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Comparison of poly(A)-mRNA prepared from membranes and free polyribosomes of mouse liver
Abstract:
We have compared poly(A)-mRNA isolated from mouse liver endoplasmic reticulum (ER) membranes (ER mRNA) with that from free polyribosomes (free mRNA) by translation in a cell-free system and by mRNA--cDNA reassociation analysis. The partitioning of certain translation products between the two fractions suggests that the physical contamination of the free mRNA by the ER is 10%, and contamination of the ER by the free is 1%. Reassociation crossreactions suggest that no sequences are totally absent from either fraction. In particular, the rare sequences are shared between the two fractions. Among the more abundant sequences, we detect three groups: (i) a group of about 10 sequences that are relatively abundant in both fractions; (ii) a group that occurs mostly in the free fraction; (iii) a group of about 12 sequences, 6 or 7 of them encoding secretory polypeptides. The members of this group are largely or entirely confined to the ER fraction. Secretory proteins are almost entirely absent from the products of th free mRNA fraction. Quantitation of individual mRNA sequences by hybridization with cDNA clones shows that a range of sequence concentrations exists within the abundant class of ER mRNA.
Insights
Researchers compared endoplasmic reticulum (ER) messenger RNA (mRNA) with free mRNA in mouse liver. Secretory protein mRNA is primarily found in ER mRNA, indicating distinct cellular localization for protein synthesis.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Messenger RNA (mRNA) localization is crucial for efficient protein synthesis and cellular function.
- Endoplasmic reticulum (ER) membranes are involved in the synthesis of secretory and membrane proteins.
- Free polyribosomes synthesize cytosolic proteins.
Purpose of the Study:
- To compare the molecular composition of poly(A)-mRNA from mouse liver ER membranes and free polyribosomes.
- To investigate the distribution of specific mRNA sequences, particularly those encoding secretory proteins, between these two cellular fractions.
- To assess potential cross-contamination between the ER and free mRNA populations.
Main Methods:
- Isolation of poly(A)-mRNA from mouse liver ER membranes and free polyribosomes.
- Cell-free translation system to analyze the protein products synthesized by each mRNA fraction.
- mRNA-cDNA reassociation analysis to quantify and compare the abundance of specific mRNA sequences.
Main Results:
- Translation products indicated approximately 10% contamination of free mRNA by ER mRNA and 1% contamination of ER mRNA by free mRNA.
- Rare mRNA sequences were found in both ER and free fractions, suggesting broad distribution.
- Abundant mRNA sequences were categorized into three groups: those common to both fractions, those enriched in the free fraction, and a distinct group largely confined to the ER fraction, including sequences for secretory polypeptides. Secretory protein synthesis was largely absent from the free mRNA fraction.
Conclusions:
- Mouse liver ER-bound and free mRNA populations exhibit distinct sequence compositions, particularly regarding secretory protein mRNA.
- Secretory protein mRNA is predominantly localized to the ER membrane fraction.
- The study provides quantitative insights into mRNA distribution and sequence concentrations within abundant mRNA classes in the ER fraction.