No 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...

No abstract

Two distinct forms of globin messenger RNA were isolated from mouse spleen cells infected with Friend erythroleukemia virus: polyribosomal messenger ribonucleoprotein particles (15S mRNP), and their corresponding protein-free mRNAs obtained by chemical deproteinization . The translation efficiencies of both messenger forms were assayed in a Krebs II ascites cell-free system . Selective removal of RNA-binding proteins from the ascites cell lysate did not affect globin synthesis when the mRNA was supplied as 15S mRNP ; deproteinized mRNA however was not translated . Only in the presence of two fractions of RNA-binding proteins was the protein-free mRNA translated . Some of the RNA-binding proteins have the same molecular weights and isoelectric points as the principal proteins of 15S mRNP .Many investigations support the concept that eukaryotic messenger RNA is associated with various proteins as messenger ribonucleoprotein particles (mRNP) (1-8) . Two classes of mRNP can be isolated from the cytoplasm, one that consists of free mRNP not bound to ribosomes, and the other that is present in polyribosomes (1,3,6,9,10) . The physiological importance of stable protein-mRNA interactions and even the existence of mRNP have sometimes been questioned, because mRNP and deproteinized mRNA stimulate the cell-free translation system equally well (11-15) . Furthermore, earlier studies by 17) indicated that the cytosol contains soluble proteins with affinity for RNA, raising the possibility that mRNPs are artifacts of cell fractionation (see discussion in Kumar and Pederson (18]). On the other hand, mRNPs can be shown to remain intact under conditions of ionic strength where nonspecific RNA-protein complexes dissociate (14,(19)(20)(21)(22)(23) . Moreover, RNA-protein cross-linking experiments indicate that mRNA is complexed with proteins in situ (24-26) .These conflicting observations can be reconciled by the hypothesis that RNA-protein associations do occur in vivo, but that the mRNP-proteins are also present as soluble RNAbinding proteins . This view is compatible with the metabolic behavior of mRNP, which indicates that the proteins shuttle on and off the mRNA (27)(28)(29) . In addition, recent data from this laboratory (30-32), as well as from others (33,34), have shown that in fact some of the RNA-binding proteins have the same electrophoretic mobilities as the proteins isolated in association with mRNAs as native mRNP in the same cells .Clearly, the possible role of mRNP proteins in translation cannot be investigated if one uses a translation system in which THE JOURNAL OF CELL BIOLOGY " VOLUME 93 JUNE 1982 893-898 c0 The Rockefeller University Press " 0021-9525/82/06/0893/06$1 .00 an extensive supply of these proteins is present in soluble form . Therefore, we have devised a method for eliminating these proteins from an ascites cell lysate, by passing the lysate through a RNA affinity column (31,32) . This treatment makes translation in the lysate dependent on protein factors that bind to the column, or on some ...

Acidic proteins with affinity to RNA from cytoplasmic fractions of Krebs II Ascites cells were isolated by means of affinity chromatography on RNA-Sepharose CN-Br-columns.Stepwise elution with 350 mᴍ [K+] and 1000 mᴍ [K+] removed two fractions of proteins both of which are required for the formation of 40S-or 80S-initiation complexes and for efficient translation.