Release of poly a(+) messenger RNA from rat liver rough microsomes upon disassembly of bound polysomes

Abstract

Several procedures were used to disassemble rat liver rough microsomes (RM) into ribosomal subunits, mRNA, and ribosome-stripped membrane vesicles in order to examine the nature of the association between the mRNA of bound polysomes and the microsomal membranes. The fate of the mRNA molecules after ribosome release was determined by measuring the amount of pulse-labeled microsomal RNA in each fraction which was retained by oligo-dT cellulose or by measuring the poly A content by hybridization to radioactive poly U. It was found that ribosomal subunits and mRNA were simultaneously released from the microsomal membranes when the ribosomes were detached by: (a) treatment with puromycin in a high salt medium containing Mg ++, (b) resuspension in a high salt medium lacking Mg ++, and (c) chelation of Mg ++ by EDTA or pyrophosphate.Poly A-containing mRNA fragments were extensively released from RM subjected to a mild treatment with pancreatic RNase in a medium of low ionic strength. This indicates that the 3' end of the mRNA is exposed on the outer microsomal surface and is not directly bound to the membranes. Poly A segments of bound mRNA were also accessible to [aH]poly U for in situ hybridization in glutaraldehyde-fixed RM.Rats were treated with drugs which inhibit translation after formation of the first peptide bonds or interfere with the initiation of protein synthesis. After these treatments inactive monomeric ribosomes, as well as ribosomes bearing mRNA, remained associated with their binding sites in microsomes prepared in media of low ionic strength. However, because there were no linkages provided by nascent chains, ribosomes, and mRNA, molecules were released from the microsomal membranes without the need of puromycin, by treatment with a high salt buffer containing Mg ++.Thus, both in vivo and in vitro observations are consistent with a model in which mRNA does not contribute significantly to the maintenance of the interaction between bound polysomes and endoplasmic reticulum membranes in rat liver hepatocytes. 414

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

“…Previous studies attempted to address whether there is translation-independent mRNA association with the ER using cellular fractionation techniques. Because harsh chemical conditions were required to disassociate ribosomes from ER derived vesicles, which might disrupt the potentially delicate mRNA-ER association, these studies were inconclusive, providing evidence for [5][6][7][8] and against 9,10 ribosomal-independent anchoring of mRNA to the ER.…”

Multifunctional Roles for the Protein Translocation Machinery in RNA Anchoring to the Endoplasmic Reticulum

“…In contrast, analyses of in situ mRNA-ER interactions conducted with rough microsomes (RM) have demonstrated that ER-associated mRNAs can be binned into two broad classes: those that are bound via ribosome-dependent (indirect) and those that undergo ribosome-independent (direct) ER-mRNA anchoring (Fig. 1A) (13,15,(45)(46)(47). These differences are significant because they suggest that mRNA localization and anchoring to the ER is selective and regulated and so may contribute significantly to gene expression.…”

“…First, tissue culture cells are permeabilized with a digitoninsupplemented cytosol buffer to permeabilize the plasma membrane and release the cytoplasmic mRNA pool (28,49). The digitonin-permeabilized (semi-intact) cells are then treated with salts, chelating agents, and/or detergents, and mRNA-ER interactions were assessed by examining the mRNA composition of the released and ER-associated fractions (13,15,(45)(46)(47). Using HeLa cells as a model, we first examined the ER association properties of two mRNAs, B2M (a secretory protein) and GRP94 (a resident protein of the ER lumen), because previous studies with RM had identified these mRNAs as undergoing either ribosome-dependent (B2M) or ribosome-independent (GRP94) membrane association (13,15).…”

Polysomal and cytoplasmic mRNP particles containing 7S(L) RNA

“…For the isolation of free and membrane-bound polysomes the procedures of Scheele et al [6], Kruppa and Sabatini [7] and Shore and Tata [8] were combined and modified as previously described [9]. The sedimented free polysomes were dissolved in 0.2 M Tris/HCI buffer, pH 8.5, 50 mM KC1, 10 mM MgCI,, 2.5 mM dithiothreitol and 20 mM EDTANa,, incubated at 0°C for 1 h, added onto a linear sucrose gradient (20 -40%) in the above buffer and centrifuged in a Beckman SW27 rotor at 27000 rpm for 27 h. The fractions of the gradient indicated in Fig.…”