Ribonucleoprotein organization of polyadenylate sequences in HeLa cell heterogeneous nuclear RNA

Kish, Valerie M.; Pederson, Thoru

doi:10.1016/0022-2836(75)90392-7

Cited by 110 publications

(48 citation statements)

References 24 publications

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“…In addition, it has been possible to demonstrate that these latter hnRNP preparations contain specific proteins bound to defined nucleotide sequences, such as poly(A) (15,20,22), and that they FIGURE 8 Northern blot hybridization of 8-globin gene transcripts in hnRNP . RNA was recovered from nuclei, cytoplasm, or purified hnRNP particles by Proteinase K-phenol deproteinization and electrophoresed in agarose gels after denaturation in glyoxal (see Materials and Methods) .…”

Section: Identification Of Covalently Intact (3-globin Mrna Precursomentioning

confidence: 99%

Messenger RNA processing and nuclear structure: isolation of nuclear ribonucleoprotein particles containing beta-globin messenger RNA precursors.

Pederson¹,

Davis²

1980

The Journal of Cell Biology

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To explore the relationships between transcription, messenger RNA (mRNA) processing, and nuclear structure, ribonucleoprotein particles containing heterogeneous nuclear RNA (hnRNP) have been purified from globin-producing mouse Friend erythroleukemia cells. These nuclear hnRNP particles sediment at 50S-2005 and contain, in addition to high molecular weight hnRNA, a specific set of nuclear proteins predominated by a major component of 38,000 mol wt . The hnRNP particles are free of histones and ribosomal structural proteins, indicating their purification from the two other major nucleoprotein components of the nucleus: chromatin and nucleolar ribosomal precursor RNP particles. The authenticity of the Friend cell hnRNP particles is demonstrated by the results of reconstruction experiments with deproteinized hnRNA, and by the resistance of the particles to dissociation during isopycnic banding in C5 2SO 4 gradients without prior aldehyde fixation . Hybridization analysis with cloned mouse 8-globin DNA demonstrates that hnRNP particles from induced Friend cells contain newly synthesized transcripts of the a-globin gene . Agarose gel electrophoresis of hnRNP particle-derived RNA denatured in glyoxal followed by "Northern" transfer to diazobenzyloxymethyl paper and hybridization with 32 P-labeled cloned mouse fl-globin DNA reveals the presence in hnRNP of two size classes of /3-globin gene transcripts, the larger of which corresponds to the pre-spliced 15S 8-globin mRNA precursor previously identified in whole nuclear RNA, and the smaller of which corresponds to completely processed 9S a-globin mRNA . These results establish, for the first time, that the nuclear transcripts of a specific, welldefined eukaryotic structural gene can be isolated in an RNP particle form, and that their RNP structure persists throughout mRNA splicing .The transcription of structural genes in the eukaryotic cell nucleus takes place at a nucleoprotein level of organization . This is true of both the templates for transcription, chromatin (reviewed in reference 36), and the products of transcription, heterogeneous nuclear RNA, which is rapidly assembled into ribonucleoprotein particles known as hnRNP (33,34). However, the functional significance of these nucleoprotein environments for transcription and hnRNA metabolism is not understood . As a step toward defining the relationships between messenger RNA (mRNA) processing and nuclear structure, hnRNP particles have been purified from globin-producing mouse Friend erythroleukemia cells and characterized with particular reference to transcripts of the 8-globin gene. The THE JOURNAL OF CELL BIOLOGY -VOLUME 87 OCTOBER 1980 47-54 © The Rockefeller University Press -0021-9525/80/10/0047/08 $1 .00 major conclusion is that both pre-spliced and spliced mRNA sequences have a ribonucleoprotein structure in the nucleus. MATERIALS AND METHODSCell Culture, Radioisotopic Labeling, and Cell Fractionation Clone 745 mouse Friend erythroleukemia cells were maintained in monolayer culture usingJoklik-mod...

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Section: Identification Of Covalently Intact (3-globin Mrna Precursomentioning

confidence: 99%

Messenger RNA processing and nuclear structure: isolation of nuclear ribonucleoprotein particles containing beta-globin messenger RNA precursors.

Pederson¹,

Davis²

1980

The Journal of Cell Biology

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“…Standard labeling conditions were as follows : cells were centrifuged and resuspended to a concentration of 2 x lo6 cells/ml in medium containing dialysed calf serum, incubated for 90 min then exposed to [5-3H]uridine (27)(28)(29)(30) Ci/mol; 10 pCi/ml) for 30 min. In most cases, actinomycin D was added to 0.04 pg/ml 20 min before radioactive uridine in order to prevent the synthesis of rRNA [27].…”

Section: Cell Growth and Labeling Conditionsmentioning

confidence: 99%

“…proteins Cells labeled as described above were mixed with a ten-fold excess of unlabeled ones and hnRNA . proteins were extracted using a slight modification of the procedure of Kish and Pederson [28]. All operations were carried out at 0 -4 "C.…”

Section: Cell Growth and Labeling Conditionsmentioning

confidence: 99%

Characterisation of an Endogenous Protein Kinase Activity in Ribonucleoprotein Structures Containing Heterogenous Nuclear RNA in HeLa Cell Nuclei

