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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 [(3)H] 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.
Insights
Messenger RNA (mRNA) does not significantly anchor polysomes to rat liver endoplasmic reticulum membranes. Ribosomal subunits and mRNA are released together, with mRNA
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Rough microsomes (RM) are crucial for protein synthesis and translocation.
- The precise interaction between mRNA and microsomal membranes is not fully understood.
Purpose of the Study:
- To investigate the role of mRNA in anchoring bound polysomes to rat liver microsomal membranes.
- To elucidate the nature of the association between mRNA and the endoplasmic reticulum.
Main Methods:
- Disassembly of rat liver rough microsomes (RM) into ribosomal subunits, mRNA, and membrane vesicles.
- Quantification of pulse-labeled microsomal RNA using oligo-dT cellulose binding or poly A content hybridization.
- In situ hybridization of glutaraldehyde-fixed RM with radioactive poly U.
- In vivo drug treatments to inhibit protein synthesis.
Main Results:
- Simultaneous release of ribosomal subunits and mRNA from membranes upon ribosome detachment.
- mRNA's 3' end is exposed on the outer microsomal surface, not directly membrane-bound.
- Poly A segments of bound mRNA are accessible for in situ hybridization.
- Ribosomes and mRNA remain membrane-associated even when translation is inhibited, unless nascent chains are absent.
Conclusions:
- mRNA does not play a significant role in maintaining polysome-membrane interactions in rat liver hepatocytes.
- Nascent polypeptide chains are critical for the stable association of polysomes with endoplasmic reticulum membranes.