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

Abstract

To explore the relationships between transcription, mRNA processing, and nuclear structure, ribonucleoprotein particles containing heterogeneous nuclear RNA (i.e., hnRNP) were purified from globin-producing mouse Friend erythroleukemia cells. These nuclear hnRNP particles sediment at 50S-200S and contain, in addition to high MW hnRNA, a specific set of nuclear proteins predominated by a major component of 38,000 MW. The hnRNP particles are free of histones and ribosomal structural proteins, indicating their purification from the 2 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 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 2 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. It is established that 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.