Structure and Function of hnRNP Proteins

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9514179 Williams Heterogeneous nuclear RNAs (hnRNA) are found in the nucleus in complexes with more than 20 hnRNP proteins. A1 hnRNP is unusual in that it appears to have several functions in addition to its structural role in hnRNP fiber formation. A1 interacts with intermediate filaments; modulates alternative 5'-splicing; can recognize a high affinity RNA target sequence that is similar to splice site consensus sequences; and facilitates annealing of complementary nucleic acids. The latter "activity" is controlled by phosphorylation of a critical serine residue in A1. A1 hnRNP also contains two conserved RNA binding domains at its NH2 terminus, and has an RGG repeat which may be a significant RNA binding domain. In these studies, the yeast two hybrid system will be used to identify other proteins that interact with A1 in vivo. Circular dichroism, fluorescence spectroscopy, and nuclear magnetic resonance will be used to probe the structure and nucleic acid binding properties of the RGG motif, and to locate sites of methylation within the domain. The methylase that carries out the posttranslational modification of the RGG will be cloned and analyzed. The results of these studies will significantly increase our understanding of a multi-functional hnRNP protein that plays a key role in pre-mRNA biogenesis. %%% Although all of the information contained within a cell is encoded in its DNA, most of the actual functions of cells and the organs and systems that they make up are carried out by proteins that are in turn composed of varying sequences of amino acids. Since proteins are made in the cytoplasm of the cell while the DNA that encodes them is in the nucleus, a molecule called messenger RNA (mRNA), structurally related to DNA, serves to transport the "message" for protein synthesis from the nucleus to the cytoplasm. In general, there is a unique mRNA for each protein. This project is directed at understanding how a particular class of nuclear proteins (hnRNP proteins) that are cri tical for the formation and transport of mRNA carry out their functions. In particular, how hnRNP proteins bind to mRNA and to other proteins will be investigated so that this critical step in gene expression will be better understood. ***