The objective of this application is to determine whether TIA-1 and TIAR, RNA-binding proteins that are essential for normal embryogenesis and germ cell maturation, regulate the cellular response to stress. Whereas TIA-1 and TIAR are concentrated in the nucleus at steady state, heterokaryon analysis reveals that both proteins shuttle between the nucleus and the cytoplasm. In response to environmental stress (e.g., heat shock, oxidative stress, UV irradiation), TIA-1 and TIAR accumulate in the cytoplasm where they co-aggregate with poly (A)+ RNA at discrete ribonucleoprotein particles that resemble the """"""""stress granules"""""""" (SGs) that harbor untranslated mRNAs in heat shocked plant cells. Preliminary results suggest that TIA-1 can promote the assembly of SGs. Because TIA-1 and TL R can function as translational repressors, their association with SGs might also contribute to the general translational arrest that accompanies environmental stress. In this capacity, the assembly and disassembly of TIA-1/R+ SGs is proposed to regulate the cellular response to stress by controlling the translation of essential survival factors.
The specific aims are: i) To identify the RNA and protein components of TIA-1+ and TLAR+ SGs, ii) To determine how stress promotes the assembly of TIA1/R+ SGs, iii) To determine how TIA-1 and TLAR function as translational repressors, and iv) To determine whether TIA-1/R+ SGs regulate the cellular response to stress.
These aims will be accomplished by comparing the RNA and protein composition of TIA-1+ and TIAR+ SGs using a combination of biochemical and immunochemical methods applied to cells derived from mutant mice lacking either TIA-1 or TIAR. We will use dominant negative mutants to determine the role of eIF-2a kinases in SG formation. We will determine how TIAR functions as a translational repressor by comparing the expression of target proteins (e.g., TNF-a, TIA-1) in wild type, TIA-1-/- or TIAR -/- cells. Finally, we will use TIA-1 mutants that either promote or inhibit SG formation to determine whether SGs regulate the cellular response to stress. By understanding the function of TIA-1/R+ SGs, we hope to learn how stressed cells decide whether to survive and repair the damage, or die by apoptosis. This information is relevant to human cancer and autoimmune disease.
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