Characterization of mRNA-protein complexes from mammalian cells

Abstract

In a previous report we described the use of oligo(dT)-cellulose for the isolation of mRNA-protein complexes from EDTA-dissociated polysomes extracted from normally growing or adenovirus infected KB-cells (I). Experiments presented here provide evidence that proteins involved in these complexes bind specifically to mRNA since: a) the proteins and mRNA cosediment through sucrose gradients, b) they adsorb and elute from oligo(dT)-cellulose together, and c) analysis of the products from ribonuclease digestion experiments show that the poly (A) end and a separate small fraction of the mRNA are resistant to the enzymes and attached to protein.

Evidence for the protection of specific RNA sequences in globin messenger ribonucleoprotein particles

“…Two identifiable regions of mRNA molecules have been proposed as putative binding sites for proteins: the 5'-terminal cap structure (30) and the 3'-OH poly(A)-stretch of the mRNA (3)(4)(5)(6)(7). We believe that other binding sites must exist since electron microscope visualization of 15 S mRNP particles showed that proteins are located at several different points along the messenger molecule (8).…”

“…The 15 S globin mRNP released from polyribosomes by EDTA has a characteristic protein composition (2) which includes a major 73,000 MW polypeptide bound to the 3' terminal poly(A) segment (3), similar to that found for a variety of other messenger molecules (4)(5)(6)(7). Electron microscopy data obtained by dark-field visualization of duck globin 15 S mRNP particles has shown that proteins are distributed al ong all of the messenger molecule although not uniformly (8).…”

mRNA polyadenylate-binding protein: gene isolation and sequencing and identification of a ribonucleoprotein consensus sequence.

“…UV cross-linking of proteins to RNA in intact cells overcomes these difficulties and allows the identification of proteins which bind heterogeneous nuclear RNA and mRNA in vivo (11,16,51,54). To learn more about these proteins and about the RNP complexes, we have immunized mice with purified UV-crosslinked RNP complexes and have produced antibodies to several of the major RNP proteins in vertebrate cells (1,12,13).Much of the knowledge of RNP proteins has come from work with higher eucaryotes (12,24,48 (7) and is one of the most conserved and extensively investigated eucaryotic RNP proteins. Although many important functions in mRNA metabolism have been attributed to this protein, little is known about its function, amino acid sequence, and metabolism.…”

“…Much of the knowledge of RNP proteins has come from work with higher eucaryotes (12,24,48 (7) and is one of the most conserved and extensively investigated eucaryotic RNP proteins. Although many important functions in mRNA metabolism have been attributed to this protein, little is known about its function, amino acid sequence, and metabolism.…”

“…Much of the knowledge of RNP proteins has come from work with higher eucaryotes (12,24,48). However, little is known about the structure of mRNPs and hnRNPs in simple * Corresponding author.…”