Design

1. Design

Myelodysplastic syndromes (MDS) are clonal marrow stem-cell disorders, characterized by ineffective haemopoiesis leading to blood cytopenias, and by progression to acute myeloid leukaemia in a third of patients. 15% of cases occur after chemotherapy or radiotherapy for a previous cancer; the syndromes are most common in elderly people. Clinical manifestations result from cytopenias (anaemia, infection, and bleeding). Diagnosis is based on examination of blood and bone marrow showing blood cytopenias and hypercellular marrow with dysplasia, with or without excess of blasts [1,2,3].

Due to Clinical features of MDS are non-specific, many MDS patients globally are delayed in treatment because of the failure to detect MDS in time. Many patients mistakenly believe that they have anemia in the early stage of MDS, thus neglecting the treatment. Therefore, it is urgent to develop more effective detection methods for MDS diagnosis. Our team is committed to design a new model based on new biomarkers that can diagnose MDS with specificity, accuracy, and easy in the future.

RNA splicing is an essential event during gene expression in eukaryote. An accumulation of evidence now suggests that aberrant splicing is strongly associated with many diseases, especially MDS [1,2], leading to the emergence of pharmacological modulation of RNA splicing as a promising therapeutic strategy. There are many RNA splicing-related genes, such as SF3B1, U2AF2, and SRSF2, which have been found to have mutations in around 50% of MDS patients [3,4]. Among these genes, SF3B1 is the most frequently mutated RNA splicing factor in MDS [5], which is present in approximately 25% of MDS patients. Notably, SF3B1 mutations tend to be associated with a good prognosis in MDS . Therefore, the analysis of RNA splicing status could be used for the diagnosis of MDS.

2. Parts Design

According to previous studies, we found that exons of mitogen-activated protein kinase kinase kinase 7 (MAP3K7) and zinc finger protein 91 (ZFN91) were skipped in MDS patients [6,7]. This mechanism gives rise to our idea of constructing a sensor based on the exon skipping of MAP3K7 and ZFN91 to the plasmids containing reporter genes, which monitor the alteration of RNA splicing in cells. Our project will help us to evaluate the effectiveness of these sensors in diagnosing MDS patients.

To measure the alteration of RNA splicing, we engineered one dual luciferase reporter system where two luciferase genes were fused with GTGAGT-recessive exon (MAP3K7 or ZNF91)-CCACAG minigenes (Fig 1). Upon transfection of the dual luciferase reporters into mammalian cells, the pre-mRNA of this dual reporter would be processed in either of two ways. During normal splicing in cells, the internal stop codon would be removed, then the upstream reporter gene (Firefly luciferase, Fluc) and the downstream reporter gene (Renilla luciferase, Rluc) will be placed in the same reading frame to generate a fusion protein Fluc-Rluc. In the case that splicing in inhibited by splicing inhibitors, the stop codon in the recessive exon would lead to the translation termination of the mRNA, thus producing the Fluc protein alone. Therefore, the Fluc gene is expressed regardless of whether splicing occurred, whereas the downstream Rluc gene can only be expressed after splicing. The ratio of Rluc+ Fluc to Rluc intensity [(Rluc+Fluc)/Rluc] represents the proportion of spliced transcripts, that is, the splicing efficiency of pre-mRNA.

Figure 1. The dual luciferase reporter system

The ratio of (Rluc+ Fluc) to Rluc intensity [(Rluc+Fluc)/Rluc] represents the proportion of spliced transcripts, that is, the splicing efficiency of pre-mRNA. The dysregulation of RNA splicing has been reported in MDS patients. We tested the sensitivity of these reporters in cell lines. We were unable to test the reporters in MDS patients at present. We propose that these splicing reporters could help us to diagnosing MDS patients through using cell-free systems or adeno-associated virus (AAV) in the future. According to our experimental design, we can diagnosis MDS based on the the ratio of (Rluc+ Fluc) to Rluc intensity (figure 2)

Figure 2. The relative luciferase activity in healthy people and patients

In addition, we construed another GFP-mCherry reporter systems which two fluorescent genes were fused with a GTGAGT-recessive exon-CCACAG minigene. A shifted in-frame termination codon (TGATG) was inserted between GFP and mCherry (Fig 3). When the reporter was transfected into mammalian cells, the pre-mRNA of this dual reporter would be processed in either of two ways. During normal splicing of the cell, the internally shifted cryptic exon will be removed, mCherry will be expressed. Because the stop codon was located in front of the GFP gene, GFP could not be translatable and red fluorescent could be detected in cells. In the case where splicing is inhibited, the internally shifted recessive exon will not be removed, the mCherry gene is shifted. The GFP is expressed, and the cells show green fluorescence. Therefore, we can monitor the level of splicing in the cell through detecting the different colors of fluorescence.

Figure 3. The GFP-mCherry reporter system

[1]Mupo, Annalisa, et al. "Hemopoietic-specific Sf3b1-K700E knock-in mice display the splicing defect seen in human MDS but develop anemia without ring sideroblasts." Leukemia 31.3 (2017): 720-727.
[2]Malcovati, Luca, et al. "Clinical significance of SF3B1 mutations in myelodysplastic syndromes and myelodysplastic/myeloproliferative neoplasms." Blood, The Journal of the American Society of Hematology 118.24 (2011): 6239-6246.
[3]Adès, Lionel, Raphael Itzykson, and Pierre Fenaux. "Myelodysplastic syndromes." The Lancet 383.9936 (2014): 2239-2252.
[4]Hirai, Hisamaru, et al. "A point mutation at codon 13 of the N-ras oncogene in myelodysplastic syndrome." Nature 327.6121 (1987): 430-432.
[5]Hosono, Naoko. "Genetic abnormalities and pathophysiology of MDS." International journal of clinical oncology 24.8 (2019): 885-892.
[6] Sato, Shintaro, et al. "Essential function for the kinase TAK1 in innate and adaptive immune responses." Nature immunology 6.11 (2005): 1087-1095.
[7]Saminathan, Thangasamy, et al. "Differential gene expression and alternative splicing between diploid and tetraploid watermelon." Journal of experimental botany 66.5 (2015): 1369-1385.