Significance of modularity in the RNA-binding hnRNP and SR proteins

RNA-binding proteins are a large group of structurally diverse molecules that associate with nascent transcripts and other proteins through their structural and functional domains. The presence of multiple copies and various arrangements of the different structural domains are important in generating functional versatility as well as defining specificity for these RNA-binding proteins. As a result, RNA-binding proteins often perform both specific and redundant or overlapping functions in multiple aspects of ribonucleic acid metabolism. The heterogeneous nuclear ribonucleoproteins (hnRNPs) and the serine/arginine-rich (SR) proteins are two of the most abundant groups of RNA-binding proteins that share similar functional properties (binding to nascent transcripts, antagonistic effect on alternative splicing, and multiple roles in various aspects of RNA metabolism). These two groups of proteins, especially the hnRNP A/B subgroup and the core SR proteins, also share common features within their protein structure, which are comprised of one or more RNA recognition motifs (RRMs) linked to an auxiliary domain that is glycine-rich or arginine/serine-rich, respectively. Despite the similarities, members from the hnRNPs and the SR proteins also perform specific RNA regulatory functions. Functional studies have repeatedly emphasized that RNA-binding specificity is achieved by the combinatorial effect of the different domains within the RNA-binding protein structure. The RRM is a well-studied RNA-binding motif that is common to many RBPs. The less-studied auxiliary domain, especially the glycine-rich domain, is known to be involved in protein-protein and protein-RNA interactions. Although specific functions are assigned for the different domains of hnRNPs A/B and SR proteins, these domains are generally studied and characterized apart. Hence, in order to understand the significance of modularity in contributing to both specific and non-specific functions of an RNA-binding protein, it is important to understand the relationships between their protein domains and how these domains cooperate as a complete unit. To examine similarities and differences between the RNA-binding proteins that contribute to both functional redundancy and specificity, sequence and phylogenetic analyses at the protein, domain and functional motif levels were performed in Chapter 3 to gain a broader understanding of their evolutionary relationships. We found that there are significant sequence similarities between the different groups of RNA-binding proteins studied, including the hnRNPs and SR proteins, which share common RNA-binding RRMs. Phylogenetic analyses of the RRM domains between these proteins however, suggest a diversification of the domain sequences in the early evolution of metazoans, and subsequently strong selective pressure to maintain overall domain structure and their consensus motif sequences. Hence, although RRMs are structurally and functionally conserved, there are limited sequence similarities within the RRMs between different RNA-binding proteins that can contribute to target selectivity. This diversification, therefore, is thought to allow RRM-containing RNA-binding proteins to have redundant as well as specific roles in co- and post-transcriptional processing. In addition, the presence of the different auxiliary domains that link with these RRMs further facilitates the functional specialization of the different RNA-binding proteins in RNA regulation. The importance of the auxiliary domains, especially the glycinenrich and the arginine/serine-rich domain from hnRNPs and SR proteins, in specifying biochemical activities of a RNA-binding protein were further addressed in Chapter 4 and 5. We created a series of chimeric proteins consisting of domain swaps between different hnRNP A/B paralogues, and between hnRNPs A/B and SR proteins, to examine the contributions of the different functional domains in sub-nuclear localization of these proteins. We found that the glycine-rich domain of hnRNPs A/B, rather than the RRMs, has a dominant role in determining the nuclear localization and the enrichment of the wild-type and heterologous proteins in different subnuclear compartments. As well, both the RRMs and the arginine/serine-rich domain are irreplaceable in specifying the functional characteristics of the SR proteins. Furthermore, we confirmed that the glycine-rich domain is important in mediating the co-localization of hnRNPs A/B with the paraspeckle protein p54nrb within the subnuclear paraspeckle compartment. Interestingly, immunoprecipitation and RT-PCR analyses suggested that the hnRNPs A/B directly interact with the paraspeckle structural component NEAT1 long non-coding transcript, but not p54nrb. Hence, our protein subnuclear localization studies have collectively suggested that the auxiliary domain is an essential determinant in conferring specificity to the RNA-binding proteins in RNA regulation. In conclusion, this study highlights the significance of the cooperation between the different functional domains, especially the RRMs and the auxiliary domain, in both redundant and specific functions of the different RNA-binding proteins. In particular, our results have reinforced the view that specific RNA-binding protein functions are determined by the combination of different structural domains function in concert as a complete unit. Altogether, this study provides a better understanding of how RNA-binding proteins benefit from the complex modular networks between the different domains that contribute to both sequence-specific and non-specific activities of these proteins in essential gene regulation.