost full translation, and thus expression of some genes, but condition also full repression at post-transcriptional and cytoplasmic levels of a majority of the activated genes. These data show the higher importance of post-transcriptional controls compared to transcriptional regulation (selection of only 1 in 10) in a highly differentiated cell and are discussed in relation to the Cascade Regulation Scheme.

Age-dependent gene induction in quail oviduct. XV. Alterations of the poly(A)-associated protein pattern and of the poly(A) chain lenght of mRNA

The effect of ageing on polyadenylate [poly(A)] metabolism of mRNA was studied in two age groups of female quails: mature (250–320 days' old) and senescent animals (3–3.5 years' old). In introductory experiments it was shown that poly(A)-associated proteins can not be recovered from cytosol by affinity chromatography. We isolated the poly(A)-associated proteins from polyribosomal poly(A)-ribonucleoprotein complex [poly(A)-RNP] and radioactively labeled them with dansyl chloride. Three main protein species were identified with molecular masses of 48 000 (P48), 35 000 (P35) and 24 000 (P24). During ageing the percental portion of P48 in poly(A)-RNP from liver (mitotic tissue) and from oviduct or heart (post-mitotic tissue) is reduced at the expense of P35 and P24. Quantitative analyses revealed that the amount of poly(A)-RNP in the different organs decreases significantly with age if the values are based on DNA. The protein content in poly(A)-RNP was found to be reduced especially in post-mitotic tissue. From this finding we assume that the number of poly(A)-associated protein molecules per poly(A) stretch drops from approximately 4.7 molecules (mature oviduct) to 1.9 molecules (senescent oviduct). Control experiments revealed that free, non-polyribosomal poly(A)RNP accounts only for 10% of total poly(A)-RNP. The size of the poly(A) segment of mRNA decreases with age. After labeling with [ 3H]dimethylsulfate, the poly(A) stretch from mature oviduct was found to consist mainly of 120–180 AMP units, and those from mature liver and mature heart of 110 and 100, respectively. In organs from senescent animals the percentage of shorter poly(A) stretches is enlarged; on the average, poly(A) 70 chains were detected. These results support the assumption that age-dependent changes occur also on the post-transcriptional level during the maturation steps of poly(A)(−) hnRNA to poly(A)(+) mRNA.

The polyadenylated RNA directing the synthesis of the rat myelin basic proteins is present in both free and membrane-bound forebrain polyribosomes.

Free and membrane-bound polyribosomes were isolated from the forebrain of actively myelinating 24-day-old rats. The poly(A)+ RNA (polyadenylated RNA) extracted from both fractions was translated in vitro in reticulocyte lysates [Hall & Lim (1981) Biochem. J. 196. 327-336] in the presence or absence of a heterologous microsomal membrane fraction from dog pancreas. The rat myelin basic proteins synthesized in vitro were isolated by CM-cellulose chromatography and by immunoprecipitation with purified anti-(myelin basic protein) antibody. The large (mol.wt. 18 500) and small (mol.wt. 16 000) myelin basic proteins were translational products of poly(A)+ RNA from both free and membrane-bound polyribosomes. The identity of the myelin basic proteins was verified by analysis of peptides generated by the cathepsin D digestion of the immunoprecipitated proteins synthesized in vitro, in comparison with authentic rat myelin basic proteins. Although several other translational products of membrane-bound polyribosomal poly(A)+ RNA were modified when microsomal membranes were present during translation, molecular weights of the myelin basic proteins themselves were unchanged. The myelin basic proteins synthesized in vitro also did not differ significantly in size from the authentic myelin basic proteins, indicating that these membrane proteins are unlikely to be synthesized as substantially larger precursor molecules. The presence of the specific mRNA species on both free and membrane-bound polyribosomes is compatible with the extrinsic location of the myelin basic proteins on the cytoplasmic surface of the myelin membrane.

The polyribosomal poly(A)-binding protein is highly conserved in vertebrate species: Comparison in duck, mouse and rabbit

