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Differential gene expression in apoptosis: identification of ribosomal protein S29 as an apoptotic inducer
N Khanna1, V G Reddy, N Tuteja
1Department of Biochemistry, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, 110029, India.
Abstract:
To identify genes that are specifically involved in apoptosis, poly(A)(+) RNAs were isolated from untreated control rat thymocytes and from adriamycin-induced apoptotic thymocytes. Directionally cloned cDNA libraries were then constructed in UNIZAP-XR vectors followed by biotin-based subtractive hybridization. Three clones were confirmed to be differentially expressed by dot blotting. Sequence analysis revealed homology to two genes previously identified, whereas one clone was novel and did not have homology to any known sequence. One clone was identical to the ribosomal protein S29, and the other was homologous to L8 ribosomal protein. Northern blot analysis revealed a marked increase in the expression of mRNA encoding ribosomal protein S29 in the apoptotic thymocytes compared to the controls. Transfection studies revealed that enhanced S29 expression resulted in increased apoptosis in rat thymocytes and HeLa cells as assessed by various morphological and biochemical characteristics, including cell shrinkage, chromatin condensation, membrane blebbing, formation of apoptotic bodies, TUNEL, FACS, and internucleosomal DNA fragmentation. This was accompanied by upregulation of p53, Caspase 3, and bax, whereas bcl-2 was downregulated as revealed by Western blotting. The current findings provide the first hint of a role for ribosomal protein S29 in the apoptotic process.
Insights
Ribosomal protein S29 enhances apoptosis. Increased S29 expression in thymocytes and HeLa cells promoted programmed cell death, affecting key apoptosis-related proteins.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Apoptosis, or programmed cell death, is crucial for development and tissue homeostasis.
- Identifying novel genes regulating apoptosis is essential for understanding cellular fate and disease.
- Previous research has identified numerous apoptosis-related genes, but the molecular mechanisms are not fully elucidated.
Purpose of the Study:
- To identify novel genes specifically involved in the process of apoptosis.
- To investigate the role of differentially expressed genes in adriamycin-induced apoptosis.
- To elucidate the functional significance of ribosomal protein S29 in programmed cell death.
Main Methods:
- Isolation of poly(A)(+) RNAs from control and apoptotic rat thymocytes.
- Construction of directionally cloned cDNA libraries and subtractive hybridization.
- Confirmation of differential expression using dot blotting, Northern blotting, and sequence analysis.
- Transfection studies in rat thymocytes and HeLa cells to assess the functional role of S29.
- Analysis of apoptosis markers (morphological, biochemical, TUNEL, FACS, DNA fragmentation) and protein expression (Western blotting).
Main Results:
- One novel differentially expressed clone was identified, showing homology to ribosomal protein S29.
- Northern blot analysis confirmed a significant increase in ribosomal protein S29 mRNA in apoptotic thymocytes.
- Enhanced S29 expression induced apoptosis in rat thymocytes and HeLa cells, evidenced by characteristic apoptotic features.
- Upregulation of p53, Caspase 3, and Bax, and downregulation of Bcl-2 were observed following S29 overexpression.
Conclusions:
- Ribosomal protein S29 plays a significant role in promoting apoptosis.
- Overexpression of S29 influences the expression of key apoptosis regulatory proteins, including p53, Caspase 3, Bax, and Bcl-2.
- These findings suggest a novel function for ribosomal protein S29 in the apoptotic pathway.