The long-term goal of this proposal is to increase our understanding of the regulatory network underlying muscle differentiation in the course of human development and aging. Genetic complementation experiments revealed that expression of the 3' untranslated regions (3'UTRs) of certain cytoskeletal genes can activate stably transfected muscle specific promoters and the endogenous myogenin gene in a differentiation-defective myoblast mutant, NMU2. The shortest and most potent, the 214 nucleotide alpha-tropomyosin 3'UTR sequence (F1), was studied further and shown to arrest proliferation of fibroblasts and suppress anchorage-independent growth and tumor formation by NMU2 cells. These effects were only observed in cells expressing a relatively high level of F1 RNA, comparable to that in human muscle tissue. Moreover, in the rare tumors that developed, the F1 construct was retained but F1 RNA expression was extinguished, further suggesting that the effects of F1 required the continued expression of 3'UTR transcripts. These findings add to the growing body of evidence indicating that untranslated RNA sequences may have a regulatory role in growth and differentiation. The goal of this proposal is to enhance our understanding of the mechanisms by which the 3'UTR RNAs exert their effects. Two independent approaches will be pursued in parallel. (I) We will test the hypothesis strongly suggested by preliminary data that 3'UTR RNAs such as F1 function as cellular double-stranded RNAs (dsRNAs) in signal transduction pathways known to play a role in growth control. These pathways are the interferon activated RNase L and dsRNA activated protein kinase (PKR) pathways. Although viral RNA activators of these enzymes are known, endogenous RNA activators have not yet been identified. (II) In parallel, a more global approach to elucidating the mechanisms of action of the 3'UTRs will be undertaken. The minimal active sequence in each 3'UTR will be determined based on functional assays, and the proteins that bind specifically to these sequences will be cloned and their functions determined. (III) The role in myogenesis of the signal transduction pathways suggested by the hypothesis in (I) will be explored. Both PKR and RNase L have been implicated as regulators of growth with a role in differentiation and tumor suppression, but these potential functions have not been directly tested. Using a tetracycline inducible expression system that allows both temporal and quantitative regulation of gene expression, the cDNAs that encode wildtype enzymes will be tested for their ability to induce myogenesis and suppress tumor formation, and cDNAs encoding dominant negative forms for the enzymes will be tested for the converse effects. The findings of the proposed studies will contribute to our understanding of the mechanisms by which untranslated RNAs can function as regulators of growth and differentiation (riboregulators). They may also prove useful in the design of therapeutic strategies directed at enhancing muscle gene expression in certain muscular degenerative diseases or suppressing the neoplastic behavior of rhabdomyosarcomas.
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