In eukaryotic cells mRNA is present in the form of messenger ribonucleoprotein complexes (mRNP particles). When polyribosomes, from a variety of cell types and tissues, are dissociated with EDTA or puromycin, the released mRNA is found tightly associated with 3 major proteins o fM r ~ 43 000-52 000 and 72 000-78 000 (review [1]). The larger of these proteins was shown to interact with the 3'-polyadenylate sequence of mRNA [2,3] since it may be recovered with poly(A) following digestion of polyribosomes or purified mRNP with ribonucleases A and T1. From density measurements of the poly(A) RNP particle it may be estimated that ~4 5 copies of this protein are associated with the average-sized poly(A) fragment [4]. The affinity of the poly(A)-binding protein for poly(A) is very high since it resists not only 0.5 M KC1 [1] but also centrifugation through Cs2SO4 density gradients [5,6] and affords protection ofpoly(A) against ribonucleases and nucleases [7 10]. Furthermore, this protein binds not only poly(A) but also regions of poly(A) adjacent and non-adjacent to poly(A) [81. A protein of the same M r as the polyribosomal poly(A)-binding protein has been observed to be associated with the 3'-poly(A)region of nuclear pre-

Developmental changes in the composition of polyadenylated RNA isolated from free and membrane-bound polyribosomes of the rat forebrain, analysed by translation in vitro.

Free and membrane-bound polyribosomes were isolated from the rat forebrain during its development. Polyadenylated RNA [poly(A)+ RNA] was isolated from both fractions, by using oligo(dT)-cellulose chromatography, and its composition studied by translating the poly(A)+ RNA in vitro in reticulocyte lysates. Electrophoretic analysis of the translation products showed that both free and membrane-bound polyribosomal poly(A)+ RNA gave many common components, but that there were also distinct differences in the protein composition of the products of the two fractions. Several proteins, of mol.wts. 39 000, 37 000, 31 000, 27 000 and 17 000, appeared to be products predominantly of free polyribosomal poly(A)+ RNA, whereas others, of mol.wt. 47 000, 33 000, 24 000 and 21 000 were specific to the membrane-bound polyribosomal poly(A)+ RNA fraction. More developmental changes were observed in the translational products of the membrane-bound poly(A)+ RNA fraction. Proteins of mol.wts. 33 000 and 21 000, which were predominant components of the translational products of this fraction when isolated from 10-day and older rats, were not present in translational products derived from preparations isolated from 3-day-old rats. The developmental appearance of these proteins as translational products of the membrane-bound poly(A)+ RNA suggests the appearance of new mRNA species. These transcriptional changes are discussed in relation to processes involved in brain differentiation, including myelination.

Characterization of a poly(A) +RNA-containing structural component directly associated with cytoplasmic membranes in dormant Artemia cysts

Poly(A) +RNA-containing material was extracted from the purified cytoplasmic membranes of dormant Artemia cysts by treatment with mild detergents. Sedimentation analysis of the extracts showed a predominant poly(A)-containing fraction at 40 S, associated with about 6% of the extracted proteins. Only limited amounts of poly(A)-containing material were found in the heavier fractions. Poly(A) +RNA extracted from the 40-S fraction sedimented around 14 S. The poly(A)-containing 40-S structures could be purified by treatment with non-ionic or zwitterionic detergents followed by resedimentation in sucrose gradients in the presence or absence of detergent. When the 40-S fraction was analyzed by isopycnic centrifugation in Cs 2SO 4 gradients, the main part of the poly(A)-containing material banded at a density of 1.27 g/ml. Electron-microscopic examination of this fraction revealed circular or slightly bullet-shaped profiles measuring 17–26 nm. When the 40-S fraction had been submitted to mild RNAase treatment prior to density gradient centrifugation, the material was displaced towards lower density and became less distinct. Purified 40-S particles showed a complex protein pattern not very similar to that of polyribosomal poly(A) +RNA-containing particles from developing embryos, but with components in common with unfractionated membranes. The particles also contained some lipids. The experiments indicate that a major part of the membrane-bound, latent poly(A) +RNA in dormant Artemia cysts occurs in the form of relatively uniform, detergent- and Cs 2SO 4-resistant structures, independent of ribosomes, but intimately associated with membrane components.

Changes in polyribosomes and poly(A)-containing polyribosomal RNA in the uterus of the ovariectomized rat in response to oestradiol-17 beta treatment.

Polyribosome formation and the characteristics of polyribosomal poly(A)-containing RNA from uteri of ovariectomized rats responding to a single dose of oestradiol-17 beta was investigated. The mean proportion of polyribosomes in the atrophic uterus was 65%. In response to 10 micrograms of oestradiol-17 beta/100 g body mass, the amount of polyribosomes increased to 88% 24 h after stimulation. Thereafter the proportion of polyribosomes decreased to a value of 48% at 72h. The pattern of amino acid incorporation in oocytes from Xenopus laevis injected with these polyribosomes was similar to the changes in polyribosome formation and degradation. The polyribosomal poly(A)-containing RNA from the controls consisted of a heterogeneous population of RNA with sedimentation values between 5S and 25S. The hormone stimulation resulted in an increase in both the amount and the size (13S to 35S) of the RNA.

