Structure And Function Of The Tandem WW Domains Of The Pre-mRNA Splicing Factor FBP21

Gene expression in eukaryotic cells involves several steps, including transcription, mRNA processing, and export. Each stage is physically and functionally connected by DNA-RNA-protein interaction network, ensuring that there is efficient and precise transfer between manipulations. Pre-mRNA splicing takes place in the spliceosome, a highly dynamic ribonucleoprotein particle that consists of five small nuclear RNAs and hundreds of proteins. Small nuclear ribonucleoproteins (snRNPs) and numerous protein factors are essential for the formation of the active spliceosome. In budding yeast, the splicing factor Prp40 participates in cross-intron bridging by interacting with the branch point-binding protein (BBP) and the U5 snRNP component Prp8. Prp40 contacts the 5'splice site and interacts with BBP, bringing the 5'splice site and the branch point in spatial proximity. These interactions are believed to be conserved in mammals. FBP21 (formin binding protein 21), the mammalian Prp40-like protein, contains a matrin-type zinc finger and two group III WW domains. FBP21 is a component of the mammalian spliceosomal A/B complex, associated with U2 snRNPs and colocalizes with splicing factors in nuclear storage sites for pre-mRNA splicing factors. Moreover, FBP21 interacts directly with the splicing factors U1 snRNP protein U1C, the core snRNP proteins SmB/SmB', and the branch point-binding protein SF1/mBBP. Although the above data strongly suggest that FBP21 has a role in pre-mRNA splicing, there are no in vivo data to support this contention.In this dissertation, we report the NMR structure of the tandem WW domains of the human spliceosomal ptotein FBP21, which represents the first available structure of WW domains of group III. The structure of the FBP21 tandem WW domains suggests a considerable flexibility in the relative orientations of WW1 and WW2 toward each other. NMR backbone relaxation experiments and 1DNH RDCs measurements indicated that the linker region between the tandem WW domains is highly flexible and lacks a well ordered structure. The linker flexibility leads to some interdomain motion between the tandem WW domains. The ligand specificity and binding interface of the tandem WW domains, as well as the importance of having two WW domains, have been explored by NMR chemical shift perturbation and isothermal titration calorimetry (ITC). Our data reveal a cooperation between the two tandem WW domains in ligand binding, which results from the conjugation of two low affinity binding sites and the presence of a highly flexible linker region between them. Although a number of structures of individual WW domains have been solved, only two structures of tandem WW domains have been determined. As for the third solved tandem WW domains structure, our study reveals that even with high sequence identity and similar domain array, the mechanism of tandem WW domains binding to ligand, the relative domain orientation, and dynamics can be very different and context-dependent.We further explored the biological implication of FBP21 and its tandem WW domains. Here we demonstrate for the first time that FBP21 is an activator of pre-mRNA splicing in vivo and its splicing activation function is mediated by the two tandem WW domains of group III. Importantly, the stimulation of pre-mRNA splicing requires both WW domains and the flexible interdomain linker. The splicing activation effect of FBP21 was nearly completely abolished by mutation of only one of its two WW domains. Shortening the linker between the two WW domains also substantially decreased the splicing efficiency of FBP21. GST pull down assays and immunoprecipitation were used to study the in vitro and in vivo interaction between FBP21 and splicing factor SIPP1. We also studied the subcellular localization of FBP21 using immunofluorescence assays. The interaction with SIPP1 and subcellular localization of FBP21 are both mediated by the two tandem WW domains. This can be explained well by our observation from structural data that each of the WW domains of FBP21 represents a relatively weak binding site and the high affinity binding of ligand requires the simultaneous interaction of both WW domains, and the linker flexibility enables their simultaneous interaction with more than one ligand. The results from functional exploration consist very well with the structural data. Our study provides a structural basis for understanding the molecular mechanism underlying the functional implication of FBP21 and the biological specificity of tandem WW domains.