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Direct association of messenger RNA with microsomal membranes in human diploid fibroblasts
Abstract:
Messenger RNA (mRNA) of membrane-bound polysomes in a membrane fraction of WI-38 cells remains associated with the microsomal membranes even after ribosomes and their nascent polypeptide chains are removed by using puromycin in a high salt buffer or by disassembling the ribosomes in a medium of high ionic strength lacking magnesium. mRNA either was specifically labeled in the presence of actinomycin D, or it was recognized by virtue of its affinity for oligo-dT. Poly A segments in bound mRNAs have an electrophoretic mobility in acrylamide gels which is characteristic of cytoplasmic mRNAs and corresponds to 150-200 adenyl residues. Extensive RNase treatment did not lead to release of the poly A segments of membrane-associated mRNA molecules either from an intact membrane fraction or from a membrane fraction previously stripped of ribosomes. On the other hand, RNase treatment led to the release and digestion of the nonpoly A segments of the mRNA molecules, indicating that the site of attachment of mRNA to the ER membranes is located near or at the 3' end of the molecule which contains the poly A. A direct association of mRNAs and endoplasmic reticulum membranes is considered in a modelto explain the assembly of bound polysomes and protein synthesis in a membrane-associated apparatus.
Insights
Messenger RNA (mRNA) remains bound to cellular membranes even after ribosome removal. This attachment, mediated by the poly-A tail, is crucial for protein synthesis in membrane-associated systems.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Membrane-bound polysomes are key sites for protein synthesis.
- Understanding mRNA-membrane interactions is vital for deciphering protein localization and function.
Purpose of the Study:
- To investigate the mechanism of messenger RNA (mRNA) attachment to microsomal membranes.
- To identify the specific regions of mRNA involved in membrane association.
Main Methods:
- Utilized WI-38 cells and isolated microsomal membrane fractions.
- Employed puromycin and high salt buffers to remove ribosomes.
- Labeled mRNA with actinomycin D or used oligo-dT affinity.
- Analyzed mRNA poly-A segments via gel electrophoresis and RNase treatment.
Main Results:
- mRNA remained membrane-associated after ribosome removal.
- Poly-A segments of bound mRNA were characteristic of cytoplasmic mRNA (150-200 adenyl residues).
- RNase treatment released non-poly-A segments but not poly-A segments, indicating attachment near the 3' end.
Conclusions:
- mRNA directly associates with endoplasmic reticulum membranes via its 3' poly-A tail.
- This association is essential for the assembly of bound polysomes and membrane-associated protein synthesis.