Investigation of the structure and function of the spliceosomal 220K/Prp8 protein

Splicing is an important stage of eukaryotic pre-mRNA maturation. During this process, the non-coding sequences, called introns, are removed and coding sequences, exons, are ligated together to create a continuous mRNA template for translation. The splicing process is performed by the spliceosome. The spliceosome is a highly dynamic RNP machine, built from several snRNP particles and numerous non-snRNP protein factors. The spliceosome assembles on the pre-mRNA in a stepwise manner directed by the conserved signal sequences: the 5' splice site, the branch-point and 3' splice site. After assembly, the spliceosome undergoes major remodeling of its RNA structure and its protein composition changes significantly. This process aims at the correct positioning of active groups in such a way that reactions of intron excision and exon ligation can occur without any requirement for external energy. However, details of the catalytic process remain unknown. One of the largest spliceosomal proteins, 220K, is the only protein which was photochemically cross-linked to all three signal sequences on the premRNA. The data previously obtained place this protein at the centre of the intricate network of protein–protein and protein–RNA interactions, suggesting that it may play an essential role in directing and regulating the splicing process. However, at the beginning of this study, information about this protein was quite scarce. In this work, we set out to investigate the domain structure of the 220K protein and to establish functions of the revealed domains. A domaindissection approach allowed us to create a fragment of 220K which could be overexpressed in E. coli in a soluble form. We were able to crystallize it and solve its structure. The structure of the domain revealed that it adopts a fold characteristic for enzymes of the RNase H family. This domain is exceptionally well suited for handling complex nucleic acid substrates and its presence in close proximity to the spliceosome active centre implies an active participation in the catalytic process. However, this 220K fragment retains only the spatial organization of the RNase H active site capable of coordinating catalytic metal ions, since the amino acid residues are different. Therefore, we set out to determine the importance of those residues for the splicing process by in vivo mutagenesis studies in yeast. In parallel, we tried to determine whether this protein domain is capable of catalyzing an RNase H-like nucleolytic reaction. Indeed we were able to find indications of such activity in the purified protein fragment. We also investigated whether this protein domain is capable of binding divalent metal cations and, thus, of supplying these metal ions to the splicing process. We were able to obtain evidence for a low-affinity binding of manganese ions to the RNase H-like domain using electron paramagnetic resonance spectroscopy. Taken together, our data suggest a more active involvement of the 220K protein in splicing catalysis than had previously been assumed and we suggest that the spliceosome is a genuine RNP enzyme. Finally, we also established a protocol for the co-expression of the fulllength human 220K and yeast Prp8 proteins in complex with the human 116K and yeast Snu114 proteins respectively. However, this expression and purification strategy still needs optimization to yield sufficient amounts of the complex for structural investigation.