Heterogeneous nuclear ribonucleoprotein (hnRNP) proteins bind nascent pre-mRNA transcripts in a sequence-dependent manner and influence the fate of these RNAs and, thus, the expression of their genes. The full range of specific tasks of hnRNP proteins is unknown and is thought to include RNA processing, transport, and localization. Determining the interactions hnRNP proteins have with other proteins and cellular structures is vital to understanding their functions. The focus of this proposal is the protein-protein interactions of the abundant human hnRNP proteins A1, A2, and C1. The first specific aim of this study is to identify and characterize proteins which interact with A1, A2, and C1. This will be done using the yeast two-hybrid system, affinity chromatography, and immunoprecipitation. Interactions will be confirmed by chromatography, far-western analysis, co-localization by Immunofluorescence, and co-immunoprecipitation.
The second aim of this study is to localize the protein domains involved in these interactions, using deletion analysis, and to possibly determine specific amino acids with in these domains required for interactions using site-directed mutagenesis. The results of these studies will be relevant to other RNA- binding proteins which share common modular domain motifs with these hnRNP proteins, several of which have recently been implicated in human diseases. These studies will provide information indispensable to the long term goal of this set of experiments, which is to understand the roles that hnRNP proteins play in the essential and fundamental areas of RNA metabolism and gene expression. Understanding these basic cellular events provides the basis for developing strategies to combat human disease.
