The U1A/U2B /SNF Family of RNA Binding Proteins: Evolution of RNA Binding Specificity and Contributions of Heterotropic Linkage to snRNP Protein Partitioning

The U1A/U2B /SNF is a family of RNA binding proteins that is a highly conserved throughout eukaryotes. These proteins are found in the U1 and/or U2 splicing snRNPs (small nuclear ribonucleoprotein particles). In humans, U1A and U2B specifically bind to the U1 and U2 snRNAs, respectively. The Drosophila genome codes for SNF, an essential protein that localizes to both the U1 and U2 snRNP. While a specific splicing functions for these proteins have not been determined, their conserved snRNP localization suggests an important splicing-related function. The difference in protein number and partitioning between Drosophila and humans suggested that these proteins may use different RNA binding mechanisms to function in their cellular contexts. This work begins by exploring some of the differences amongst human U1A, U2B , and Drosophila SNF. The thermodynamics of the RNA-protein interactions also reveal substantial differences in the RNA binding mechanisms of these proteins. Further studies investigate the evolution of this protein family in metazoans. Reconstructing the protein phylogeny permitted resurrection of ancestral proteins. This led to the discovery that the last common ancestor of humans and Drosophila had a single U1A/U2B /SNF family homolog. This protein had RNA binding properties that most closely resemble those of Drosophila SNF. Evolution of protein motions and RNA binding specificity toward the defining characteristics of modern vertebrate proteins is also examined. Finally, linkage effects between protein-protein and protein-RNA interactions are analyzed. U2A\u27; is a U2 snRNP-specific protein that binds to U2B ; in humans and SNF in Drosophila. In Drosophila, large, positive linkage was only seen between U2A\u27-SNF and SNF-U2 snRNA binding. The RNA dependence of enhancement for SNF binding to U2A\u27 can explain the observed protein partitioning of U2A\u27 in vivo. For the more complicated human system, which contains two SNF homologs, substantial contributions to protein partitioning come from differences in both intrinsic RNA-protein binding affinities and differences in protein-U2A\u27 binding affinities. RNA dependence of the linkage parameter also contributes to protein partitioning. The binding parameters can explain U2A\u27 protein partitioning, and the presence of U2A\u27 reinforces U1A and U2B partitioning to their respective snRNAs. These linkage studies have important implications for the assembly of RiboNucleoprotein Particles, macromolecular complexes that are fundamental to many cellular activities