Messenger RNA processing and nuclear structure: isolation of nuclear ribonucleoprotein particles containing beta-globin messenger RNA precursors.
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-200S and contain, in addition to high molecular weight hnRNA, a specific set of nuclear proteins predominated by a major component of approximately 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. Th authenticity of the Friend cell hnRNP particles is demonstrated by the results of reconstruction experiments with deproteinized hnRNA, and by the resistance of the articles to dissociation during isopycnic banding in Cs2SO4 gradients without prior aldehyde fixation. Hybridization analysis with cloned mouse beta-globin DNA demonstrates that hnRNP particles from induced Friend cells contain newly synthesized transcripts of the beta-globin gene. Agarose gel electrophoresis of hnRNP particle-derived RNA denatured in glyoxal followed by "Northern" transfer to diazobenzyloxymethyl paper and hybridization with 32P-labeled cloned mouse beta-globin DNA reveals the presence in hnRNP of two size classes of beta-globin gene transcripts, the larger of which corresponds to the pre-spliced 15S beta-globin mRNA precursor previously identified in whole nuclear RNA, and the smaller of which corresponds to completely processed 9S beta-globin mRNA. These results establish, for the first time, that the nuclear transcripts of a specific, well-defined eukaryotic structural gene can be isolated in an RNP particle form, and that their RNP structure persists throughout mRNA splicing.
D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D005622:Friend murine leukemia virus; D005914:Globins / Q000235:genetics*; D004915:Leukemia, Erythroblastic, Acute / Q000378:metabolism; D051379:Mice; D008970:Molecular Weight; D009698:Nucleoproteins / Q000378:metabolism*; D012332:RNA, Heterogeneous Nuclear / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000302:isolation & purification / Q000378:metabolism*; D014158:Transcription, Genetic
D009698:Nucleoproteins; D012332:RNA, Heterogeneous Nuclear; D012333:RNA, Messenger; D012261:Ribonucleoproteins; D005914:Globins