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Novel SRPK 1 inhibitors specifically target alternative splicing in human primary retinal epithelial cells
Author(s) -
Rowlinson J.,
Batson J.,
Toop H.,
Allen C.,
Gibbons B.,
BabaeiJadidi R.,
Zhang J.,
Wearmouth S.,
Knapp S.,
Morris J.,
Bates D.
Publication year - 2015
Publication title -
acta ophthalmologica
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.534
H-Index - 87
eISSN - 1755-3768
pISSN - 1755-375X
DOI - 10.1111/j.1755-3768.2015.0625
Subject(s) - rna splicing , alternative splicing , sr protein , biology , gene isoform , microbiology and biotechnology , rna , biochemistry , gene
Purpose Specifically altering the splicing of VEGF ‐A from pro‐angiogenic VEGF ‐A 165 a to anti‐angiogenic VEGF ‐A 165 b could prove effective in the treatment of wet AMD . VEGF ‐A alternative splicing is controlled through RNA binding protein SRSF 1 which when phosphorylated by the serine kinase SRPK 1 induces pro‐angiogenic VEGF ‐A 165 a expression. We aimed to use novel SRPK 1 inhibitors that specifically alter splicing in retinal pigment epithelial ( RPE ) cells. Methods RPE cells were treated with novel compounds, synthesized based on the structure of SRPK 1, followed by extraction of RNA and protein. RT ‐ PCR and digital PCR was used to examine splicing of genes expressed in the eye or known targets of SRPK 1, and ELISA and immunoblotting were used to detect VEGF ‐A isoform expression. Results Novel SRPK 1 inhibitors dose‐dependently increased the expression of the anti‐angiogenic VEGF ‐A165b isoform. RT ‐ PCR showed that SRPK 1 inhibition altered alternative splicing of MKNK 2, a known splicing target of SRSF 1, resulting in a 2 fold change to isoform a in RPE cells. No change in splicing was observed for BCL 2L1, ARR 1, CAMK 2D, RAC 1, FN 1 or hn RNPB 1/A2. Other SRSF 1 targets such as TEAD 1, BIM and MST 1R were not expressed in RPE cells. Conclusions We have developed novel SRPK 1 inhibitors that specifically target splicing within the retina and offer an alternative more specific therapeutics for patients with exudative AMD .