Results

1. Construction of the plasmids

A previous study indicated exons of MAP3K7 and ZFN91 were skipped in MDS patients. In order to detect the dysregulation of RNA splicing, we designed two exon-skipping LUC reporters (MAP3K7-LUC and ZNF91-LUC) which contains parts of the MAP3K7 or ZNF91 intronic and exonic sequences in the luciferase gene.

1.1 Construction of MAP3K7-LUC and ZNF91-LUC

The plasmids of MAP3K7-LUC and ZNF91-LUC were synthesized by Nanjing Genscript Biotechnology Corporation. The map of these two plasmids was listed below (Fig 1). When we got these plasmids, we transformed them into DH5α bacteria and screened the positive clones on LB solid plates containing ampicillin (Fig 2). To test the quality of these plasmids, MAP3K7-LUC and ZNF91-LUC were analyzed on 1% Agarose Gel (Fig 3). Moreover, MAP3K7-LUC and ZNF91-LUC were also confirmed by using sequencing (Fig 4).

Fig 1. Map of the pGL-duel plasmid vector containing parts of the MAP3K7 or ZNF91 intronic and exonic sequences

Fig2. Solid plates of MAP3K7-LUC sensor and ZNF91-LUC sensor

Fig 3. Electrophoresis of MAP3K7-LUC sensor (1) and ZNF91-LUC sensor (2).

Fig 4. The MAP3K7-LUC (up) and ZNF91-LUC (below) plasmids sequencing

2. Functional verification of MAP3K7-LUC and ZNF91-LUC plasmid sensors

In order to test the ability of MAP3K7-LUC and ZNF91-LUC to detect dysregulation of RNA splicing, Pladienolide B (PB, an RNA splicing inhibitor) was used. The plasmids MAP3K7-LUC and ZNF91-LUC were transfected into 293T cells, respectively. PB (final concentration: 1 ng/μl) was added to disturb the process of RNA splicing. According to the principle of our plasmid sensors, the fusion proteins (Fluc-Rluc) were expressed in 293T cells. However, when RNA splicing was interrupted, only the Fluc proteins were induced (more details about the principle were listed in the project description). After transfection, cells were lysed and the expression of luciferase was measured by using plate reader (SpectraMax i3). In normal 293T cells, the expression of the fusion proteins (Fluc-Rluc) was high (Table 1 and 2, Fig 5). PB treatment induced the downregulation of the fusion proteins (Table 1 and 2, Fig 5). The ratio of (Rluc+ Fluc) to Rluc intensity [(Rluc+Fluc)/Rluc] was significantly decreased in cells treated with PB, compared with normal cells (Table 1 and 2, Fig 5). These results suggested MAP3K7-LUC and ZNF91-LUC sensors can detect the alteration of RNA splicing in cells.

Table 1. The value of fluorescence in cell transfected with MAP3K7-LUC

Table 2. The value of fluorescence in cell transfected with ZNF91-LUC

Fig 5. The relative luciferase activity in cells. Cells were transfected with MAP3k7-LUC or ZNF91-LUC and treated with PB for 48 h.

3. The sensitivity of MAP3K7-LUC and ZNF91-LUC sensors

In order to test the sensitivity of MAP3K7-LUC and ZNF91-LUC sensors, different concentrations of plasmids (final concentration: 1 ng/μl、0.6 ng/μl、0.2 ng/μl) were transfected into cells. PB treatment decreased the ratio of (Rluc+ Fluc) to Rluc intensity [(Rluc+Fluc)/Rluc] in cells transfected with MAP3K7-LUC or ZNF91-LUC plasmids. Our data indicated even a small number of plasmid sensors can detect the changes of intracellular splicing, suggesting the sensitivity of our sensors is high (Table 3 and 4, Fig 6 and 7).

Table 3. The value of fluorescence in cell transfected different concentrations of MAP3K7-LUC

Fig 6. The relative luciferase activity in cells with different concentrations of transfected MAP3K7-LUC.

Table 4. The value of fluorescence in cell transfected with different concentrations of ZNF91-LUC

Fig 7. The relative luciferase activity in cells transfected with different concentrations of ZNF91-LUC.

