Improvement
The introduction of our experiment:
Our improved experiment is based upon the YiYe-China iGEM project from 2022(https://2022.igem.wiki/yiye-china/). The project aims to provide a plasmid sensor to detect intracellular alternative splicing for early diagnosis of myelodysplastic syndromes(MDS). The project had established a dual luciferase reporter system to monitor the alteration of RNA splicing in cells These reporters incorporate segments of both the intronic and exonic sequences of MAP3K7 or ZNF91 within the luciferase gene (Fig 1). The activity of luciferase can be used to measure the changes of intracellular splicing, so as to play an auxiliary role in the diagnosis of MDS.
Fig .1 The dual luciferase reporter system
The dual luciferase reporter system works as follow: fusion proteins (Fluc-Rluc) were expressed in cells, but when RNA splicing was disrupted, only Fluc proteins were induced. In normal cells, the expression of fusion proteins (Fluc-Rluc) was notably high. Treatment with PB (dysregulate RNA splicing) led to the downregulation of these fusion proteins. The ratio of (Rluc+ Fluc) to Rluc intensity [(Rluc+Fluc)/Rluc] was significantly decreased in cells treated with PB compared to normal cells. These findings strongly indicate that the MAP3K7-LUC and ZNF91-LUC sensors can effectively detect alterations in RNA splicing in cells. Through these results, we found that the MAP3K7-LUC plasmid exhibited higher luciferase activity after transfection, which could potentially make the system more sensitive. Consequently, we focused on evaluating the performance of the MAP3K7-LUC sensor in detecting cellular RNA splicing abnormalities. Our goal is to improve the plasmid by improving the recognition ability of inserted introns, thereby increasing the sensitivity and splicing efficiency of this MAP3K7-LUC sensor and further improving its role in MDS diagnosis.
Improved part：
The Branch point site of introns play a key role in the alternative splicing of RNA. Using the https://hsf.genomnis.com/home tool, we predicted the branch point site of the MAP3K7-LUC inserted intron. The website identified two possible branch point motifs, "agctaAg" and "atctgAg." However, the "atctgAg" motif had a higher predicted value of 84.49(Fig 2). Additionally, considering the definition of the branch point site, this region is typically located approximately 30 nucleotides upstream of the 3 splice site (3 SS), and "atctgAg" is closer to the 3 SS. Therefore, we concluded that the A of "atctgAg" motif is the branch point site of the MAP3K7-LUC inserted intron.
Fig .2 MAP3K7-LUC inserts introns branch point site prediction
According to literature reports, the sequence of intron branch point sites is highly conserved. For example, the fourth and seventh positions are mostly pyrimidines, and the second and fifth positions are mostly purine. The heptanucleotide with the highest score (TACTA*C) based on the energy motif is completely complementary to the conserved motif (GTAGTA) in the U2 snRNP, which complies with the finding that TACTAAC is the most efficient BPS for mammalian mRNA splicing(Fig 3).
Fig .3 BPS motifs, the larger the letter, the greater the probability. (a)BPS motifs based on high energy efficiency;(b)BPS motif inferred through database
Based on the literature reports mentioned earlier, we mutated the "G" in the seventh position of the "atctgAg" motif to "C."
Subsequently, we entered the mutated intron sequence into the prediction website once more to determine the branch point site. After the improvement, we observed that the "atctgAc" motif obtained a higher predictive value of 87.21, which is higher than the value of the "atctgAg" motif (84.49) (Fig 4). We hypothesized that this improvement would enhance the splicing efficiency of the intron, thereby improving the recognition efficiency of MAP3K7-LUC receptors. We conducted the following experiment to test this hypothesis.
Fig. 4 MAP3K7-LUC IMPROVED inserts introns branch point site prediction
To introduce the desired point mutation into the MAP3K7-LUC plasmid, we initiated the process by designing a point mutation primer. Utilizing the original plasmid as a template, we performed PCR amplification. Subsequently, the amplified product was treated with the DpnI enzyme to digest the original template DNA, and the resulting product was then introduced into DH5α for transformation. After transformation, we plated the cells onto agar plates containing ampicillin to select for colonies with ampicillin resistance.(Fig 5)
Fig. 5 Solid plates of MAP3K7-LUC IMPROVED
From these plates, we isolated single colonies and sequenced. These results confirmed that the ATCGAG sequence at the branch point site of the intron insertion in MAP3K7-LUC had been successfully mutated to ATCGAC(Fig 6、7). And then we proceeded to amplify it from a single colony and extracted the newly constructed MAP3K7-LUC IMPROVED plasmid. This improved plasmid was then used in subsequent cell transfection experiments.
Fig .6 The MAP3K7-LUC plasmids sequencing
Fig .7 The MAP3K7-LUC IMPROVED plasmids sequencing
In order to test the ability of MAP3K7-LUC and MAP3K7-LUC IMPROVED to detect dysregulation of RNA splicing, Pladienolide B (PB, an RNA splicing inhibitor) was used. The plasmids MAP3K7-LUC and MAP3K7-LUC IMPROVED 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. We observed noticeable differences in the cell culture medium as well. The medium with PB exhibited slower consumption and a redder color, while the medium without PB remained more yellow (Fig 8). This observation aligns with the principles of our plasmid sensors. After transfection, we lysed the cells and measured luciferase expression using a plate reader (SpectraMax i3).
In normal cells, the expression of the fusion proteins (Fluc-Rluc) was high. Treatment with PB downregulated the expression of fusion proteins. Specifically, the ratio of (Rluc+ Fluc) to Rluc intensity [(Rluc+Fluc)/Rluc] was significantly reduced in cells treated with PB compared to normal cells. Notably, regardless of the presence of PB, the luciferase activity of the MAP3K7-LUC IMPROVED plasmid was consistently higher than that of MAP3K7-LUC. These results demonstrate that the plasmid sensor MAP3K7-LUC IMPROVED can effectively detect intracellular RNA alternative splicing (Table 1). Furthermore, our improvements have enhanced the splicing efficiency of the introns in the plasmid sensor, resulting in a dual luciferase reporter system with increased detection efficiency and sensitivity（Fig 9).
In conclusion, the ability of MAP3K7-LUC IMPROVED plasmid to detect dysregultion is more efficient than that of previous plasmid, which may provide the stronger power to dignosis MDS.
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 1. The value of fluorescence in cell transfected MAP3K7-LUC or MAP3K7-LUC IMPROVED with or without PB treatment
Fig .9 The relative luciferase activity in cells transfected with MAP3K7-LUC or MAP3K7 IMPROVED and with or without PB treatment.
Reference
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