Studies on the intracellular segregation of polyribosome-associated messenger ribonucleic acid species in the lactating guinea-pig mammary gland.

1. Free and membrane-bound polyribosomes were isolated and the associated mRNA species characterized by cell-free protein synthesis, RNA-complexity analysis and polyribosome run-off in vitro. 2. Of the recovered polyribosomal RNA 85% was associated with membrane-bound polyribosomes and contained 87--93% of the total milk-protein mRNA species as assessed by cell-free protein synthesis or RNA-complexity analysis. 3. RNA-complexity analysis showed that the abundant (milk-protein mRNA assumed) species constituted 55% of the post-nuclear poly(A)-containing RNA population, the remainder consisting of a moderately abundant population (18%) and a low abundance population (27%). Calculations suggest that each population contained up to 2, 48 and 5000 different species respectively. 4. RNA-complexity analysis of the free polyribosomal poly(A)-containing RNA demonstrated that all the species in the post-nuclear fraction were present, though in different proportions, the abundant, moderately abundant and low-abundance groups representing 38, 30 and 32% of this population. 5. RNA-complexity analysis of the membrane-bound polyribosomal poly(A)-containing RNA revealed a more limited population, 72% consisting of the abundant (milk-protein mRNA) species, and 28% a population of up to 900 RNA species. 6. Polyribosome run-off confirmed that milk-protein mRNA was associated with the membrane-bound and free polyribosomes, but represented only a small fraction of the total protein synthesized by the latter. 7. Comparative analysis of milk proteins synthesized in mRNA-directed cell-free systems, or by run-off of free and of membrane-bound polyribosomes, is consistent with the interpretation that in vivo the initiation of protein synthesis occurs on free polyribosomes, followed by the attachment of a limited population to the endoplasmic reticulum. After attachment, but before completion of peptide synthesis, the detachable N-terminal peptide sequence of one of these(pre-alpha-lactalbumin) is removed. 8. The results are discussed in terms of the mechanisms involved in the intracellular segregation of mRNA species in the lactating guinea-pig mammary gland.

The proportion of ribosomes active in protein synthesis and the content of polyribosomal poly(A)-containing RNA in adult and senescent rat livers.

The proportion of ribosomes active in protein synthesis and the content of polyribosomal poly(A)-containing RNA are unchanged when adult and senescent rat livers are compared. Therefore, the decreased protein synthesizing activity of senescent liver microsomes observed previously is not due to a deficiency in total mRNA content.

Epstein-Barr Virus-Specific RNA III. Mapping of DNA Encoding Viral RNA in Restringent Infection

Namalwa and Raji cells, originally obtained from a Burkitt tumor biopsy, grow as continuous cell lines in vitro and contain the Epstein-Barr virus (EBV)-related nuclear antigen EBNA (B. M. Reedman and G. Klein, Int. J. Cancer 11:499-520, 1973) and RNA homologous to at least 17 and 30% of the EBV genome, respectively (S. D. Hayward and E. Kieff, J. Virol. 18:518-525, 1976; T. Orellana and E. Kieff, J. Virol. 22:321-330, 1977). The polyribosomal and polyadenylated [poly(A)+] RNA fractions of Namalwa and Raji cells are enriched for a class of viral RNA homologous to 5 to 7% of EBV DNA (Hayward and Kieff, J. Virol. 18:518-525, 1976; Orellana and Kieff, J. Virol. 22:321-330, 1977). The objective of the experiments described in this communication was to determine the location within the map of the EBV genome (D. Given and E. Kieff, J. Virol. 28:524-542, 1978) of the DNA which encodes the viral RNA in the poly(A)+ and non-polyadenylated [poly(A)-] RNA fractions of Namalwa cells. Hybridization of labeled DNA homologous to Namalwa poly(A)+ or poly(A)- RNA to blots containing EcoRI, Hsu I, or Hsu I/EcoRI double-cut fragments of EBV (B95-8) or (W91) DNA indicated that these RNAs are encoded by DNA contained primarily in the Hsu I A/EcoRI A and Hsu I B/EcoRI A fragments and, to a lesser extent, in other fragments of the EBV genome. Hybridizations of Namalwa poly(A)+ and poly(A)- RNA in solution to denatured labeled EcoRI A or B fragments, Hsu I A, B, or D fragments, and Hsu I A/EcoRI A or Bam I S fragments and of Raji polyribosomal poly(A)+ RNA to the EcoRI A fragment indicated that (i) Namalwa poly(A)+ RNA is encoded primarily by 6 x 10(5) daltons of a 2 x 10(6)-dalton segment of DNA, Bam I S, which is tandemly reiterated, approximately 10 times, in the Hsu I A/EcoRI A fragment and is encoded to a lesser extent by DNA in the Hsu I B, EcoRI B, and Hsu I D fragments. Raji polyribosomal poly(A)+ RNA is encoded by a similar fraction of the EcoRI A fragment as that which encodes Namalwa poly(A)+ RNA. (ii) The fraction of the Bam I S fragment homologous to Namalwa poly(A)- RNA is similar to the fraction homologous to Namalwa poly(A)+ RNA. However, Namalwa poly(A)- RNA is homologous to a larger fraction of the DNA in the Hsu I B, Hsu I D, and EcoRI B fragments.

