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2018 OUC-China
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Csy4 (Csy6f), a member of CRISPR family.
Csy4 is a 21.4 kDa protein that binds and cleaves at the 3' side of a stable RNA hairpin structure via sequence- and structure-specific contacts. Csy4 binds its substrate RNA with extremely high affinity and functions as a single-turnover enzyme. Tight binding is mediated exclusively by interactions upstream of the scissile phosphate that allow Csy4 to remain bound to its product. Substrate specificity is achieved by RNA major groove contacts that are highly sensitive to helical geometry, as well as a strict preference for guanosine adjacent to the scissile phosphate in the active site. A highly basic a-helix docks into the major groove of the hairpin and contains multiple arginine residues that form a network of hydrogen.
 Fig.1 The Csy4/Hairpin complex. Background of 2018 OUC-China' project
This year, we design a toolkit focused on translational regulation, which is composed of a RNA endoribonuclease (Csy4) and a RNA module (hairpin). In our project, the cleavage function of Cys4 releases a cis-repressive RNA module (crRNA, paired with RBS) from the masked ribosome binding site (RBS), which subsequently allows the downstream translation initiation. A Ribosome Binding Site (RBS) is an RNA sequence to which ribosomes can bind and initiate translation.
We want to achieve precise expression of proteins by using different Csy4 mutants. The aim is using one system to realize diverse expression. We focus on the sites which play an important role in binding and cleavage. Gln104 is located in the linker segment connecting the body of Csy4 to the arginine-rich area, which makes sequence-specific hydrogen bonding contacts in the major groove of the RNA stem to nucleotides G20 and A19. His29 is in its deprotonated form and functions as a general base during cleavage by activating the 2′-hydroxyl nucleophile through proton abstraction. The side chain of Tyr176 points into the active site and stacks on top of the His29 imidazole group, which plays a role in orienting His 29. Phe155 is to recognize the ssRNA-dsRNA junctions in RNA hairpin. Based on the molecular simulation and the theory of fluctuations, four mutants are chosen rationally: Q104A, H29A, Y176F, F155A.
 Fig.2 Four key sites of wild type Csy4.
This year, we used point mutations to redesign four mutants on the basis of Csy4-WT(BBa_K1062004) which are Csy4-Q104A(BBa_K2615004), Csy4-Y176F(BBa_K2615005), Csy4-F155A(BBa_K2615006) and Csy4-H29A(BBa_K2615007). The capabilities of cleavage and recognition are different for each Csy4 mutants, and we name them the Csy4 family. The combination of the Csy4 family members and the miniToe family members constitute a post-transcriptional regulatory toolkit for achieving different expression levels of target genes.
Csy4-WT, the wild type, is a member of the CRISPR family, and also the core member of our project. Csy4-WT can specifically recognize and cleave a 22nt hairpin structure, known as the miniToe-WT. We confirmed that Csy4-WT is the strongest of the Csy4 family through the analysis of the results of our fluorescence microscopy, flow cytometry and microplate reader experiments. And the strength of the remaining members of the Csy4 family shows a staircase pattern.
Csy4-Q104A, which is second only to Csy4-WT in strength in the Csy4 family, coming from point mutation, and we change the CAG(encoding Gln) to GCG(encoding Ala) on the 104th site based on Csy4-WT. It can also recognize and cleave the 22nt miniToe, regulating the expression of downstream genes. When we conducted experiments with the miniToe-WT combination and used sfGFP as the downstream target gene, we could see the experimental results that the sfGFP expression level of Csy4-Q104A was about half that of Csy4-WT.
Csy4-Y176F, the third-strongest in the Csy4 family. It is designed in the same way as Csy4-Q104A, but with the 176th site changed from TAC(encoding Tyr) to TTT(encoding Phe). It can be seen from the experimental results that the expression of downstream genes regulated by Csy4-Y176F is correlated with the stepwise decline of Csy4-WT and Csy4-Q104A.
Csy4-F155A, strength is the fourth in the Csy4 family. At point mutation, we changed its 155th site from TTC(encoding Phe) to GCG(encoding Ara). It has a weaker cleavage and recognition capability.
Csy4-H29A, the most special one of our Csy4 family, whose 29th site is changed from CAC(encoding His ) to GCG(encoding Ara). Csy4-H29A has a high binding affinity but has the lowest capacity of cleavage, so we call it dead-Csy4. There is no doubt that its downstream gene expression is the lowest in the family.
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