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Identification of mRNAs associated with programmed cell death in immature thymocytes
G P Owens1, W E Hahn, J J Cohen
1Department of Cellular and Structural Biology, University of Colorado School of Medicine, Denver 80262.
Abstract:
Programmed cell death is an essential cellular process that occurs in epithelial turnover, neural development, and regulation of cell populations of the immune system. Thymocytes undergo programmed cell death in response to several inductive stimuli, including exposure to glucocorticoids or radiation. This program can be blocked by inhibitors of RNA or protein synthesis; this implies that new proteins are required to execute the death programs. To search for possible death-associated mRNAs, we directionally cloned cDNA representing mRNA from control and dexamethasone-treated thymocytes. These libraries were used to produce ample amounts of DNA and RNA used in subtractive hybridization for the removal of sequences present in both control and induced cells. The remaining unhybridized sequences were selectively amplified by polymerase chain reaction and cloned to produce a library enriched for sequences expressed in death-induced cells. From this library we isolated cDNAs of death-associated mRNAs. One of these mRNAs, RP-8, appears within 1 h after exposure to gamma radiation, and a second mRNA, RP-2, is observed within 2 h. Both of these mRNAs accumulate during a period when a reference mRNA, actin, is declining. RP-2 and RP-8 are no longer detectable after 6 h postinduction, when apoptosis and mRNA degradation are evident in the culture. Sequence analysis of RP-8 cDNA indicates the presence of a zinc finger domain suggestive of a possible DNA regulatory role for the RP-8 protein. cDNA sequence results on RP-2 classify the corresponding protein as an integral membrane protein. We conclude that RP-2 and RP-8 are death-associated mRNAs that should be functionally evaluated in the context of the death process. As previously suggested, it may be that a family of "death genes" is activated by various stimuli depending on the type of cell, in a manner somewhat analogous to the induction of heat shock (stress) protein genes.
Insights
Researchers identified novel mRNAs, RP-2 and RP-8, crucial for programmed cell death in thymocytes. These "death genes" are rapidly expressed following radiation or glucocorticoid exposure, indicating their role in initiating apoptosis.
Area of Science:
- Cell Biology
- Molecular Biology
- Immunology
Background:
- Programmed cell death (apoptosis) is vital for tissue homeostasis and immune system regulation.
- Thymocytes initiate apoptosis in response to stimuli like glucocorticoids and radiation.
- Apoptosis execution requires the synthesis of new proteins, suggesting the involvement of specific mRNAs.
Purpose of the Study:
- To identify and characterize mRNAs associated with programmed cell death in thymocytes.
- To investigate the temporal expression patterns of these death-associated mRNAs.
- To explore the potential functions of newly identified death-associated genes.
Main Methods:
- Directional cloning of cDNA from control and dexamethasone-treated thymocytes.
- Subtractive hybridization to remove common sequences.
- Polymerase chain reaction (PCR) amplification and cloning of differentially expressed sequences.
- Sequence analysis of isolated cDNAs (RP-2 and RP-8).
Main Results:
- Identified two novel death-associated mRNAs, RP-2 and RP-8.
- RP-8 mRNA appeared within 1 hour, and RP-2 within 2 hours post-induction.
- Expression of RP-2 and RP-8 peaked as actin mRNA levels declined.
- RP-8 contains a zinc finger domain, suggesting a DNA regulatory role.
- RP-2 encodes an integral membrane protein.
Conclusions:
- RP-2 and RP-8 are key death-associated mRNAs involved in thymocyte apoptosis.
- These findings support the hypothesis of a family of "death genes" activated by various stimuli.
- Further functional evaluation of RP-2 and RP-8 in apoptosis is warranted.