Structure of nuclear ribonucleoprotein: identification of proteins in contact with poly(A)+ heterogeneous nuclear RNA in living HeLa cells.

Abstract

The processing of heterogeneous nuclear RNA into messenger RNA takes place in special nuclear ribonucleoprotein particles known as hnRNP. We report here the identification of proteins tightly complexed with poly(A) + hnRNA in intact HeLa cells, as revealed by a novel in situ RNA-protein cross-linking technique. The set of cross-linked proteins includes the A, B, and C "core" hnRNP proteins, as well as the >42,000 mol wt species previously identified in noncross-linked hnRNP. These proteins are shown to be cross-linked by virtue of remaining bound to the poly(A) + hnRNA in the presence of 0.5% sodium dodecyl sulfate, 0.5 M NaCl, and 60% formamide, during subsequent oligo(dT)-cellulose chromatography, and in isopycnic banding in CS2S04 density gradients . These results establish that poly(A) + hnRNA is in direct contact with a moderately complex set of nuclear proteins in vivo . This not only eliminates earlier models of hnRNP structure that were based upon the concept of a single protein component but also suggests that these proteins actively participate in modu ;ating hnRNA structure and processing in the cell .Eukaryotic messenger RNA is derived from precursors collectively known as heterogeneous nuclear RNA (hnRNA) . The conversion of hnRNA to mRNA involves a number of welldefined steps, including the addition of poly(A) and 5'-caps, and the removal of intervening DNA sequence transcripts through a series of coupled cleavage-ligation reactions known as splicing . These mRNA processing steps take place while hnRNA is tightly complexed with nuclear proteins in ribonucleoprotein particles known as hnRNP (reviewed in reference 1). hnRNP particles were first seen as nascent RNP fibrils on the lateral loops of amphibian oocyte lampbrush chromosomes (2, 3) and later by electron microscopy in a variety ofeukaryotic cells (4-14) . hnRNP particles have been isolated (15, 16), and their constituent RNA and proteins have now been characterized in some detail (15-32).It is likely that the structure of hnRNP is functionally related to mRNA processing (1) . We have been studying the nucleoprotein structure of defined RNA sequences in hnRNP using nucleases and other probes. For example, the use of nucleases specific for either single-or double-stranded RNA has revealed that intramolecular double-stranded regions of hnRNP are essentially free of associated protein, in contrast to singlestranded hnRNA which is extensively complexed with protein (27). Recently, we have found that messenger RNA-homologous sequences in hnRNP are extensively complexed with protein as revealed by nuclease protection experiments (31, 33) . The recent demonstration that hnRNP from mouse Friend erythroleukemia cells contains the prespliced 15S precursor of ß-globin mRNA (30) supports the idea that hnRNP particles are the sites of mRNA processing in the cell.In this investigation we used a novel methodology (32) to address another important aspect of hnRNP structure : the number of different protein species that are complexed with hnRNA . Beca...

Physical change in cytoplasmic messenger ribonucleoproteins in cells treated with inhibitors of mRNA transcription.

“…10C) (5 ,ug/ml for 2 h); T, total cell material; P, polyribosomal fraction after selection on an oligo(dT)-cellulose-_ column as detailed in the legend to Fig. 8. the ability of UV light to generate photoreactive species of RNA and thus bring about cross-linking of mRNA to pro--teins which are in direct contact with it in the intact cell (10,34,35,50,51,53 (39,42) and in vitro (29) have also been described. The relationship between these observations and the altered interaction of 38K with mRNA is not known, but it may be that 38K is involved in mRNA stabilization.…”

“…This is due to the fact that nonspecific interactions between RNA and proteins are likely to occur in vitro (2,14,38). UV cross-linking of RNA to protein in intact cells overcomes these difficulties and allows the identification of proteins which are in direct contact with hnRNA and mRNA in vivo (10,34,35,50,51,53).…”

“…This is due to the fact that nonspecific interactions between RNA and proteins are likely to occur in vitro (2, 14, 38). UV cross-linking of RNA to protein in intact cells overcomes these difficulties and allows the identification of proteins which are in direct contact with hnRNA and mRNA in vivo (10,34,35,50,51,53).The in vivo UV cross-linking approach relies on the fact that UV light of sufficient intensity generates photoreactive species of RNA which react virtually indiscriminately with molecules, including proteins, which are in direct contact with it. Cross-linked complexes of polyadenylated [poly(A)+] mRNA were isolated under protein-denaturing conditions to ensure that only proteins covalently linked to mRNA were purified with it, eliminating nonspecific associations of proteins with mRNA (51, 53).…”

Immunopurification of heterogeneous nuclear ribonucleoprotein particles reveals an assortment of RNA-binding proteins.

“…A more stringent definition of genuine hnRNP proteins has been provided by UV light-induced covalent cross-linking of proteins to RNA in vivo, which allows identification of proteins that are bound to the RNA in the cell (Mayrand et al 1981;Dreyfuss et al 1984a, b). These studies showed numerous proteins, including those that correspond in SDS-PAGE mobility to A, B, and C proteins, but the proteins are no longer useful for biochemical studies.…”

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).…”