Pre-mRNA/mRNA-binding proteins play critical and multifunctional roles in the post-transcriptional regulation of gene expression, including pre-mRNA processing, mRNA stability, transport and translation. Most RNA-binding proteins contain RNA-binding motifs for RNA-protein interactions as well as auxiliary domains for protein-protein interactions. This work began with the investigation of the RNA binding and the protein-protein interaction properties of the heterogeneous nuclear ribonucleoprotein C1 (hnRNP C1), which is an abundant nuclear-restricted RNA-binding protein involved in splicing. Using random PCR mutagenesis coupled with phage display screening methods, we identified essential residues in the RNP motif RNA-binding domain (RBD) for C1 RNA-binding and found a leucine-rich coiled-coil domain that mediates C1-C1 protein interactions (CID). Mutations in CID abolished C1 protein-protein interactions as well as reduced RNA-binding, indicating that oligomerization has an effect on the RNA-binding activity of the protein. While C1 represents the non-shuttling hnRNP proteins, hnRNP A1 exemplifies the shuttling pre-mRNA/mRNA-binding proteins that accompany mRNAs from the nucleus to the cytoplasm, and it is therefore a potential mRNA export mediator. Therefore, we studied the interactions between hnRNP A1 and the components of the nucleo-cytoplasmic transport machinery. We identified RanBP(L), a novel interacting protein with RanGTPase, the key regulator of nuclear transport directionality. We further characterized RanBP(L) and delineated a novel RanGTP binding domain. Finally, we studied another RNA-binding protein, the fragile X mental retardation syndrome protein (FMR1). FMR1 is a cytoplasmic RNA-binding protein likely playing a role on translation in the cytoplasm. We isolated and characterized the first invertebrate FMR1/FXR gene family member from Drosophila melanogaster ( dfmr1). Compared to the vertebrate FMR1/FXR proteins, dFMR1 has conserved amino acid sequence, similar RNA-binding and protein-protein interaction activities, and a similar embryonic expression pattern. Overexpression transgenic flies were constructed to study analogous point mutations in the RNA-binding domains to those found in patients. We conclude that, both in the nucleus and in the cytoplasm, RNA-binding proteins have diverse functions and play essential roles in gene expression by interacting with target RNAs and protein factors. Perturbation of such interactions may lead to the pathogenesis of human genetic disorders.
Thesis (Ph.D. in Cell and Molecular Biology) -- University of Pennsylvania, 2001. Source: Dissertation Abstracts International, Volume: 62-11, Section: B, page: 4960. Supervisor: Gideon Dreyfuss.