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In vitro deadenylation of mammalian mRNA by a HeLa cell 3' exonuclease
J Aström1, A Aström, A Virtanen
1Department of Medical Genetics, Biomedical Center, Uppsala University, Sweden.
Abstract:
We have identified a 3' exonuclease in HeLa cell extracts which deadenylates mammalian mRNA and leaves the mRNA body intact after poly(A) removal. Only homopolymeric adenosine tails located at the 3' end were efficiently removed by the exonuclease. The poly(A) removing activity did not require any specific sequences in the mRNA body either for poly(A) removal or for accumulation of the deadenylated mRNA. We conclude that the poly(A) removing activity is a 3' exonuclease since (i) reaction intermediates gradually lose the poly(A) tail, (ii) degradation is prevented by the presence of a cordycepin residue at the 3' end and (iii) RNAs having internally located poly(A) stretches are poor substrates for degradation. The possible involvement of the poly(A) removing enzyme in regulating mRNA translation and stability is discussed.
Insights
Researchers found a 3' exonuclease that removes poly(A) tails from mRNA in HeLa cells. This enzyme degrades only adenosine tails, leaving the mRNA intact, and may regulate gene expression.
Area of Science:
- Molecular Biology
- Gene Regulation
- Enzymology
Background:
- Messenger RNA (mRNA) stability is crucial for gene expression regulation.
- The poly(A) tail of mRNA plays a significant role in its stability and translation.
- Mechanisms of poly(A) tail removal (deadenylation) in mammalian cells are not fully understood.
Purpose of the Study:
- To identify and characterize enzymes responsible for poly(A) tail removal from mammalian mRNA.
- To investigate the substrate specificity and mechanism of the identified deadenylating enzyme.
Main Methods:
- Enzymatic assays using HeLa cell extracts.
- Analysis of RNA intermediates during poly(A) tail removal.
- Testing substrate specificity with modified RNA molecules.
Main Results:
- A 3' exonuclease activity was identified in HeLa cell extracts that specifically removes homopolymeric adenosine tails from the 3' end of mRNA.
- The mRNA body remained intact after poly(A) tail removal.
- The enzyme's activity did not depend on specific mRNA sequences.
- Evidence supporting a 3' exonuclease mechanism, including progressive tail shortening and inhibition by 3' cordycepin, was obtained.
Conclusions:
- A novel 3' exonuclease capable of deadenylating mammalian mRNA has been identified.
- This enzyme efficiently removes poly(A) tails without degrading the mRNA body.
- The enzyme's activity suggests a role in regulating mRNA translation and stability.