Blanchard

Brunel²,

Jeanteur³

1977

European Journal of Biochemistry

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Ribonucleoprotein particles containing heterogeneous nuclear RNA (hnRNA) were isolated from sonically disrupted HeLa cells nuclei by sedimentation through a triple sucrose cushion. We found that these particles were able to catalyse the incorporation of radioactive phosphate from [Y-~~PIATP into phosphoserine and phosphothreonine residues of endogenous proteins. Optimal conditions for enzymatic activity were defined after investigation of the influence of several parameters of the reaction. The requirement for a divalent cation was most efficiently met by 10 mM Mg', to a lower extent by Mn2+ and not at all by Ca2'. An apparent K, value for ATP of 0.02 mM was determined and the enzyme was found to be sensitive to p-hydroxymercuribenzoate. Neither cyclic AMP nor cyclic GMP stimulated the reaction over a wide range of concentrations and the same protein substrates were phosphorylated in their presence.After excluding possible contamination by other subcellular fractions, considerable evidence for the association of kinase and phosphate acceptor proteins with ribonucleoprotein structures containing hnRNA was gathered along several lines by showing that the enzymatic activity closely followed tritiated RNA pulse-labeled in the presence of low doses of actinomycin D in the following circumstances : (a) sedimentation in sucrose gradients, (b) isopycnic banding in metrizamide density gradients in the presence or absence of M g ' ions and (c) adsorption to oligo(dT)-cellulose columns under conditions where poly(A)-containing material is adsorbed.Analysis of phosphorylated substrates by electrophoresis on polyacrylamide gels containing sodium dodecylsulphate revealed that, although they spanned a wide range of molecular weights, the most intensely labeled species were localized in bands corresponding to molecular weights of 37000 and 28 000.The above pattern of polypeptides phosphorylated in vitro is compared with that of phosphoproteins labeled in vivo after exposure of cells to [32P]phosphate.The most widely accepted model for messenger RNA formation in eucaryotic cells postulates that it is derived by post-transcriptional modification of the rapidly labeled heterogeneous nuclear RNA (hnRNA, for review see [l]). However, only in few instances could a direct precursor-product relationship between this hnRNA and cytoplasmic messenger RNA be established with good evidence [2,3].Abbreviations. hnRNA, heterogenous nuclear RNA; mRNA, messenger RNA; hnRNA . proteins, heterogenous nuclear ribonucleoprotein particles; CAMP and cGMP, adenosine and guanosine 3': 5'-monophosphates.

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“…This protein, with a M , ranging from 73 000 to 80000 depending upon the authors, has been identified in polyribosomal mRNP isolated from different cellular systems (see review in [21]) and was shown to interact with the poly (A) segment of mRNA [18,201. Moreover, it has been detected in nuclear RNP particles [50]. Thus, a general role of this protein in mRNA metabolism could be supposed.…”

Section: Discussionmentioning

confidence: 96%

Identification and Characterization of the Translationally Repressed Cytoplasmic Globin Messenger‐Ribonucleoprotein Particles from Duck Erythroblasts

Víncent

Civelli

Maundrell

et al. 1980

European Journal of Biochemistry

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Globin messenger ribonucleoprotein (mRNP) particles which have been isolated from duck erythroblast post-polyribosomal supernatant are translationally inactive in vivo and in uitro but contain translatable mRNA active after deproteinisation. They were characterized following pruification by successive sucrose gradient sedimentation in a buffer containing 0.05 M KCl. The complex, which sediments homogeneously at about 20 S, has a density of 1.39 g/cm3 and thus consists of four parts protein to one part RNA; 40% of this RNA is globin mRNA and no other mRNA could be detected.Sedimentation of the purified globin mRNP on sucrose gradients in 0.5 M KCl produced four components while polyacrylamide gel electrophoresis in non-denaturing conditions and in the presence of EDTA resulted in the separation of three components. Hybridization to globin cDNA and translation in vitro of the RNA extracted from these subparticles revealed the existence of two core particles containing globin mRNA with nominal sedimentation coefficients of 13 S and 16 s.Analysis of the protein components of the isolated sub-complexes by dodecyl sulfate and bidimensional gel electrophoresis indicated a very characteristic protein composition for each of these complexes. The 16-S and 13-S globin mRNPs differed essentially by the presence in the 13-S mRNP only of a group of major polypeptides. Of the other two sub-complexes, one consisted of 90% small RNA in the 4-S range; the second sedimented ahead of the globin mRNP core particles at about 19s and consisted of a very characteristic set of about 14 polypeptides. The polyrisobomal73OOO-M, poly(A)-binding protein was not detected in the purified free globin mRNP although the mRNA in the untranslatable particle is polyadenylated.The presence in the cytoplasm of duck erythroblasts of two forms of untranslated globin messenger ribonucleoprotein particles, distinct in their protein composition from polyribosomal globin mRNP. suggests that they may have a specific role in the regulation of translation of globin mRNA.

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Ribonucleoprotein organization of polyadenylate sequences in HeLa cell heterogeneous nuclear RNA

Cited by 110 publications

References 24 publications

Messenger RNA processing and nuclear structure: isolation of nuclear ribonucleoprotein particles containing beta-globin messenger RNA precursors.

Messenger RNA processing and nuclear structure: isolation of nuclear ribonucleoprotein particles containing beta-globin messenger RNA precursors.

Characterisation of an Endogenous Protein Kinase Activity in Ribonucleoprotein Structures Containing Heterogenous Nuclear RNA in HeLa Cell Nuclei

Identification and Characterization of the Translationally Repressed Cytoplasmic Globin Messenger‐Ribonucleoprotein Particles from Duck Erythroblasts

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