Fox-1 RBD: a protein domain binding strongly and specifically to RNA sequence UGCAUGU.
Sequence and Features
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Usage and Biology
In many projects involving RNA approaches, the availability of a library of proteins able to bind strongly and specifically to an RNA target sequence represents a valuable and versatile set of BioBricks. Here we describe Fox-1 RBD, a protein domain able to recognise and bind a specific RNA sequence.
Fox-1 stands for Feminizing Locus on X, a protein acting as a numerator element to count the number of X chromosomes relative to ploidy. In worms, Fox-1 is thought to determine sex by post-transcriptional repression of Xol-1. In mammalian genes, it strongly influences the splicing of alternative exons by binding to “UGCAUG” RNA sequence . Orthologs are present in a wide range of multicellular eukarya species like zebrafish (Q642J5, Uniprot), mice (Q9JJ43, Uniprot), rats (A1A5R1, Uniprot), bulls (A6QPR6, Uniprot), frogs (Q66JB7, Uniprot), chicken (F1NCA4, Uniprot), polar bear (A0A384DA29, Uniprot), cats (A0A337SR52, Uniprot), platypuses (F6SR19, Uniprot), sunfishes (A0A3Q4B4Y5, Uniprot), tasmanian devils (G3VXH1 , Uniprot) and humans (Q9NWB1 , Uniprot).
This protein has a domain of interaction with RNA called RNA Binding Domain (RBD), also commonly referred as RNA Recognition Motif (RRM) and Ribonucleoprotein (RNP) domain, referring to the domain formed by the residues 109 to 208 of Fox-1 (13.6 kDa when his-tagged). Its RBD has shown a high affinity (Kd = 0.49 nM at 150mM NaCl) to “UGCAUGU” RNA sequence where residues F126 and F160 play a crucial role in its binding capacity . Interestingly, it has also shown a high specificity to the mentioned sequence where single mutations can reduce from 4,8 to more than 1.500 fold the affinity with its substrate , becoming a powerful BioBrick where a highly specific and affine interaction is needed between a protein and RNA. As shown in its crystal structure, C-terminal of the RBD is distant from the binding site and can accept a His-tag without showing a negative effect on the binding capacity , probably providing an adequate locus to fuse other domains or proteins. A mutated version of the protein domain has been expressed and tested as a control, where mutations F160A & F126A deplete its binding capacity  (BBa_K3407005).
Fox-1 RBD and RBD* (mutated) overexpression and purification
In order to check the overexpression of Fox-1 RBD and its mutated version (BBa_K3407005), we incubated the E. coli BL21 (DE3) (Negative control) and E. coli BL21 (DE3) transformed either with the pBbB7a_Fox-1_RBD (BBa_K3407020) or pBbB7a_Fox-1_RBD* (BBa_K3407024) at 37ºC until the OD600 reached ~ 0.6. We induced the cultures with a final IPTG concentration of 1 mM and incubated them 4h at 37ºC and overnight at 30ºC. The total protein content of the cells was analysed by SDS-PAGE electrophoresis (Figure 2A). As the recombinantly expressed Fox-1 RBD and RBD* (mutated) contain a His-tag at their N-terminal part, we purified them using affinity chromatography with the HisLink protein purification system (Promega). Samples obtained from the different purification steps were analysed by SDS-PAGE electrophoresis (Figure 2B).
Additional protein bands corresponding to molecular weights of 13.6 kDa (Fox-1 RBD) and 13.4 kDa (Fox-1 RBD*, mutated) were observed after induction (Figure 2A), indicating that both proteins were overexpressed. Moreover, a clear band is obtained in the elution samples (E) (Figure 2B), demonstrating that both Fox-1 RBD and RBD* were successfully purified.
Fox-1 RBD binds to its target sequence located in a shRNA loop
To study RNA-protein interaction, an Electrophoretic mobility shift assay (EMSA) was performed (Figure 3). When a protein binds to RNA, the complex migrates slower in the gel, shifting the RNA band to a higher position. shRNA (BBa_K3407006), containing “UUGCAUGUU” sequence in its loop, and Fox-1 RBD (BBa_K3407004) were first incubated in 1:1 and 1:2 molar ratios shRNA:Fox in binding buffer. 400ng of RNA were loaded in each lane corresponding to 10 fmol, along with 10 fmol of Fox-1 RBD (1:1 ratio) or 20 fmol (1:2 ratio) in a total of 15 μL reaction. Samples were then loaded in a native polyacrylamide gel to avoid protein denaturation.
To demonstrate binding specificity, the same experiment was performed with Fox-1 RBD* (BBa_K3407005), a mutated version of Fox-1 with a reduced affinity for its binding sequence. In addition, the assays were also carried out with shRNA* (BBa_K3407007), a version of the shRNA with two mutations in the loop, which inhibits binding to Fox-1 RBD  (Figure 3).
The non-bound shRNA and shRNA* appear on the gel as ~30bp dsRNA (negative control, Figure 4). Incubation of shRNA and ahRNA* with wild-type Fox-1 RBD leads to a notable shift of the RNA band at both 1:1 and 1:2 molar ratios. This upper band corresponds to the shRNA-Fox-1 RBD complex, showing that both purified compounds are active. The lower fraction of free shRNA in the 1:2 molar ratio indicates that increasing the concentration of Fox-1 RBD yields a higher amount of complex formed.
Interestingly, incubation of shRNA with mutated Fox RBD* does not lead to complex formation. This results corroborates literature data showing that the introduced mutations strongly reduce the affinity of the protein to the target RNA sequence. Surprisingly, Fox-1 RBD (denoted as Fox), but not Fox-1 RBD* (denoted as Fox*), is also able to bind to mutated shRNA*, suggesting that under the studied conditions the mutations in the RNA loop sequence do not abolish complex formation (Figure 5). Different results were previously obtained using surface plasmon resonance , which may be explained by differences in the experimental conditions.