The proposed research will rest two hypotheses concerning hyperthermia. The first hypothesis is that the interactions of hsc 70 protein, its ribotron U14 snRNA, and the nucleolar protein, fibrillarin, are essential to nucleolar structure and function under normal growth conditions are disrupted by elevated temperatures. The second hypothesis is that the increased expression of hsc 70 and U14 snoRNA results in increased resistance to heat-induced cell death. Understanding these roles will help explain intrinsic cellular heat resistance, which is one of the limiting factors to the efficacy of hyperthermia in the clinic. Specifically we will 1) determine the pattern of expression of U14 snoRNA in mammalian cells exposed to various stress conditions. We will examine the regulations and control of the expression of this gene product in terms of its transcription, processing and turnover. These regulatory characteristics will be compared with the regulatory characteristics associated with the expression of the hsc 70 protein. 2) Determine the function of U14 snoRNA in ribosomal biogenesis. This task will be accomplished by generating two types of inducible antisense expression vectors containing the U14-5 ribotron, transfecting them into hamster cells and then determining the effect of the expression of such antisense constructs on the metabolism of ribosomal RNA. 3) Determine the functional aspects of the interaction of hsc 70 with nucleolar proteins and U14 snoRNA in cells. First. we will systematically investigate the interactions among hsc. 70. several nucleolar proteins and U14 RNA using immunofluorescence, immunofluorescence, immunoprecipitation and in-situ hybridization. Second, we will construct deletion mutants of various sizes in the hsc 70 by examining the altered hsc 70 in terms of cellular localization, interaction with nucleolar proteins and with U14 snoRNA, in both normal and stressed cells. 4)Determine whether or not U14 snoRNA in concert with hsc 70 and nucleolar proteins play a role in both heat-resistance and heat-sensitivity by examining the processes delineated in specific aims 1 and 2 in transiently and permanently heat resistant cells and heat sensitive cells. This task will be accomplished by determining both the heat-induced alterations in U14 snoRNA in the phenomenon of protein synthesis independent thermotolerance, a thermotolerant state which is different from that which will be studied in 4, in that it is only induced by heat shock and develops in the complete absence of protein synthesis. This task will be accomplished by comparing the metabolism of hsc 70 transcripts in cells recovering from conditions which generate protein synthesis independent thermotolerance and from conditions which do not.
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