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

Abstract

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...

Heat shock alters nuclear ribonucleoprotein assembly in Drosophila cells.

“…One obvious possibility is hyperphosphorylation, which might reduce electrostatic interactions of hnRNP proteins with hnRNA. The effect could also be due to a sudden cessation of hnRNP protein translation upon heat shock, so that the preexisting nuclear pool of free hnRNP proteins (which appears to be small in the first place [41]) becomes rapidly depleted after heat shock. We do not think that the effect on hnRNP assembly is actually dependent on the heat shock proteins themselves, because it can be observed within 10 min after heat shock (Fig.…”

“…These heterogeneous nuclear RNP (hnRNP) particles contain protein and RNA in a mass ratio of about 4:1 (7,38,42). A remarkable property of hnRNP particles is their ability to withstand isopycnic banding in Cs2SO4 without prior aldehyde fixation (7,41). We have isolated hnRNP particles from Drosophila KcO cells (S. Mayrand and T. Pederson, unpublished data) and have found that they also are stable during centrifugation in Cs2SO4 and band at 1.34 g/cm3, which indicates the same 4:1 protein-RNA composition as mammalian hnRNP (Fig.…”

“…The precautions taken with respect to temperature control during cell harvesting and labeling are described in further detail below. HeLa and mouse erythroleukemia cells were maintained as previously described (37,41).…”

“…For the experiments with mammalian cells, HeLa or mouse erythroleukemia cells were harvested, pretreated with low concentrations of actinomycin (38,41), and then incubated with [3H]uridine for 20 min at either 37°C or the desired temperature (39 to 45°C). Cell fractionation and hnRNP isolation were performed as previously described (38,41).…”

Serine/threonine phosphorylation regulates binding of C hnRNP proteins to pre-mRNA.

“…The first described spliceosome proteins were called hnRNP proteins, based on their association with large heterogeneous nuclear RNA transcripts now known to include unspliced pre-mRNAs (6)(7)(8)(9)(10)(11)(12). The C-group hnRNP proteins are abundant nuclear proteins of Mr 42,000-44,000 that have been implicated in splicing (13,14) and are known to be phosphorylated in vivo (8,(15)(16)(17) by a casein kinase II-type activity (18,19).…”