The Structural And Functional Research Of Schizosaccharomyces Pombe Dis312

Exonucleases are large enzymes that participate in RNA processing, which plays important roles in many life processes. Exonucleases can alter RNA levels by digesting aberrant RNA molecules, thereby contributing to RNA surveillance. Additionally, some precursors of RNA require trimming by exonucleases during their maturation process.The exosome is the most significant complex involved in the cleavage of RNA chains; the chains are cleaved repeatedly from the 3’to 5’ends one nucleotide at a time. Dis312 localizes in the cytoplasm and is involved in genetic interactions with components that function in the cytoplasmic mRNA-degradation pathway. For example, Dis312 can degrade uridylated pre-let-7 to block the expression of let-7 microRNAs in mouse embryonic stem cells. Human Dis312 mutations can cause Perlman syndrome, which results in overgrowth and is related to Wilm’s tumour. Moreover, the variability in chromosome number and mitotic errors in HeLa cells are increased after dis312 knockdown.Recently, many structural studies have focused on Dis312 and its homolouges. In 2014, the crystal structure of mouse Dis312-RNA complex has been solved by Faehnel (PDB code 4pmw). It adopts a similar conformation like those of the Rrp44-RNA complex (PDB code 2vnu) and the RNase Ⅱ-RNA complex (PDB code 2ix1):the three OB-fold domains (CSD1, CSD2 and S1) lie on the top face of the catalytic RNB domain to form the entrance to the RNA path, and the conserved RNA-binding residues in the RNB domain contribute to the formation of the catalytic region. However, there are also differences in the RNA path among the three homologous protein-RNA complexes. For example in the mouse Dis312-RNA complex the three RNA-binding domains adopt different orientations and form a funnel that defines a novel RNA recognition path that is more open and straight.The structure of mouse Dis312-RNA complex explains the mechanism of interaction between Dis312 and RNA substrate firstly, and find the answer why Dis312 specific choose poly U as substrate. However, there are still questions needed to answer, for example, the RNA-free structure of Dis312, and the whole RNA binding process of Dis312. For better understand the catalytic mechanism of Dis312, we solved the structure of RNA-free form of fission yeast Dis312. In our structure, CSDs is oriented on the side of RNB domain, instead of forming the entrance of the RNA channel with the S1 domain as observed in the mouse Dis312-RNA complex. The structural deviations are also observed between RNA-free form Dis312 and Rrp44/RNase Ⅱ-RNA complex. After eliminating the possibilities of sequence difference and crystal packing, we conclude that the different RNA binding state is the most likely reason that causes such a conformational change. The results of FPA indicate that the RNB and S1 domains contribute the major binding affinity for the RNA substrate, and the CSDs also paticipates in the interaction process. Combining the structural comparisons with mutagenic experiments, we speculate that fission yeast Dis312 could have a conformational change when binding RNA substrate.So far, there are two Dis312 structures have been solved, one is mouse Dis312-U14 complex, and the other is our fission yeast Dis312 structure in RNA-free form. In order to elucidate the catalytic mechanism of Dis312, we also have solved the crystal structure of fission yeast Dis312-U14 complex.Although the complex structure we solved is incomplete, which lacks the CSDs, the RNB and S1 domains fit the electron density map well, which means the conformation and mutual positions of the two domains are basically correct. The informations provided by RNA-free form Dis312 structure and partial Dis312-U14 structure indicate that, before interacting with RNA substrate, Dis312 presents a more open conformation with a large distance between CSDs and S1 domain. During the RNA-capturing process, CSD would need to have a orientational change to anchor the RNA substrate and enhance the affinity between fission yeast Diss312 and RNA substrate.