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HeLa cell poly(A)- mRNA codes for a subset of poly(A)+ mRNA-directed proteins with an actin as a major product
Abstract:
Poly(A)+ and poly(A)- mRNA from HeLa cells were separated and translated in heterologous messenger-dependent protein synthesizing systems. Two-dimensional electrophoretic analysis revealed three classes of polypeptides. At the level of detectability in the electropherograms, a small number (about 10) of proteins were detected only among the poly(A)- mRNA products, a larger number (about 40) were produced by both poly(A)- and poly(A)+ mRNA, and a large number of polypeptides were found exclusively in the poly(A)+ mRNA products. The major product of both poly(A)+ and poly(A)- mRNA was shown to be the beta form of actin.Previous cross hybridization measurements suggested little homology between poly(A)+ and poly(A)- mRNA populations. In view of the apparent identity of many poly(A)- products with those of poly(A)+, the homology between poly(A)+ and poly(A)- mRNA sequences was examined in greater detail. cDNA complementary to only the most abundant poly(A)+ message sequences was prepared. About 10% of this cDNA hybridized to abundant sequences in the poly(A)- fraction. This corresponded to only 2% of the total mass of poly(A)+ mRNA and accounted for the failure to detect cross hybridization in previous experiments. Thus, a small number of poly(A)+ sequences appear to be present in relatively high concentration in poly(A)- mRNA as evidenced by both the translation products and the cross hybridization results.
Insights
Polyadenylated (poly(A)+) and non-polyadenylated (poly(A)-) mRNA from HeLa cells produce distinct and overlapping proteins. A small fraction of poly(A)+ mRNA sequences are highly concentrated in poly(A)- mRNA.
Area of Science:
- Molecular Biology
- Gene Expression
- Eukaryotic mRNA
Background:
- Polyadenylation of mRNA is a crucial post-transcriptional modification in eukaryotes.
- Polyadenylated (poly(A)+) and non-polyadenylated (poly(A)-) mRNA populations differ in stability and translation efficiency.
- Previous studies suggested limited homology between poly(A)+ and poly(A)- mRNA populations.
Purpose of the Study:
- To compare the protein products of poly(A)+ and poly(A)- mRNA from HeLa cells.
- To investigate the sequence homology between poly(A)+ and poly(A)- mRNA populations in detail.
Main Methods:
- Separation and in vitro translation of poly(A)+ and poly(A)- mRNA from HeLa cells.
- Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) for polypeptide analysis.
- Preparation of complementary DNA (cDNA) to abundant poly(A)+ mRNA sequences and subsequent hybridization with poly(A)- mRNA.
Main Results:
- Three classes of polypeptides were identified: exclusive to poly(A)- mRNA (approx. 10), common to both (approx. 40), and exclusive to poly(A)+ mRNA.
- Beta-actin was a major translation product for both mRNA populations.
- Approximately 10% of abundant poly(A)+ cDNA hybridized to poly(A)- mRNA, indicating a small overlap in sequences present at high concentration in the non-polyadenylated fraction.
Conclusions:
- Despite previous assumptions of low homology, a subset of poly(A)+ mRNA sequences is present at high concentrations within the poly(A)- mRNA fraction.
- This overlap in mRNA populations explains the shared protein products and necessitates a re-evaluation of mRNA classification based solely on polyadenylation status.