4. The effect of MAP3K7-LUC and ZNF91-LUC sensors as monitors to detect the alteration of RNA splicing.

To test the effect of MAP3K7-LUC and ZNF91-LUC sensors as monitors to detect the alteration of RNA splicing, cells were transfected with plasmid sensors following treated with different concentration of PB (0.6 ng/μl, 0.2 ng/μl). The treatment with different concentration of PB represents the different degrees of RNA splicing alteration. In the presence of PB, the luminescence activity was significantly decreased in a dose dependent (Table 5 and 7, Fig 9 and 11). Moreover, based on the luciferase values in cell treated with different concentration of PB, the standard curve of MAP3K7-LUC and ZNF91-LUC sensors were made by EXCEL (Table 6 and 9, Fig 10 and 12). We find that the value of luciferase is dependent on the concentration of PB in cells. Based on the formula, the correlation coefficient (R2 value) of MAP3K7-LUC and ZNF91-LUC was 0.9915 and 0.998, respectively. The R2 value of ZNF91-LUC is better than MAP3K7-LUC. Importantly, we found that the slope of the standard curve of ZNF91-LUC was greater than MAP3K7-LUC, which indicates ZNF91-LUC is more sensitive than MAP3K7-LUC.

Fig 8. Cells in 24-well plates. The left is the cells before transfection and the right is the cells transfected with plasmids for 48 h.

Table 5. The value of fluorescence in cell transfected MAP3K7-LUC with different PB treatment

Fig 9. The relative luciferase activity in cells transfected with MAP3K7-LUC and treated with different concentration of PB.

Table 6. The original date of the standard curve of MAP3K7-LUC

Fig 10. The standard curve of MAP3K7-LUC

Table 7. The value of fluorescence in cell transfected with ZNF91-LUC and treated with different concentration of PB

Fig 11. The relative luciferase activity in cells transfected with ZNF91-LUC and treated with different concentration of PB.

Table 8. The original date of the standard curve of MAP3K7-LUC

Fig 12. The standard curve of ZNF91-LUC

5. Construction of MAP3K7-GFP

In order to visualize the situation of RNA splicing, we designed a new fluorescent plasmid sensor. The plasmid of MAP3K7-GFP was synthesized by Nanjing Genscript Biotechnology Corporation. MCherry gene was fused with a GTGAGT-recessive exon-CCACAG mingene. And a shifted in-frame termination codon (TGATG) was inserted between GFP and mCherry. The fused genes were respectively inserted into the pcDNA3.1(+)-C-eGFP plasmid vector which digested by BamH I and EcoR V (Fig 13). When we got these plasmids, we transformed them into DH5α bacteria and screened the positive clones on LB solid plates containing ampicillin (Fig 14). To validate these plasmids, MAP3K7-GFP was digested with BamH I and EcoR V and analyzed on 1% Agarose Gel (Fig 15).

Fig 13. Map of the pcDNA3.1(+)-C-eGFP plasmid vector

Fig 14. Solid plates of MAP3K7-GFP sensor

Fig 15. Electrophoresis of MAP3K7-GFP sensor

6. Functional verification of MAP3K7-GFP plasmid sensors.

Using the same method, we tested the ability of MAP3K7-GFP to detect dysregulation of RNA splicing in 293T cells with or without Pladienolide B treatment. The plasmid MAP3K7-GFP was transfected into 293T cells. PB (final concentration: 0.3 ng/μl) was added to disturb the process of RNA splicing. According to the principle of our plasmid sensor, during normal splicing of the cell, mCherry will be expressed but GFP could not be translatable and red fluorescent could be detected in cells. In the case where splicing is inhibited, the GFP is expressed, and the cells show green fluorescence (more details about the principle were listed in the project description). After 48 h cell culturing, we observed the fluorescence expression of cells under fluorescence microscope (10x eyepiece, 20x objective). We found that the group transfected with plasmid only had more red fluorescence than the group treated with PB. After transfection with MAP3K7-GFP plasmid, the GFP fluorescence was relatively less and dark. The GFP fluorescence was significantly enhanced in cells treated with PB, indicating that the inhibition of cell splicing was well detected by the plasmid sensor(Fig 16). After the observation of the cells, we digested them and added into a 96-well plate, and the fluorescence values of mCherry and GFP were measured by microplate reader (SpectraMax i3). We used the ratio of fluorescence values of mCherry and GFP to measure the inhibition of cell splicing. We found that the ratio of mCherry and GFP was 0.73 in cells transfected with MAP3K7-GFP. And the ratio of mCherry and GFP reached to 0.16 in cells following PB treatment (Table 9, Fig 17). There results were also consistent with the images of fluorescence in Fig 16. In conclusion, MAP3K7-GFP plasmid sensor can monitor the level of splicing in the cell well by detecting the different color of fluorescence.

Fig 16. Fluorescence images of cells transfected with MAP3K7-GFP

Table 9. MCherry and GFP fluorescence values of cells transfected with MAP3K7-GFP

Fig 17. The change of fluorescence value ratio in cells transfected with MAP3K7-GFP after PB treatment