The overall objections of this proposal are to study the biochemical mechanisms involved in the regulation of gene expression during growth and differentiation of limb bud. It is currently believed that transcriptional control is the major site of regulation of gene expression in eukaryotic cells. However, translational controls involving a variety of mRNAs, particularly in very early stages of embryonic development are known to operate. These controls are mediated through interaction of mRNAs with cellular macromolecules and alterations in components of the translation machinery. We have recently isolated and characterized a novel cytoplasmic translation inhibitory 10S ribonucleoprotein (iRNP) containing a 4S heterogeneous RNA (iRNA) species from 13-14 day old chick embryonic muscle. The iRNA and iRNP are potent inhibitors of mRNA translation in vitro. Their site of action is a single step in the initiation phase of protein synthesis, namely by blocking mRNA binding to the 43 S preinitiation complex. The iRNA appears to act as an """"""""anti-messenger RNA"""""""" and its in vivo function may involve modulation of cellular translation patterns. Translation inhibitory RNA and RNP particles, similar to iRNA and iRNP, have also been reported in a wide variety of eukaryotic cells and embryonic tissues, although they have not been well characterized. The objectives of this project are: to probe whether iRNA and iRNP-like """"""""regulators"""""""" function in limb bud and somites; if iRNA and iRNP-regulated translational control is operating in limb bud, to further characterize the iRNA species with respect to resolution and characterization of subspecies, delineation of their sequences; quantitation of their levels as a function of limb bud growth and correlation of their cellular levels with those of specific components of the exctracellular matrix e.g. fibronectin and type 2 collagen; to define the mechanism of their interaction with mRNA and to isolate and characterize their genes. Together with comparative studies on iRNA and iRNP particles of chick embryonic muscle, the proposed studies will increase our understanding of the structure of iRNA and iRNP and their role in the regulation of gene expression during embryonic development.
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