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Cytoplasmic poly(A) polymerases mediate cellular responses to S phase arrest
Rebecca L Read1, Rui G Martinho, Shao-Win Wang
1Cancer Research UK Molecular Oncology Laboratory, University of Oxford Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DS, United Kingdom.
Abstract:
The S-M checkpoint delays mitosis until DNA replication is complete; cells defective in this checkpoint lose viability when DNA replication is inhibited. This inviability can be suppressed in fission yeast by overexpression of Cid1 or the related protein Cid13. Fission yeast contain six cid1/cid13-like genes, whereas budding yeast has just two, TRF4 and TRF5. Trf4 and Trf5 were recently reported to comprise an essential DNA polymerase activity required for the establishment of sister chromatid cohesion. In contrast, we find that Cid1 is not a DNA polymerase but instead uses RNA substrates and has poly(A) polymerase activity. Unlike the previously characterized yeast poly(A) polymerase, which is a nuclear enzyme, Cid1 and Cid13 are constitutively cytoplasmic. Cid1 has a degree of substrate specificity in vitro, consistent with the notion that it targets a subset of cytoplasmic mRNAs for polyadenylation in vivo, hence increasing their stability and/or efficiency of translation. Preferred Cid1 targets presumably include mRNAs encoding components of the S-M checkpoint, whereas Cid13 targets are likely to be involved in dNTP metabolism. Cytoplasmic polyadenylation is known to be an important regulatory mechanism during early development in animals. Our findings in yeast suggest that this level of gene regulation is of more general significance in eukaryotic cells.
Insights
Fission yeast Cid1 and Cid13 proteins are cytoplasmic poly(A) polymerases, not DNA polymerases. Overexpression of these enzymes rescues cells with defective DNA replication checkpoints, suggesting a role in mRNA stability.
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- The S-M checkpoint ensures DNA replication completion before mitosis.
- Defects in this checkpoint lead to cell death upon DNA replication inhibition.
- Overexpression of Cid1 or Cid13 suppresses this inviability in fission yeast.
Purpose of the Study:
- To investigate the function of fission yeast Cid1 and Cid13 proteins.
- To determine if Cid1 and Cid13 possess DNA polymerase or poly(A) polymerase activity.
- To elucidate the role of cytoplasmic polyadenylation in DNA replication checkpoint pathways.
Main Methods:
- Biochemical assays to determine enzyme activity (DNA vs. RNA substrates).
- Analysis of protein localization (nuclear vs. cytoplasmic).
- Investigating the effect of gene overexpression on cell viability under replication stress.
Main Results:
- Cid1 functions as a poly(A) polymerase with RNA substrates, not a DNA polymerase.
- Cid1 and Cid13 are localized in the cytoplasm, distinct from known nuclear poly(A) polymerases.
- Substrate specificity suggests Cid1 polyadenylates cytoplasmic mRNAs, potentially including those for the S-M checkpoint.
Conclusions:
- Fission yeast Cid1 and Cid13 are cytoplasmic poly(A) polymerases with roles in mRNA regulation.
- Cytoplasmic polyadenylation is a significant gene regulatory mechanism in eukaryotes, extending beyond animal development.
- These findings highlight a conserved regulatory mechanism impacting cell cycle control and gene expression.