Messenger RNA synthesis in synchronized Chinese hamster ovary cells

Chinese hamster ovary cells were synchronized without inhibitors by mitotic selection and labelled in G 1, S or G 2 phase by incubation for 90 min with [ 3H]-or [ 14C]uridine. Purified polyribosomes were extracted with phenol and the polyadenylated mRNA prepared by poly(U)-Sepharose chromatography. Polyadenylated [ 3H]uridine-labelled mRNA from the G 1 phase of the cell cycle was compared by exponential polyacrylamide gel electrophoresis in formamide with [ 14C]uridine-labelled polyadenylated mRNA from the S or G 2 phase. The electrophoretic patterns obtained correspond to the size range expected for mRNA (7–28 S). No prominent differences were detected between mRNAs synthesized in different phases of the cell cycle. From these data we conclude that the major size classes of polyribosomal poly(A)-containing mRNA are synthesized in equal ratios throughout the cell cycle.

Terminal completion of poly(A) synthesis in sea urchin embryos

The kinetics of accumulation of nuclear and cytoplasmic poly(A) have been determined in sea urchin blastulas and gastrulas, stages when essentially all mRNA is synthesized de novo in the nucleus. A majority of the labeled poly(A) is found in the cytoplasmic fraction after a brief pulse. The ratio of radioactive AMP to adenosine in pulse-labeled nuclear, cytoplasmic, and polyribosomal poly(A) is considerably less than the number average length of the labeled poly(A), indicating that there is 3′-terminal addition of adenosine to previously synthesized poly(A). The size distribution of pulse-labeled, terminally elongated poly(A) in the cytoplasm is similar to that of the largest nuclear poly(A) rather than the steady-state size distribution of cytoplasmic poly(A), which is smaller and more heterogeneous. The most likely interpretation of these results is that there is a predominant 3′ terminal addition of short tracts of adenosine to poly(A) attached to nuclear RNA just before or during entrance of this RNA into the cytoplasm. In this respect, much of the 3′ terminal addition may be thought of as terminal completion of poly(A) synthesis.

The effect of tryptophan on nucleocytoplasmic translocation of RNA in rat liver

The effect of the administration of tryptophan on the transport of nuclear poly(A)-containing mRNA to the cytoplasm in rat liver was investigated. Administration of tryptophan to fasted rats pretreated with cordycepin and actinomycin D led to decreased levels of nuclear poly(A)-mRNA and a concomitant increase in the levels of polyribosomal poly(A)-mRNA in the cytoplasm as determined by measuring in vivo incorporation of labeled precursors into hepatic RNA. Using isolated hepatic nuclei of rats prelabeled in vivo with [ 14C]orotic acid, there was greater release of labeled poly(A)-mRNA into the incubation medium from nuclei of tryptophan-treated rats than from nuclei of control animals. The increased release of RNA from hepatic nuclei of tryptophan-treated animals was not related to the cell sap present in the media since cell saps from livers of control and experimental rats gave similar results. These results support earlier findings which suggest that in the rat tryptophan increases the rate of translocation of hepatic poly(A)-mRNA from nucleus to cytoplasm.

Nuclear and polyribosomal ribonucleoprotein particles containing poly (A) in rat liver cells.

Poly(A) containing ribonucleoprotein particles were prepared from rat liver nuclei and polyribosomes. The particles have sedimentation coefficients of 14 S and 9 S, respectively. In CS2SO4 density gradients the particles banded at densities of 1.28-1.29 g cm-3. Both nuclear and polyribosomal poly(A)-RNP contain in addition to some minor polypeptides, two main polypeptides having molecular weights of 63 000 and 90 000 dalton, respectively indistinguishable from each other according to their electrophoretic mobilities.