Currently, our group engages in four specific research themes:

1. Involvement of various ribosomal proteins in a p53-dependent checkpoint response
As mention above, we previously demonstrated that RPS6 deficiency activates a p53-dependent checkpoint response. However, it is unknown to what extent this checkpoint response is triggered by a deficiency of any RP, or just a subset of these proteins. In order to overcome the paucity of animal models for RP deficiencies, we will perform gene silencing in cell culture. It could be assumed that some RP deficiencies will fail to activate p53. Indeed, RPL11 or RPL5 deficiency do not trigger the p53 response, but instead, prevent activation of p53 in response to inhibition of ribosome biogenesis by a low dose of actinomycin D.

2. Identification of cancer-associated mutations in p53-activating RP genes.
We will test in vivo importance of p53-activating RPS in p53-mediated tumor suppression (RPL5, RPL11 etc.). We hypothesize that mutations in these genes could arise in human cancers, granting an advantage to cells in escaping p53 surveillance. To test this hypothesis, we will systematically search for somatic mutations in p53-activating RP genes in various human cancer types. We predict that mutations these genes are likely to be found in cancers carrying wild-type p53 gene. It has been shown that some hemotherapeutics trigger p53 via specific RPS. Thus, it is possible that cancer cells could become resistant to chemoterapy by selecting mutations in these genes. To gain an insight into this issue, we will also sequence p53-activating RP genes in tumor samples from patients who received chemotherapy.

3. Characterization of the molecular steps of a p53-dependent checkpoint in response to RPS6 and RPL24 deficiencies. Our recent experiments revealed that silencing of either RPL24 or RPS6 induced a p53-dependent checkpoint response in cell culture. The prevailing dogma assumes that ribosome-induced checkpoint is triggered by defects in the synthesis or processing of rRNA in the nucleolus. This model has been based on analyses of cells, whose rRNA synthesis or processing was inhibited by actinomycin D or deficiency of RPS6, respectively. However, activation of a p53-dependent checkpoint by RPL24 deficiency is somewhat surprising, as we failed to detect a defect in rRNA synthesis and processing in the nucleolus upon deficiency of this protein. We intend to understand how these two ribosomal protein deficiencies activate a p53-dependent checkpoint at the molecular level.

4. Involvement of the p53 tumor suppressor in the phenotype of RPL24-deficient mice. The Belly spot and tail (Bst) mouse is a semi-dominant, hypomorphic mutation caused by an intronic deletion in the RPL24 gene, affecting RPL24 mRNA splicing. RPL24Bst/+ mice are characterized by reduced body size, a white ventral middle spot, white hind feet, retinal abnormalities, a kinked tail and other skeletal abnormalities. Since RPL24Bst/+ MEFs (mouse embryonic fibroblasts) from these mice showed a significant reduction in the rate of overall protein synthesis, it has been suggested that their phenotype result exclusively from faulty translation of mRNAs in tissues that depend on rapid and flawless protein synthesis. As mentioned above, we observed that RPL24 deficiency, which does not affect the nucleolar steps of ribosome biogenesis, also triggered the p53 response. This observation led us to consider the possibility that p53 is also up-regulated in RPL24Bst/+ mice. Indeed, we showed that the p53 tumor suppressor is aberrantly up-regulated during the restricted period of embryonic development. Our current experiments are aimed at understanding whether the p53 tumor suppressor is responsible for the Bst phenotype.