Messenger RNA (mRNA) in eukaryotic cells appears to be associated with a set of proteins to form ribonucleoprotein (RNP) complexes. These complexes are considered to be the true functional units of mRNA and their proteins are widely believed to play a central role in mRNA synthesis, processing, storage and translation. However, little is known about the proteins which are in direct contact with mRNA in vivo. Recent observations also suggest that most of the mRNA is associated with a complex cytoplasmic proteinacious network of fiber-forming and contractile proteins referred to as the cytoskeleton. This association seems to be important for mRNA translation and suggests there may be a link between cellular skeletal structures and regulation of gene expression. This hypothesis must be tested through detailed molecular examination of the proteins which are associated with mRNA. The study of the complexes of mRNA with specific cytoplasmic proteins is the focal point of this proposal. To identify mRNP proteins in vivo, UV irradiation of intact cells is used to induce crosslinking of mRNA to its associated proteins. The attributes of this technique make it extremely suitable for this purpose. Preliminary experiments enable the identification of a small subset of proteins which become crosslinked to poly(A)+mRNA in HeLa cells. The crosslinked complex will be used to raise monoclonal antibodies to the major proteins of the bulk of the cytoplasmic mRNPs. These will be used to study the intracellular localization of mRNP proteins, to obtain additional information about them and to begin to examine their possible function. The possible association of mRNPs with cytoskeletal elements will also be investigated. The proteins which are associated with a small, distinct set of mRNAs--VSV mRNAs--will also be determined.
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